Three-dimensional nodule bio-platform and uses thereof

Abstract

Provided herein is a 3-dimensional biological platform comprising immune cells and Mycobacterium that form Mycobacterium within spheroids and develop into tubercles with granulomatous lesions. Also provided are methods of making the biological platform and uses of the biological platform in screening methods.

Description

Statement regarding federally funded research or development

[0001] This invention was conducted with government support, in part, through internal grants granted by the Centers for Disease Control and Prevention (CDC) [Combating Antibiotic-Resistant Bacteria, Laboratory Safety Science and Innovation, and Division of Tuberculosis Elimination Intramural Research Funds]. The government holds certain rights to this invention. Technical Field

[0002] This invention relates to a 3D biological platform comprising immune cells and mycobacteria, which forms a co-culture of mycobacteria within a spheroid and develops into a tuberculoma with a cluster of granulomatous lesions. The invention also relates to methods for preparing fresh or cryo-stable biological platforms and the use of said biological platform in screening methods. Background Technology

[0003] Tuberculosis (TB) is one of the leading infectious killers worldwide, and current strategies for its prevention and treatment are insufficient to achieve eradication this century. A key strategy for achieving this goal is to develop shorter treatment regimens for both drug-sensitive and drug-resistant TB. While effective, standard treatment for drug-sensitive TB is lengthy, requiring 4 to 9 months, and for drug-resistant TB, treatment extends to 18–24 months. The combination of prolonged treatment duration and drug toxicity complicates treatment completion, thus promoting the development of resistance. Extensive efforts are underway to develop new antibiotics for treating TB, but conventional pathogen-targeting strategies have serious drawbacks that contribute to microbial resistance. To avoid these problems, a new treatment paradigm has emerged that involves the therapeutic modulation of the host immune response to improve pathogen eradication, known as host-directed therapy (HDT). HDT has the potential to not only shorten treatment but also reduce the likelihood of developing resistance. Furthermore, HDT can alleviate immune symptoms in target organs, promote immune memory, and prevent disease relapse.

[0004] Initially, most novel therapeutics were screened in vitro against specific pathogens, using materials such as agar or liquid cultures, without the presence of any host immune cells. However, this is not the optimal method for targeting intracellular pathogens such as Mycobacterium tuberculosis (Mtb). Animal models can be used to identify new therapeutics, but these models are very expensive and limit the number of compounds that can be screened. Furthermore, animal models may not accurately represent latent TB infection (LTBI) and TB disease in humans. Using animal models of TB for such screenings also presents significant challenges due to the need for animal biosafety level 3 (ABSL-3) facilities for these studies.

[0005] Macrophages are the natural target host cells for Mtb infection and serve as the primary microenvironment for infection and as frontline executors of protective immunity, acting as both innate and adaptive immunity, to control or eliminate the pathogen. However, Mtb presents numerous challenges by manipulating host signaling pathways to evade, tolerate, and disrupt innate and adaptive immune responses. This, however, also creates opportunities for novel therapeutic strategies such as HDT. Recent progress has been considerable in demonstrating the feasibility of HDT approaches targeting TB. However, little is known about the potential targets of HDT in humans. To advance HDT to clinical applications, a better understanding of the host-pathogen interaction is needed, particularly at the level of TB lesions known as granulomas. While granulomatous structures have traditionally been considered beneficial to the host by limiting infection, recent studies using animal models have revealed that they promote the growth and spread of mycobacteria and may be detrimental to the host. One of the more severe clinical manifestations of TB is the formation of multiple adjacent tuberculous granulomas in the infected organ (most commonly the lungs and brain) into structurally organized 3-dimensional (3D) masses, known as “tuberculomas.”

[0006] This field faces numerous challenges in accelerating research to identify Mtb-manipulated host pathways that HDT or immunotherapies can target. Most importantly, we lack a rapid, robust, and widely applicable biological platform for the rapid screening of host-targeted compounds and other therapeutics against TB.

[0007] Traditional 2D cell cultures (also known as macrophage "monolayers") can be used to identify therapeutics that modulate host-pathogen interactions. However, this approach is limited by poor predictability of drug efficacy and toxicity, a lack of accurate insight into the mode of action of selected compounds, and the inability of the system to mimic the microenvironment within TB granulomatous lesions. Some drugs that are effective against TB in the granulomatous environment cannot be identified using 2D systems because they are ineffective in the in vitro 2D environment. However, many published studies have used 2D cell cultures of human or mouse monocyte / macrophage lines, typically after mitogen stimulation and differentiation (which leads to alterations in cell signaling and other host cell processes). Primary human macrophages are generally preferred over monocyte / macrophage lines for studying host-pathogen interactions in vitro. However, even bronchoalveolar lavage fluid (BAL) or leukapheresis typically cannot generate sufficient lung or blood monocyte-derived macrophages from human donors for high-throughput screening. To circumvent many of these problems and mimic the microenvironment in human tuberculous granulomas (TB), in vitro cell culture models of granuloma formation have been developed using human peripheral blood mononuclear cells (PBMCs) infected with Mtb. Many of these granuloma models have been developed in 2D systems, such as flat-bottomed cell culture microplates. These 2D models exhibit some characteristics of granulomas, such as macrophage aggregation, multinucleated giant cell formation, and necrosis; however, these structures are poorly organized, small in size, susceptible to disturbance during culture replenishment, and lack key granulomatous properties because they do not undergo the full range of changes associated with the structure of human 3D tuberculous granulomas. These 2D granuloma models are typically developed using human PBMCs differentiated from granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF). However, due to this in vitro differentiation and activation process, they are not suitable for elucidating the precise mechanisms of action of HDT compounds and immunotherapeutic agents.

[0008] Human TB granulomatous lesions are tissue-organized 3D aggregates of immune cells, primarily composed of a central core of tissue-resident and recruited blood mononuclear cell-derived macrophages, surrounded by lymphocytes, in the infected lung. Therefore, in vitro granuloma models have also been developed using human PBMCs and / or lung cell lines (embedded in a collagen matrix in flat-bottomed microplates to form 3D structures). In these 3D models, loose, small granulomatous aggregates of macrophages form in response to Mtb infection, and the bacteria exhibit dormancy characteristics. While useful for studying host-pathogen interactions during latency and recovery, this model is typically time-consuming, labor-intensive, and has low throughput. Furthermore, there are technical challenges in adding more host cells to maintain dynamic granulomatous structures over extended time periods, and enzymatic treatment with collagenase is required to release cells from the matrix for downstream applications.

[0009] Another recently developed in vitro model uses a 3D system based on encapsulating Mtb-infected PBMCs and collagen matrix within alginate microspheres via bioelectrospray. This encapsulated system is then imaged and used in 2D microplates to study host-pathogen interactions or the effects of anti-TB drugs. While applicable and helpful in studying different aspects of host-pathogen interactions and the effects of anti-mycobacterial agents, the cell aggregates formed in this model are small and lack well-organized solid or cavitary granulomatous lesion formation and associated microenvironment, including central necrosis, cavity formation, and gradients of oxygen, nutrients, and drugs due to the porous nature of the microspheres. The effect of the voltage applied during bioelectrospray on host cell responses is also not fully understood. The development of this 3D system requires technical expertise and instrumentation in bioelectrospray at the BSL-3 facility. Further addition of cells within the hardened microspheres to simulate cell recruitment is not feasible, and cells need to be released from the microspheres for downstream assays (such as flow cytometry or RNA sequencing), which could affect transcriptomic and immunological characterization. Recently, a novel in vitro 3D culture system has been described. In this technique, fully tissueed granulomas from an adult zebrafish model infected with Mycobacterium marinum are microscopically dissected and maintained in 3D cell culture. While this technique facilitates high-resolution imaging and manipulation of granulomas, only a small number of granulomas (20-100) can be microscopically dissected per animal, which is inefficient for high-throughput compound screening, and it also requires specialized facilities, specialized instruments, and technical expertise to accommodate infected animals.

[0010] As mentioned above, available 2D and 3D cell culture systems and in vitro granuloma models lack key features and microenvironments in human TB lesions, such as the formation of tuberculoma structures, well-tissued granulomatous lesions, biochemical and physicochemical gradients, and the generation of enhanced hypoxia, necrosis, acidosis, and cavitation. Limited 3D bioplatforms (such as collagen-embedded human PBMC models or alginate microsphere models encapsulating human PBMCs mixed with the extracellular matrix) are complex, lack adaptability and throughput for drug screening, and releasing live cells from these bioplatforms for further downstream studies is challenging. The size and density of microcellular aggregates formed in these models vary greatly between blood donors, and these aggregates are highly unstable. In addition to reducing bacterial load, an ideal aspect of human TB treatment is the reduction in the number and size of granulomatous lesions (immunopathology), as a measure of treatment success, cure, and preservation of tissue structure. Current in vitro models and biological platforms lack the ability to provide multidimensional assessments of therapeutic efficacy, including, in addition to reductions in bacterial load, drug penetration into granulomatous lesions and the reduction in the number and size of these lesions. Furthermore, the successful preservation, maintenance, and long-term storage of screening biological platforms are ideal for commercial and research applications, and the storage stability of currently available granulomatous platforms is unknown. Therefore, there is a need in the art for improved, robust, and storage-stable 3D biological platforms for investigating host-pathogen interactions involving TB and screening TB therapeutics. Summary of the Invention

[0011] This article provides a three-dimensional (3D) cell co-culture comprising a variety of human immune cells and a variety of mycobacteria. The 3D co-culture can be prepared by: (a) suspending the human immune cells in Roswell Park Memorial Institute cell culture medium further comprising one or more of L-glutamine, FBS, sodium pyruvate, and HEPES buffer; (b) mixing the mycobacteria with the human immune cells at an MOI of 0.005–0.5; wherein the mixture is contained in polystyrene U-bottom ultra-low adsorption wells of a microplate, wherein the wells contain a hydrogel coating, and wherein the hydrogel is hydrophilic, neutrally charged, and bioinert; and (c) incubating the mixture at 37ºC for a time sufficient to allow the human immune cells and mycobacteria to form mycobacterial structures and granulomatous lesions within spheroids.

[0012] This article also provides a method for preparing a 3D cell coculture comprising multiple human cells and multiple mycobacteria. The method may include (a) suspending the human immune cells in a cell culture medium further comprising one or more of L-glutamine, FBS, sodium pyruvate, and HEPES buffer; (b) mixing the mycobacteria with the human immune cells at an MOI of 0.005-0.5; wherein the mixture is contained in the wells of a microplate; and (c) incubating the mixture at 37ºC for a time sufficient to allow the human immune cells and mycobacteria to form mycobacterial structures and granulomatous lesions within spheroids.

[0013] The mycobacterial structures within spheroids disclosed herein can be formed by human immune cells and various mycobacteria. The human immune cells may include cells selected from monocytes, macrophages, and peripheral blood mononuclear cells (PBMCs). The monocytes may include THP-1 cells, U937 tissue cells, and purified CD14. + Monocytes, or monocyte subsets within PBMCs. The mycobacteria may include Mycobacterium marinum (Mm), Mycobacterium tuberculosis (Mtb), or a Mycobacterium tuberculosis complex. The Mm may include strain 1218 or strain M. The Mtb may include strain H37Rv, strain Erdman, strain CDC1551, strain Beijing F2, or clinical isolates of drug-sensitive or drug-resistant Mtb.

[0014] The 3D coculture may contain 500-5000 colony-forming units (CFU) of mycobacteria / 10 5 Personal immune cells result in a multiplicity of infection (MOI) of 0.005-0.05. The 3D co-culture may include Mm strain 1218 or strain M, and the MOI may be about 0.006-0.012. The 3D co-culture may include Mtb, and the MOI may be about 0.012-0.05, or particularly about 0.025. The 3D co-culture may include Mtb strain H37Rv, and the MOI may be about 0.015-0.05. The MOI may be about 0.025. The 3D co-culture may include Mtb Erdman or Beijing F2, and the MOI may be about 0.12-0.05. The 3D co-culture may include Mtb Erdman, and the MOI may be about 0.05. In the methods disclosed herein, the mycobacteria may be mixed with human immune cells at an MOI.

[0015] The mycobacteria can express green fluorescent protein (GFP), red fluorescent protein (RFP), fluorescent markers, or luminescent markers. The RFP may include tdTomato or far-red fluorescent protein. The 3D cell co-culture may be contained in U-shaped wells, which may contain a hydrogel coating. The hydrogel may be one or more of the following: hydrophilic, neutrally charged, and bioinert. The hydrogel may contain perfluorinated polymers, olefins, or combinations thereof. The hydrogel may include CORNING® PURMAPTRIX™ peptide hydrogel. The hydrogel may contain one or more of collagen, fibrin, and alginate. The wells may be CORNING® ultra-low adsorption spherical microplates or S-BIOPRIMESURFACE® 3D culture spherical plates. The 3D cell coculture may further comprise one or more additional cell types selected from the following: A549 human lung epithelial cells, HUVEC-1 human fetal endothelial cells, HULEC human lung endothelial cells, and MRC-5 human lung fibroblasts, or primary human epithelial cells, endothelial cells, and fibroblasts.

[0016] In the method disclosed herein, the mycobacterial structure enclosed in spheroids can be brought into contact with extracellular matrix (ECM). Three days after mixing the human immune cells and the mycobacteria, the ECM can be added to the mycobacterial structure enclosed in spheroids. The method may further include, within 30 minutes of mixing the human immune cells and the mycobacteria, placing and storing the mixture at -80ºC and optionally at ≤-160ºC. The method may further include, after mixing the human cells and the mycobacteria, incubating the mixture at 37ºC for 16-72 hours; removing the cell culture medium; adding a cryopreservative to the co-culture of human cells and mycobacteria; and freezing the spheroid co-culture. The cryopreservative may contain 5% (v / v) dimethyl sulfoxide (DMSO) in RPMI-1640 or 3D cell culture medium. The cryopreservative may further contain heat-inactivated fetal bovine serum.

[0017] This article provides a method for screening molecules capable of preventing, treating, or reducing mycobacterial infections. The method may include (a) contacting the 3D bioplatform with the molecule; (b) measuring one or more properties of the mycobacterial structure within the spheroid; and (c) comparing one of the one or more properties with a control, wherein a change in at least one of the one or more properties indicates that the molecule is capable of treating or reducing the mycobacterial infection or one or more pathological features thereof. The one or more properties may include one or more of the following: (a) the amount of fluorescence produced by the mycobacteria, wherein the mycobacteria express fluorescent molecules; (b) granuloma integrity; (c) mycobacterial growth; (d) granuloma formation; (e) granuloma growth; (f) granuloma number and size; (g) autophagy-lysosome formation; (h) inflammasome and pyroptosis induction; (i) hypoxia induction or inhibition; (j) lysosomal acidification; (k) host cytotoxicity; and (l) the expression level of one or more gene or protein biomarkers expressed by cells of the 3D bioplatform.

[0018] The one or more properties may include the amount of fluorescence produced by the mycobacteria. The amount of fluorescence may indicate the mycobacterial load. A reduction in the mycobacterial load of the molecule to a threshold, compared to a control, may indicate that the molecule is capable of treating or reducing mycobacterial infection or one or more of its pathological features. The threshold may be a reduction of at least 25%. The threshold may be a z-score less than -2 or less than -4.

[0019] The one or more properties may include the expression levels of one or more genes expressed by cells of the 3D bioplatform. Changes in the expression levels of the one or more genes, proteins, or immune processes compared to a control may indicate that the molecule can treat or reduce mycobacterial infection or one or more of its pathological features. The control may comprise uninfected human immune cells or infected but untreated human immune cells. The molecule may be added approximately 0–6 days after co-incubation of the mycobacteria and the human immune cells. The one or more properties may be observed once or multiple times within 1–14 days after contacting the 3D bioplatform with the molecule, optionally once on days 7, 9, and 12.

[0020] This article provides a method for treating or reducing tuberculomatous lesions in a subject of need. The method may include administering an antimycobacterial agent to the subject. An antimycobacterial agent for treating or reducing tuberculomatous lesions is also provided, as well as the use of the antimycobacterial agent in the manufacture of a medicament for treating or reducing tuberculomatous lesions. The antimycobacterial agent may be selected from antiCD11a antibody, anti-α4β7 integrin antibody, antiCD30 antibody, antiIGF1R antibody, antiIL-6R antibody, AT9283, tinizonide (the active metabolite of nitrozonide), dasatinib, quinacrine dihydrochloride, all-trans retinoic acid (ATRA), vorinostat, sitagliptin, H89, and lansoprazole. The antimycobacterial agent may be selected from anti-CD11a antibody, anti-α4β7 integrin antibody, anti-CD30 antibody, anti-IGF1R antibody, anti-IL-6R antibody, AT9283, tinizonide (the active metabolite of nitrozonide), dasatinib, quinacrine dihydrochloride, all-trans retinoic acid (ATRA), vorinostat, sitagliptin, H89, and lansoprazole. The subject may have a mycobacterial infection.

[0021] This article provides a method for screening candidate agents that induce training immunity in human immune cells. The method may include measuring the growth of pathogenic mycobacteria in a 3D platform after co-culturing human immune cells and pathogenic mycobacteria. The human immune cells may have already been contacted with the candidate agent. Reduced growth of the pathogenic mycobacteria or reduced granulomatous lesions through co-culturing can indicate that the candidate agent induces training immunity in the human immune cells. The candidate agent may be a vaccine, immunotherapeutic agent, biologic, or host-targeted therapeutic agent. Attached Figure Description

[0022] This patent or application document contains at least one color drawing. Upon request and payment of the necessary fees, the official authority will provide a copy of this patent or patent application publication with one or more color drawings.

[0023] Figures 1A-1D This diagram illustrates a workflow type of "Mycobacteria in spheroids" co-culture in a 96-well 3D cell culture microplate for generating a human 3D tuberculoma bioplatform. Figure 1A A workflow for generating congenital solid 3D tuberculoma-like structures using freshly cultured immortalized human THP-1 mononuclear cells and pathogenic mycobacteria. Figure 1B An improved workflow for incorporating THP-1 monocytes and pathogenic mycobacteria into the human extracellular matrix to generate 3D tuberculoma-like structures with cavitary features. Figure 1C Purified primary human blood CD14 +A workflow for generating human donor-specific neoplastic 3D in vitro tuberculomas with innate and adaptive immune cells from monocytes and pathogenic mycobacteria (which subsequently include lymphocyte-rich autologous PBMC subsets). Figure 1D A workflow for a cryo-stable 3D tuberculoma bioplatform using human THP-1 mononuclear cells and pathogenic mycobacteria, said bioplatform being cryopreserved for future use and revived as needed to generate solid 3D tuberculomas. 3D refers to three-dimensional structures; THP-1 is a mononuclear cell line isolated from peripheral blood of human leukemia patients; ECM is extracellular matrix; and PBMCs are peripheral blood mononuclear cells.

[0024] Figures 2A-2D The results show the results of studying different multiplicity doses of two fluorescent Mycobacterium marinum strains in a co-culture workflow of "Mycobacteria in spheroids" in order to optimize the 3D tuberculoma biological platform. Figures 2A-2B The results show the effects of using eight different MOI doses (1 to 6000 CFU). Figure 2A Mycobacterium marinum 1218 (GFP) and ( Figure 2B THP-1 mononuclear cells (1×10⁻¹⁰) produced by Mycobacterium tdTomato strain "M" 5 The kinetics of mycobacterial proliferation, granulomatous lesion formation, and subsequent pathogenesis in co-cultures of "mycobacteria in spheroids" were studied. Representative images are from one of two experiments conducted for each strain, and are images of one of six 3D spheroids produced using eight MOI doses or an infection-free control, taken at five different time points within three weeks of growth. Scale 2000 µm. Figures 2C-2D This demonstrates the use of THP-1 mononuclear cells (1×10⁻⁶). 5 ) and nine different progressively increasing MOI doses (1 to 12,000 CFU) Figure 2C Mycobacterium marinum 1218 (GFP) and ( Figure 2DPercentage of area affected by mycobacterial proliferation and granulomatous lesions in 3D cocultures of *Mycobacterium tdTomato* strain “M”. Circles represent the degree of lesioning in a 3D spheroid. Bars represent the mean percentage of area affected by lesioning. Data are the mean ± SD from one of two experiments (n = 6 spheroids). Coculturing *Mycobacterium tdTomato* strain with a low MOI of 0.006 (600 CFU) resulted in well-tissued granulomatous lesions, with <50% and 75% of 3D tuberculoma area affected by mycobacterial growth by day 12 and day 21, respectively. The percentage of affected area and induced lesioning after low MOI 0.006 were compared with the percentage of affected area and induced lesioning after standard MOI 0.12 and the uninfected control. *p < 0.05 and ****p < 0.0001 were determined using the Kruskal-Wallis and Dunn post-hoc tests. NS, not significant.

[0025] Figures 3A-3D This demonstrates a tuberculoma form developed using a 3D “mycobacteria in spheroids” co-culture workflow with freshly cultured human immortalized THP-1 or purified primary blood mononuclear cells and pathogenic mycobacteria. Figure 3A Solid tuberculomas generated and imaged on day 12 after 3D co-culture of freshly cultured THP-1 mononuclear cells with pathogenic Mycobacterium marinum “M”, Mycobacterium tuberculosis (M. tuberculosis) H37Rv, Beijing F2, CDC1551, or Erdman strains expressing tdTomato fluorescent protein. Scale: 2000 µm. Figure 3B Representative images of solid tuberculomas or uninfected control spheroids stained with commercially available staining kits following manufacturer's instructions for necrosis (green), hypoxia (red), and acidosis (red) generated after 3D co-culture of THP-1 monocytes and wild-type Mycobacterium marineis "M". Figure 3C This demonstrates the generation of 3D tuberculomas following a modified 3D co-culture workflow of freshly cultured THP-1 mononuclear cells and a pathogenic Mycobacterium marinum strain 1218 expressing GFP, with the incorporation of human ECM (0 µl, 10 µl, or 30 µl) composed of type I human collagen and fibronectin. The scale was 2000 µm. A cavitary feature was generated in the 3D tuberculomas using 30 µl ECM, but no cavitary feature was generated in the control 3D spheroids. Figure 3D Following 3D co-culture of total human PBMCs with Mycobacterium marineis 1218 (GFP) or Mycobacterium marineis “M” (tdTomato) or with purified CD14 from human blood supplemented with PBMC fractions rich in autologous lymphocytes. +Solid tuberculomas derive from 3D co-culture of monocytes and Mycobacterium tuberculosis Erdman. 3D co-culture of total PBMCs and Mycobacterium tuberculosis produces small cellular granuloma aggregates, while purified CD14... + 3D co-culture of monocytes and Mycobacterium tuberculosis Erdman produced well-tissued granulomatous lesions. Figures 3A-3D The representative images in the study come from two or more independent experiments with at least three 3D spheres.

[0026] Figures 4A-4F This illustrates a solid tuberculoma formed after thawing and resuscitation of a cryopreserved 3D "mycobacteria in spheroids" co-culture produced using THP-1 mononuclear cells and Mycobacterium tdTomato. Figures 4A-4B The effects of three different cryomediums on granuloma formation in 3D cocultures were shown. Compared with FBS-based or L15-based cryomediums, 3D cocultures frozen 16 hours after co-culturing in RPMI 1640-based cryomedium and thawed 6 weeks or 16 months after freezing produced significantly more well-tissued granulomatous lesions. Figure 4C The kinetics of granulomatous lesion formation following thawing and freezing of 3D cocultures were demonstrated. Comparable trends in granulomatous lesion formation were observed in 3D cocultures that were co-cultured in RPMI 1640-based cryogenic medium, frozen for 30 minutes or 16 hours, and then thawed 6 weeks after freezing. Figures 4D-4E The effect of freezing duration on granulomatous lesion formation was demonstrated. No significant difference in granuloma counts was found in 3D cocultures that were frozen in RPMI 1640-based cryo-medium 30 minutes after co-culture and thawed after 3 days, 6 weeks, or 16 months of freezing. Figure 4F The effect of freezing time after 3D co-culture on histiolated granuloma formation was shown. Compared with 72 hours after co-culture in RPMI 1640-based freezing medium, freezing time at 30 minutes or 16 hours after co-culture was significantly reduced. Figure 4A and Figure 4C (Middle) Frozen and thawed 3D cocultures showed numerous well-tissued granulomatous lesions. Scale: 2000 µm. Representative images and data (mean ± SD) are from one to three independent experiments (n = 6–16 3D spheroids / group). Circles in the histogram represent the granulomatous lesion count for each 3D coculture. *** and **** indicate p < 0.001 and < 0.0001, respectively, using Kruskal-Wallis and Dunn post-hoc tests. NS, not significant.

[0027] Figures 5A-5E The homogeneity and reproducibility of the 3D tuberculoma and biological platform are demonstrated. Figures 5A-5D This shows the diameter of a solid 3D tuberculoma developed in a 96-well microplate by two separate assay performers using freshly cultured THP-1 mononuclear cells and Mycobacterium tdTomato. Figure 5A ),area( Figure 5B ), Granulomatous lesion count ( Figure 5C ) and bacterial load ( Figure 5D Consistency. Figure 5E The isolation of bacterial load in 3D tuberculomas treated with nitrozonidine or rifampin (positive control) and untreated or DMSO-treated (negative control) is shown. Data in the box plots in AD (whisker lines: 10–90 percentile) are from 72–144 tuberculomas per treatment in independent experiments. Circles represent the response of each 3D tuberculoma. No significant differences were found in parameters of 3D tuberculomas developed by the two performers using the Mann-Whitney test. ND, not performed. Figure 5E The bacterial load data in the data were derived from 3–6 tuberculomas per treatment, which were derived from a total of 48 3D cell culture plates (24 plates per executor) developed for screening potential pathogen-targeting and host-directed compounds using the 3D tuberculoma biotechnology platform. Rifampin was not tested in plates 25–48 developed by executor 2.

[0028] Figures 6A-6D The assay quality and high-throughput screening compatibility of the 3D tuberculoma biological platform determined by the Z' factor are demonstrated. Figures 6A-6C This study demonstrates the effect of infection dose (MOI) on the Z' factor in drug screening assays within a 3D tuberculoma bioplatform developed using THP-1 mononuclear cells and different infection doses of Mycobacterium tdTomato in 96-well microplates. Three different MOI doses were investigated: 0.0059 ( Figure 6A ), 0.0068 ( Figure 6B ) and 0.0091 ( Figure 6C The top inset shows the Z' factor in assays using 8–10 microplates / MOI, with each plate including negative and positive controls. Relative fluorescence units (RFU) data indicating bacterial load are presented as box plots, where whisker lines (minimum to maximum) show all data points, with individual circles representing the RFU level in a single solid 3D spheroid. Fluorescence intensity was measured using automatic gain, and the range of automatic gain values ​​for the microplates used in the assays is shown. The bottom inset shows the Z' factor in individual plates assayed by the bioplatform, with a single symbol representing the Z' factor in a single microplate. Assays using an MOI of 0.0091 indicate excellent to good assay quality (mean Z' factor ≥ 0.5, indicated by a red dashed line). Figure 6DA plate diagram showing the typical locations of control and test wells on a 96-well plate is provided. In the primary screening of the compound library, a total of 16 compounds can be screened in triplicate on each plate. Figures 7A-7J This study demonstrates a simple and easy-to-use in vitro tuberculoma model established through 3D cell culture of human monocytes and pathogenic mycobacteria. Figure 7A The use of pathogenic fluorescent mycobacteria and human THP-1 or primary CD14 in Corning 3D cell culture microplates for generating 3D tuberculoma models and for treatment screening and characterization is shown. + A schematic overview of the workflow for co-culturing blood mononuclear cells with mycobacteria in spheroids. Magnetic-associated cell isolation (MACS) – purified human primary blood CD14. + Monocytes or THP-1 monocytes were co-cultured with fluorescently pathogenic mycobacteria in rationally selected 3D cell culture microplates. On day 3, CD14 cells were introduced... + Supplementing autologous human CD14 in cocultures of monocytes and mycobacteria - PBMC subpopulations. Alternatively, reporter THP-1 cells and mycobacteria will be used to generate co-cultures of "mycobacteria in spheroids" to study the microenvironment and immune mechanisms. 3D tuberculoma-like structures generated from aggregated granulomas will be used for fluorescence-based, continuous, high-content imaging and to study host-pathogen interactions. The resulting HTS-compatible biological platform will be used to rapidly screen biologics and chemical compounds to quantitatively evaluate drug efficacy in host-directed immune mechanisms of bacterial growth inhibition and granuloma regression, cytotoxicity, and in situ drug action. Animal models can then be used to validate the identified "preferred lead" compounds.

[0029] Figure 7B The use of human THP-1 mononuclear cells (1×10⁻⁶) was shown. 5 Microscopic examination of co-cultures produced by Mm 1218 GFP (MOI 0.006) showed 3D spheroid formation (day 2) and mature structures (days 16 and 28) of cell aggregates including well-organized florid granulomatous lesions (mean diameter 375 µm) similar to those in solid tuberculomas (mean diameter 2024 µm). Figure 7C This image shows a series of images over three weeks in co-cultures produced using either strain Mm "M" (tdTomato) or 1218 (GFP) (MOI 0.006). Representative images are from one of two experiments investigating the growth kinetics of co-cultures produced in 200 µl of medium and subsequently supplemented with 50 µl of fresh medium (days 6 and 14). Figure 7DThe growth of Mm“M”(tdTomato) in a 3D co-culture is shown, measured using relative fluorescence units (RFU) and colony forming units (CFU) counts. Figure 7E The cytotoxicity of host cells as measured by CytoTox Glo in 3D cell cultures infected or uninfected with Mm“M”(tdTomato) is shown. Figure 7D and Figure 7E Data in this study were derived from one of two experiments (for RFU, n = 204–282 infected spheroids and 18 uninfected spheroids; for CFU, n = 6 infected spheroids at each time point; and for cytotoxicity, n = 12 infected spheroids and 12 uninfected spheroids at each time point). Error bars indicate SD. Comparison with uninfected controls was performed using two-way ANOVA with the Dunnett test. Figure 7D ) and Welch t-test ( Figure 7E ), ****p < 0.0001. Figure 7F 3D co-cultures of THP-1 mononuclear cells and virulent Mtb strain (tdTomato) using MOI 0.01 (ad) or attenuated Mycobacterium bovis BCG strain (WT or GFP) using MOI 0.01 (i and iii) or ten times MOI 0.1 (ii and iv) are shown. Figure 7G and Figure 7H The results on day 9 showed the effects on pancadherin and E-cadherin ( Figure 7G ) or ICAM-1 ( Figure 7H 3D co-culture of THP-1 monocytes and WT Mm“M” (MOI 0.004) subjected to immunostaining (red). Figure 7I 3D spheroid cultures of intact human PBMCs with or without mycobacterial infection are shown. Representative co-culture images were created using Mm 1218 GFP (MOI 0.01). Figure 7J Primary CD14 purified using MACS is shown. + Blood mononuclear cells (2×10) 5 / hole) and Mtb Erdman (tdTomato) (MOI 0.05) and then supplemented with autologous CD14 on day 3. - PBMC subgroup (4×10) 5 3D co-cultures ( / well).

[0030] Figures 8A-8D A 3D co-culture of "mycobacteria in spheroids" using human or mouse macrophage cell lines and Mycobacterium fluorescens strains is shown. Figure 8AThe growth of strains Mm“M” (tdTomato) and 1218 (GFP) at 30ºC or 37ºC in Middlebrook 7H9 or RPMI-1640 medium is shown by spectrophotometry. Data are for growth in 5 ml of medium in culture tubes using 100 µl of inoculum (1 × 10⁻⁶) from a frozen stock. 7 Optical density (OD) at 600 nm of pure cultures (n = 3) started at CFU / ml. Error bars indicate SD. Strain growth at two temperatures was compared by two-way ANOVA with Tukey's test (black asterisk for three-week growth curves) or Welch's t-test (colored asterisk for growth at different time points), **p < 0.01, ***p < 0.001, and ****p < 0.0001. Figure 8B and Figure 8C The growth of Mm“M” (tdTomato) and Mm 1218 (GFP) in a 3D coculture of monocyte-macrophage cell lines generated at 37ºC in RPMI-1640 medium in a 96-well Corning 3D cell culture microplate is shown. Five different cell lines were investigated: THP-1 monocytes (human leukemia), U937 pleural fluid macrophages (human histiocytic lymphoma), J744 ascites macrophages (BALB / c mouse reticulum cell sarcoma), RAW-264.7 macrophages (BALB / c mouse leukemia virus transformed), and AMJ2 alveolar macrophages (C57BL / 6J mice). Figure 8B Representative images of 3D spheroids infected with Mm or uninfected control 3D spheroids are shown on day 12. Figure 8C The results show that despite exchanging phenol red RPMI-1640 medium with PBS (pH 7.2), the fluorescence intensity, measured in relative fluorescence units (RFU), still reveals background autofluorescence in the GFP channels of uninfected 3D spheroids. Data are presented for 5–8 infected spheroids and 4 control spheroids per cell line. Error bars indicate SD. Multiple comparison tests were performed using Brown-Forsyth and Welch ANOVA, with *p < 0.05, **p < 0.01, and ****p < 0.0001. Figure 8D Linear relationships between the number of red fluorescent bacteria and RFU (reactive protein urinate) in 3D spheroids, 7H9 medium, or RPMI 1640 medium are shown. Error bars indicate SD (sigma index). Pearson correlation coefficient R is shown. 2 And the two-tailed P-value.

[0031] Figures 9A-9D The results show that the formation of well-organized tuberculoma-like structures in co-culture depends on the cell culture vessel and growth conditions. Figure 9AThe co-culture of THP-1 mononuclear cells and Mm "M" (tdTomato) in 96-well 3D and 2D cell culture plates with different micropore geometries and cell culture surfaces is shown. Figure 9B The co-culture of THP-1 monocytes and Mm "M" (tdTomato) in a polymer-encapsulated 3D "cell-in-box" system is shown. Regardless of whether the THP-1 cells were mixed with bacteria or infected with bacteria (overnight) before being encapsulated in natural cellulose polymer microparticles, small granulomatous aggregates rather than well-tissued lesions were formed in the encapsulated 3D system. Figure 9C This study shows the lowest background noise and autofluorescence detected in isolated RPMI-1640 and uninfected controls in Corning 3D Ultra-Low Adsorption (ULA) plates, although a significant reduction in fluorescence was observed in rifampicin (1 µg / ml)-treated co-cultures relative to untreated or DMSO-treated controls across all plate types. Data were presented in 3–21 replicates per treatment or control. RFU, Relative Fluorescence Units. Figure 9D The study showed that the bacterial load most significantly observed in Corning 3D ULA plates gradually decreased with increasing rifampicin dosage. Data were presented as 21 untreated spheroids (0 µg / ml) and 3 treated spheroids per dosage. Figure 9C and Figure 9D Error bars in the data indicate SD. Multiple comparison tests were performed using Brown-Forsyth and Welch ANOVA, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Low MOI (0.006) was used in all experiments, and representative images were taken on day 12 after co-culture. Figure 9A and Figure 9B ) and fluorescence intensity readings ( Figure 9C and Figure 9D ).

[0032] Figures 10A-10C A 3D cell culture model of dynamic granulomatous lesion generation and expression of characteristic markers is shown. Figure 10A The spatiotemporal dynamics of host and bacterial growth, as well as the development of granulomatous lesions, are illustrated in 3D spheroid co-cultures produced using red fluorescent THP-1 mononuclear cells and green fluorescent mycobacteria, or green fluorescent THP-1 cells and red fluorescent mycobacteria. Arrows are used to indicate examples of coalescing granulomatous lesions (white arrows), rapidly progressing lesions (blue arrows), and relatively stable lesions (green arrows). Figure 10BThe macrophage epithelialization and adhesion junction markers pancadherin, E-cadherin, N-cadherin, ICAM-1 (all in red) and γ-catenin (green) are shown as detected by immunostaining in neogranuloma aggregates on day 10 in a 3D co-culture of THP-1 cells and WT Mm“M” (MOI 0.004). Figure 10C The study presents 3D co-cultures immunized with isotype control antibodies against the specific immune target investigated in the study, demonstrating the absence of nonspecific staining in the resulting granulomatous lesions.

[0033] Figure 11 A- Figure 11C The model shows the classic features and environment of human tuberculoma, and indicates the structural tissue. Figure 11 A shows increased expression of the angiogenesis marker VEGFR-2 (a), the microtubule marker indicating cytoskeleton remodeling (b), and the immunomodulatory and intimal injury marker galactagogue 9 (c) in granulomatous macrophages, detected by immunostaining (red) in 3D models of THP-1 monocytes and WT Mm “M”. (d) 3D coculture of autophagy reporter THP-1 monocytes indicates blockade of autophagy flux (yellow-green spots), where autolysosome formation in permissive macrophages of granulomatous lesions is inhibited. (e) 3D coculture of inflammasome reporter THP-1 monocytes shows inflammasome activation in framework macrophages (green spots). (f) and (e) are magnified views of increased inflammasome activation in epithelioid macrophages attempting to limit Mtb (yellow) granulomas (box i) or signs of inflammasome and pyroptosis inhibition in lesions allowing Mtb (red) growth (box ii). (g) Increased staining (red) in the core of infected globules after staining with Lyso-ID dye, but not in the granulomatous lesion area, which stains for acidic organelles or environment resulting from cell death. Staining with image-iT hypoxia reagent (h), apoptosis / necrosis detection reagent (i), and live / dead staining reagent (intracellular cytoamine sensor dye for detecting dead cells) (j) shows increased hypoxia, necrosis, and cell death in the center of infected globules. The investigated 3D cocultures were developed using THP-1 monocytes and WT Mm“M” (ac and hj), WT Mtb Erdman (d and g), or Mtb Erdman tdTomato (e and f), with uninfected spheroids used as controls. Figure 11BThe study showed that cavity-like features were generated in a 3D model of THP-1 cells infected with Mm 1218 (GFP) after deposition with 30 µl ECM, but this was not observed after deposition with 5 µl ECM. Figure 11C The image shows that in a 3D model of Mtb Erdman (tdTomato) infection, treatment with anti-TNF-α and CD11a biologics or mAb biosimilars (62.5 ng) on ​​day 6 resulted in disruption of tissue integrity in neogranulomas on day 10 (blue arrows).

[0034] Figures 12A-12B The 3D model shows key features and microenvironment of human tuberculoma, including mycobacterial biofilm formation. Figure 12A The expression of microtubules (green), VEGFR-2, type 3 collagen, and galactagogue-9 (red), as determined by immunostaining, is shown in uninfected control 3D spheroids, THP-1 monocytes, and WT Mm M (MOI 0.004) 3D tuberculomas. Cell nuclei were stained blue using Hoechst 33342 dye. Increased expression of the macrophage differentiation marker CD68 (red) is also shown in the core of granulomas and 3D tuberculomas. Figure 12B Mtb biofilm formation in 3D tuberculomas of THP-1 monocytes and Mtb Erdman (WT) cells, as detected by Fun-1 staining with Calcofluor white (CW) for the biofilm-matrix component cellulose, is shown. Fun-1 (a unique dual-color fluorescent probe that readily diffuses into cells) fluoresces in the red (Texas Red) and green (GFP) channels, and CW labels chitin and cellulose (β(1,4)-D-glucopyranosyl units) exhibit blue fluorescence. Composite images from the red, green (Fun-1), and blue (CW) fluorescence channels, with or without overlay from the bright field channels, are shown. No fluorescence was detected in uninfected 3D spheroids except for diffuse green (background) fluorescence.

[0035] Figures 13A-13B Autophagy and inflammasome activation are shown in a 3D model of THP-1 reporter cells infected with Mtb Erdman. Figure 13AAutophagy induction in 3D tuberculomas is shown in THP-1-Difluo hLC3 reporter cells infected with Mtb Erdman (WT). The left inset shows basal expression of hLC3-GFP-RFP in reporter cells grown as 2D cell cultures (uninfected) in Corning ULA flat-bottomed microwells. The right side shows uninfected 3D cell cultures and tuberculoma co-cultures treated with DMSO (drug carrier), rapamycin (mTOR inhibitor and autophagy inducer), chloroquine (lysosomal inhibitor), and rapamycin plus chloroquine. On day 1 and day 6 post-treatment (i.e., day 7 and day 12 post-co-culture, respectively), untreated or DMSO-treated 3D co-cultures showed accumulation of yellow-green spots in macrophages in granulomatous zones and numerous cell clusters, indicating blockage of autolysosomal formation and incomplete autophagy flux. In these 3D co-cultures, increased red fluorescence on day 6 post-treatment indicates delayed autophagy induction (Mtb-induced and starvation-induced). In contrast, rapamycin-treated 3D cocultures exhibited relatively rapid induction of autophagy (red fluorescence) throughout the entire 3D coculture (≤ day 1 post-treatment). Chloroquine-treated cocultures showed increased autophagy on day 6 compared to day 1. Chloroquine has been shown to inhibit autophagy by blocking the fusion of autophagosomes with lysosomes and slowing lysosomal acidification. However, chloroquine-induced lysosomal inhibition can inhibit mTORC1 and secondary inducing autophagy. It can also induce LC3-II formation independently of autophagy. Figure 13B The diagram illustrates inflammasome and pyroptosis induction in 3D tuberculomas of THP-1-ASC-GFP reporter cells infected with Mtb Erdman (tdTomato). The left inset shows basal ASC-GFP expression in reporter cells grown as uninfected 2D cell cultures in Corning ULA flat-bottomed microwells. ASC-GFP expression is driven by an NF-κB inducible promoter. Therefore, GFP signal is barely detectable in resting cells. The right side shows uninfected 3D cultures and 3D co-cultures treated with DMSO (drug carrier), nigericin (an NLRP3 inflammasome inducer), and MCC950 (an NLRP3 inhibitor). The untreated or DMSO-treated 3D co-cultures show that Mtb (red) induces the assembly of ASC-dependent inflammasomes (green) in THP-1 cells, ultimately leading to cell death. 3D co-cultures treated with Nigerian styraxin showed inhibition of ASC spot (green) formation, relatively rapid macrophage death (pyroptosis), and Mtb (red) growth. Conversely, compared to DMSO, MCC950 treatment, known to inhibit inflammasome and ASC spot formation, did not reduce Mtb loading in 3D co-cultures by day 6 post-treatment. Mtb MOI 0.05 ( Figures 13A-13B ).

[0036] Figures 14A-14C This shows hypoxia and necrosis occurring in the center of a 3D tuberculoma. Figure 14A Increased hypoxic red staining is shown in the centers of 3D tuberculomas in THP-1 monocytes and WT Mtb Erdman (MOI 0.05). 3D tuberculomas or control spheroids were exposed to a pre-titrated hypoxic red fluorescent probe (ROS-ID Hypoxia / Oxidative Stress Detection Kit, Enzo Life Sciences) in 100 µl RPMI-1640 medium and cultured in a Co2 incubator for 24 or 48 h. After incubation, the RPMI-1640 medium in the wells was carefully exchanged twice with sterile PBS to remove the hypoxic red probe post-incubation. The stained 3D spheroids were imaged using the Texas Red channel in a Cytation-5 cell imager. The red hypoxia detection reagent (probe) is a non-fluorescent or weakly fluorescent aromatic compound containing a nitro (NO2) moiety. Because hypoxic cells possess nitroreductase activity, the nitro group is converted to hydroxylamine (NHOH) and amino group (NH2) in a series of chemical steps. The original molecule then degrades, releasing a fluorescent probe that stains the hypoxic cells red. The hypoxia inducer deferoxamine (200 µM, for 3 or 6 h) and the hypoxia inhibitor nizonitrate (20 µM, for 16 h) were used as positive and negative controls, respectively. Figure 14B Increased cell death (red) in the center of 3D tuberculomas compared to control 3D spheroids is shown. Cell nuclei were stained blue using Hoechst 33342 dye. Following the manufacturer's instructions, 3D spheroids were stained for 30 min in RPMI-1640 medium with a fixable red dead cell staining kit (Thermofisher). To confirm the results, live / dead cell imaging was also performed using a live / dead cell imaging kit containing esterase and BOBO-3 iodide. Esterase staining (green) indicates live cells, and BOBO-3 iodide staining (red) indicates free DNA or dead / damaged cells. Apoptosis and necrosis staining showed that the increased cell death in the center of 3D tuberculomas compared to control spheroids was due to increased monocyte / macrophage necrosis (green) rather than apoptosis (red). Staining was performed using the Abcam Apoptosis / Necrosis Kit, which includes Apoxin (a phosphatidylserine (PS) sensor for apoptosis (red), Nuclear Green DCS1 (a membrane-impermeable dye that stains the nuclei of damaged or necrotic cells (green)), and CytoCalcein dye (which stains live cells (blue / purple)). Figure 14CIncreased cell necrosis was observed in 3D tuberculomas co-cultured for 12 days.

[0037] Figure 15 A- Figure 15 D shows cavity formation following increased extracellular matrix deposition in 3D tuberculomas. Figure 15 A shows a cavity-like feature on day 21 in a 3D co-culture of THP-1 monocytes and Mm 1218 GFP (MOI 0.005) following deposition of an incremental (5–50 µl) mixture of extracellular matrix (ECM) containing human type 1 collagen and fibronectin. Figure 15 B illustrates the kinetics of cavity formation in a 3D co-culture of THP-1 monocytes and Mm 1218 GFP (MOI 0.005). 3D co-cultures deposited with 10 or 50 µl ECM are shown. In the 3D co-culture deposited with 50 µl ECM, cavity formation began at approximately day 12 and continued to grow over time. Figure 15 C shows that despite the deposition of a high dose of ECM, cavitation transformation did not occur in uninfected control 3D spheroids. A representative 3D cell culture deposited with 30 µl of ECM is shown. Figure 15 D shows that despite the deposition of high doses (≥ 30 µl) of ECM, no cavitation transformation occurred in 3D cocultures of THP-1 mononuclear cells generated using 1 / 10 MOI (0.0005).

[0038] Figures 16A-16F The study demonstrates that screening biologics and biosimilars in 3D co-culture identified immunotherapeutic agents that affect the structural tissue and integrity of granuloma-producing organisms. Figure 16A This study demonstrates that, upon treatment with anti-CD11a or anti-TNF (but not anti-CD52 or allotype Abs) (regardless of the dose or treatment duration), by day 10, 3D co-cultures of THP-1 and Mtb Erdman (tdTtomato) (MOI 0.01) exhibited loss of structural integrity in neogranulomas. The names of the biologics or biosimilars studied are described in parentheses. Figure 16B The study showed that, up to day 12 following treatment with the anti-TNF adalimumab biosimilar, 3D co-cultures of THP-1 and Mm M(tdTomato) (MOI 0.006) exhibited the formation of numerous small (miliary) granulomas (rather than large, well-organized, and dense granulomas). Figures 16C-16DThe study showed that although structural integrity was disrupted in the early stages, by day 14, anti-CD11a or anti-TNF treatment in 3D co-cultures of THP-1 and Mtb Erdman (tdTomato) resulted in the formation of mature granulomas, compared to isotype Ab treatment or culture medium alone (without Ab) control. Figure 16C The production of bacterial load is comparable to that of bacteria. Figure 16D When comparing bacterial load (RFU) in cocultures treated with anti-CD11a or anti-TNF to controls treated with the same type of Ab or controls in media alone (without Ab), the results were not significant using analysis of variance. Figure 16E The effects of biologics and biosimilars (n = 15) other than anti-CD11a or anti-TNF on the structural histogenesis of granulomas were shown up to day 10 in a 3D coculture of THP-1 and Mtb Erdman (tdTomato). Figure 16F The effects of biologics and biosimilars (n = 18) on bacterial load in 3D co-cultures of THP-1 and Mtb Erdman (tdTomato), measured on day 14, are shown. *p < 0.05, **p < 0.01 and ***p < 0.001, as determined by one-way ANOVA and post-hoc Hiddink test.

[0039] Figures 17A-17C Differentially expressed immunoproteins in host cell lysates and culture supernatants of 3D tuberculomas, quantified using human Kiloplex proteomics assays, are illustrated. Culture supernatants and cell lysates from 3D co-cultures of THP-1 monocytes and Mm 1218 GFP (MOI 0.005) with or without extracellular matrix (ECM) solution (30 µl) were isolated on day 16 and subjected to a Quantibody human Kiloplex array (QAH-CAA-X00-1) containing a human immunology kit with 1000 protein biomarkers. Each sample was analyzed in quadruplicate, and protein expression levels (pg / ml) in the cell lysate samples were determined after normalizing the amount relative to the total protein concentration present in the lysates. The relative fold change in protein expression levels was calculated, taking into account the limit of detection (LOD) and expression levels in control and infected samples, or samples with or without ECM. Use the ggplot2 package in R to plot the relative fold change of selected protein biomarkers using a dot plot. The size of the dots indicates the relative fold change of the protein, and the fold change value is shown at the bottom of each dot. Figure 17A The relative fold change in the expression of selected cell lysate proteins in Mm-infected 3D cocultures compared to uninfected control 3D cultures is shown. Figure 17BThe relative protein expression in selected culture supernatants of Mm-infected 3D co-cultures is shown compared to uninfected control 3D cultures. (Top small figure) Figure 17A and Figure 17B The upper figure (C) shows proteins with relative expression fold changes ranging from 50 to 500 fold, and the lower inset shows proteins with relative expression fold changes ranging from 5 to 50 fold. (ECM) + Instructions for adding ECM to 3D co-cultures, and ECM - Indicates 3D co-cultures without ECM. Figure 17C The effects of ECM addition on protein expression levels in cell lysates (left inset) or culture supernatant (right inset) are shown. In each inset, the left column (titled "Untreated") shows the fold change in protein expression levels between uninfected control 3D cultures containing ECM and uninfected 3D cultures without ECM. The right column (titled "Infected") shows the fold change in protein expression levels between infected 3D co-cultures containing ECM and infected 3D co-cultures without ECM. Figures 17A-17C In the diagram, red dots indicate upregulation of protein expression, and blue dots indicate downregulation of protein expression.

[0040] Figure 18 This study demonstrates confirmation of the secretion of five selected proteins in 3D tuberculomas infected with Mm 1218 THP-1 monocytes, quantified using enzyme-linked immunospot (ELISPOT) assays. A plot of every 10... 5 The image shows spotted-forming units (SFUs) of five key chemokines, cytokines, or MMPs secreted by THP-1 cells, and also shows representative images of triplicate wells from an ELISPOT assay performed on day 16 after 3D culture.

[0041] Figures 19A-19E Transcriptomic analysis of the 3D cocultures provides insights into macrophage-mycobacterial interactions that lead to granuloma formation. THP-1 cells isolated from 3D cocultures infected with Mm“M”tdTomato (MOI 0.005) or uninfected control 3D cultures were RNA-seqed at days 3, 6, 9, and 12 post-3D culture. Figure 19A Principal component analysis (PCA) plots of all RNAseq samples are shown using the DESeq2 tool in R. Figure 19BVolcano plots show the genes that were significantly upregulated and downregulated in infected 3D co-cultures compared to uninfected 3D cultures at days 3, 6, 9, and 12 after 3D culture. DEG: Differentially expressed genes. Red dots represent upregulated DEGs with log2 (fold change) > 1 and adjusted p-value < 0.05, blue dots represent downregulated DEGs with log2 (fold change) < -1 and adjusted p-value < 0.05, and dark gray dots represent less significant genes (not DEGs) with adjusted p-value ≥ 0.05. These plots were generated using the DESeq2 package in R. Figure 19C Venn plots of DEG upregulation and downregulation common at days 3, 6, 9, and 12 after 3D culture are shown. The plots were generated using an online program (bioinformatics.psb.ugent.be / webtools / Venn / ). ShinyGo (v.0.77) was used. ShinyGO 0.77 (sdstate.edu) The shared DEGs (n = 539 in the Venn diagram center, containing both upregulated and downregulated DEGs) underwent gene set enrichment analysis (GSEA). Using the ggplot2 package in R, the top-selected enriched KEGG pathways were plotted as lollipop plots. The color of the lines or dots indicated -log2(FDR), the length of the bars indicated the enrichment score, and the size of the dots indicated the number of enriched genes. Figure 19D The diagram shows the enriched classical pathways that changed before, during, and after granuloma formation in infected 3D cocultures compared to the 3D control. DEGs were subjected to gene set enrichment analysis using the Qiagen IPA classical pathway analysis tool, with absolute |log2(fold change)| > 1 and adjusted p-value < 0.05. The length of the bars in the figure represents the Z-score (n = 45) of the most enriched classical pathways, ranked based on scores on day 3 post-culture. Bars facing left indicate pathway downregulation, and bars facing right indicate pathway activation. Red circles represent neural signaling pathways, purple triangles represent cholesterol-related pathways, and blue asterisks indicate pathways primarily activated on early culture days.

[0042] Figure 19E This study shows the enriched KEGG pathways that were altered before, during, and after granuloma formation in infected 3D cocultures compared to the 3D control. The clusterprofiler package in R was used to extract KEGG pathways from... Figure 19D The same DEG set underwent gene set enrichment analysis (GSEA). The top 40 most enriched KEGG pathways are presented as a bar chart. The length of the bar indicates the normalized enrichment score (NES). Bars facing left indicate pathway inhibition, and bars facing right indicate pathway activation. Red dots represent pathways shared with IPA analysis. Figure 19D (The blue asterisk indicates a meaningful pathway identified by GSEA.)

[0043] Figure 20 A- Figure 20 G illustrates a schematic workflow for transcriptomic characterization of the 3D tuberculoma model used to generate the data shown in Figure 19. Figure 20 A shows 3D cultures of THP-1 cells generated with or without Mm“M”tdTomato infection (MOI 0.005), and total RNA was isolated from THP-1 cells at 0, 3, 6, 9, and 12 days after culture using the Qiagen RNeasy Plus kit following the manufacturer’s instructions. Figure 20 B shows the preparation of a cDNA library using the Illumina Truseq chain-specific mRNA library kit, followed by Illumina Nextseq sequencing (75 × 2 cycles). Figure 20 C shows the RNA sequencing data being quality checked by FastQC (v.0.11.5) and trimmed by the Trimmomatic (v.0.39) tool. Figure 20 D shows the mapping of high-quality reads to the human reference genome (GRCh38.p13) using the STAR alignment software (v.2.5.2b), and annotation via the GRCh38.gtf file. Figure 20 E shows that reads mapped to genes are counted using the featureCounts function in the RSubread package in R. A read count table is generated using the DESeq2 package and normalized by sequencing depth. Principal component analysis (PCA) plots and sample distance matrices are generated to visualize sample clustering. Figure 20 F shows the identification of differentially expressed genes (DEGs) using the DESeq2 package and visualization via MA and volcano plots. A Venn diagram was used to identify the core DEGs shared across different infection days. Figure 20 G shows gene set enrichment analysis (GSEA) performed using the KEGG database in R with IPA software (Qiagen. Inc.) or the clusterprofiler package. The expression changes (log2 fold changes) of core genes in the selected pathways are then presented as a heatmap.

[0044] Figure 21A and Figure 21B Further RNA sequencing data analysis of the 3D cell culture samples is shown. Figure 21A Heatmaps and hierarchical clustering of 3D cocultures and uninfected 3D controls at days 0, 3, 6, 9, and 12 post-culture are shown. Figure 21BThe MA plot is shown, representing the relationship between the fold change in log2 for each gene in the 3D co-culture and the baseline mean (basemean) compared to the 3D control. Red dots indicate upregulated genes with log2FC > 1 and adjusted p < 0.05, blue dots indicate downregulated genes with log2FC < -1 and adjusted p < 0.05, and gray dots indicate genes with no significant change in expression and adjusted p ≥ 0.05.

[0045] Figures 22A-22G The expression of core genes differentially expressed in selected pathways is shown. A heatmap of the log2 (fold change) of expression of core genes involved in the pathways was plotted using the ggplot2 package in R. The four horizontal blocks represent gene expression levels at 3, 6, 9, and 12 days after culture. The color of the blocks indicates the log2 (fold change). Cell adhesion molecules (…) are also shown. Figure 22A ), angiogenesis ( Figure 22B ), necroptosis ( Figure 22C ), autophagy and phagosomes ( Figure 22D Inflammatory bodies and pyroptosis (Fig. 21E), proteases, cathepsins, and metallo-metalloproteinases (Fig. 21F), and neuroactive ligand-receptor interactions ( Figure 22G ) as well as chemokines and cytokines ( Figure 22H )way.

[0046] Figure 23 This demonstrates the use of a 3D tuberculoma model to study training immunity induced by a vaccine against secondary pathogenic mycobacterial infection. THP-1 monocytes (1 × 10⁻⁶) were used. 6 THP-1 monocytes were infected with live attenuated BCG Danish vaccine strain (5000 CFU) in cell culture tubes at 37ºC in a Co2 incubator for 16 h. The next day, free BCG bacteria were removed from the THP-1 cell suspension by low-speed centrifugation (50 g for 5 min). Alternatively, infected THP-1 monocytes could be physically separated from free BCG using anti-CD32-biotin antibody and anti-biotin microbeads via a MACS column. The BCG-infected THP-1 monocytes (1 × 10⁻⁶ cells / ml 3D cell culture medium) were then incubated for 16 h in a Co2 incubator at 37ºC. 7 10 cells / 50 ml 3D cell culture medium were cultured in tissue culture flasks for 5 days to induce immune training. Simultaneously, control THP-1 monocytes (1×10⁶ cells / 50 ml 3D cell culture medium) were cultured. 7THP-1 cells were trained in 3D cell culture medium (i.e., RPMI-1640 only). 3D tuberculomas were then generated in 96-well plates (Corning 3D) with or without Mm1218-GFP (MOI 0.005) infection, using either BCG-trained or RPMI-trained cells. BCG-trained, but not RPMI-1640-trained, THP-1 monocytes / macrophages inhibited the growth of pathogenic Mm1218-GFP and suppressed the formation of granulomatous lesions (green), as imaged on day 15 after 3D cell culture.

[0047] Figures 24A-24D The identification of HDT compounds that inhibit pathogenic mycobacteria in a 3D tuberculoma bioplatform is shown. Figure 24A The screening of potential HDT compounds from a custom library at 20 µM in THP-1-Mm“M”(tdTomato) 3D tuberculomas is shown, and the results are presented as normalized bacterial load (%). Data were obtained from three independent experiments (n = 9 tuberculomas / compounds and 36–72 tuberculomas / controls), and solid circles represent the bacterial load in individual tuberculomas. Figure 24B This study demonstrates a "preferred seed" identified in chemical screening that reduced the bacterial load in 3D tuberculomas of four separate Mm and Mtb strains (tdTomato) by >50% compared to the untreated control. Figure 24C The results of screening for the inhibitory effects of compounds on four different mycobacterial strains are shown in a 3D tuberculoma bioplatform. Rifampin was used as a positive control for bacterial load reduction. The mean z-scores and standard errors for each compound and control screened in 2–4 experiments for each mycobacterial strain are shown by four symbols. The dashed line depicts the cutoff value when the z-score is -4. Figure 24D This shows the presence of CD14 in human PBMCs (supplemented with CD14). - CD14 of subgroup + Identification of HDT compounds that inhibit Mtb Erdman (tdTomato) in 3D tuberculomas (monocytes) or THP-1 monocytes. Data were obtained from two THP-1 assays (n = 6 tuberculomas / compound) and four human donor PBMC assays (n = 12 tuberculomas / compound). Figure 24A , Figure 24C and Figure 24D The mean ± SEM values ​​are shown. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to the DMSO control were determined by Kruskal-Wallis and Dunn post-hoc tests. Figure 24DThe green and black asterisks in the text represent the significance levels in the THP-1 and PBMC co-cultures, respectively.

[0048] Figures 25A-25C The compound screening assay using the 3D tuberculoma biological platform demonstrates excellent quality and performance. Figure 25A The effect of Mm“M”(tdTomato) MOI on the quality of screening assays in the THP-1 tuberculoma bioplatform is shown, as assessed by Z' factor statistics. The Z' factor describes the degree of separation between positive and negative controls and indicates the probability of false positives or false negatives. In 96-well plate format, assay controls were used in the absence of test compound intervention. One infection dose and five doses (MOI range 0.001 to 0.012) were investigated per plate (Corning 3D). In the assays, nitrozonide (20 µM / well) and rifampin (1 µg / ml) were used as positive controls for bacterial reduction, and DMSO and culture medium only (no drug or untreated) were used as negative controls. FI was measured on day 12, and the data shown are from one of two experiments conducted (n = 12 tuberculomas per positive control per assay plate, and 12–24 tuberculomas per negative control). Dashed lines indicate the cutoff value when the Z' factor is 0.5. A Z' factor value of 1.0 indicates an ideal assay, values ​​between 0.5 and 1.0 indicate an excellent assay, and values ​​between 0 and 0.5 indicate an acceptable assay. Values ​​below 0 indicate that the assay conditions have not been optimized and the assay is unlikely to produce useful data. For assays using Mm "M", the optimal MOI range for identification is 0.007 to 0.012 to exhibit excellent Z' factor and quality. Figure 25B This study demonstrates the optimal MOI for Mtb (tdTomato) strains in the THP-1 bioplatform, used for excellent screening assay quality. FI measurements at day 14 were considered indicative of slow-growing Mtb strains. The optimal MOI range for identifying Mtb strains H37Rv, Erdman, and Beijing strains was 0.025 to 0.05. For Mtb strain CDC1551, excellent assay quality could not be achieved, even at a higher MOI of 0.05. Increased Mtb-induced THP-1 cell death was observed at day 14 in assays of virulence Mtb strains with an MOI of 0.1; therefore, MOI > 0.05 was not considered. Figure 25CThe performance and quality of a screening assay using 3D tuberculomas infected with MtbH37Rv(tdTomato) THP-1 cells were demonstrated in several 96-well plates. The assay was performed in 10 plates using an optimal MOI of 0.025, and fluorescence (FI) was measured on day 14. Solid circles represent fluorescence in individual 3D tuberculomas or uninfected spheroids. RFU, relative fluorescence units. A total of 126 untreated, 180 DMSO-treated, and 120 tuberculomas treated individually with either nitrozonidine or rifampin were investigated. 54 uninfected spheroids were included to determine background fluorescence. The assay demonstrated excellent quality and performance (Z' factor > 0.5). Performance in individual plates was also evaluated, and a Z' factor ≥ 0.5 in all ten plates further confirmed the suitability of the assay for HTS applications.

[0049] Figure 26A- Figure 26D This demonstrates the inhibition of granulomatous lesions after HDT compound treatment in a 3D tuberculoma bioplatform infected with THP-1 strains of Mm or Mtb. In contrast, infection with Mm M (Fig. 26A) and Mtb H37Rv (…) alone… Figure 26B Mtb Beijing F2 ( Figure 26C Sixty-five potential HDT compounds were screened in the 3D tuberculoma bioplatform of Mtb Erdman's THP-1 cells (as shown in Figure 24), and the number of granulomas in the 3D cocultures was counted and expressed as a normalized lesion count (%) relative to the untreated control. For granuloma lesion counting, Z-projection images of 3D spheres were subjected to cell analysis and granuloma lesion counting using Gen 5 software, followed by manual lesion counting by three blinded readers for quality control, and the average of the three readers' counts was considered. Data are displayed as box plots with whisker lines (minimum to maximum), with all data points (repeated) shown as circles. Dashed lines indicate 25% inhibition of granuloma lesions. Data were obtained from four ( Figure 26B ), three (Figure 26A and Figure 26C ) and two ( Figure 26D Independent experiments. Each assay plate contained tuberculomas treated with untreated (untreated, n = 6), DMSO (n = 6), rifampin (n = 3–6), and the test compound (n = 3 for each compound). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to the DMSO control were determined by Kruskal-Wallis and Dunn post-hoc tests.

[0050] Figure 27Representative images of granulomatous lesion suppression following treatment with a potential HDT compound are shown in 3D tuberculomas infected with THP-1 alone, either Mm or Mtb strains. 3D tuberculomas were treated with the compound alone on day 6 post-co-culture, and images were captured using a Cytation-5 cell imager on day 6 (Mm 3D) or day 8 (Mtb 3D) post-treatment.

[0051] Figures 28A-28D This study demonstrates that the "preferred precursor" HDT compound induces a key innate immune mechanism in 3D tuberculomas. On day 6 post-treatment, at 20 µM, hypoxia, autophagy, and lysosomal acidification were induced in 3D tuberculomas by the following: Figure 28A Non-cytotoxic "first-choice leader" compounds, ( Figure 28B Representative non-cytotoxic "non-leader" compounds, ( Figure 28C ) cytotoxic "first-choice precursor" compounds, and ( Figure 28D This study exhibited species-specific heterogeneity in growth inhibition, but showed cytotoxic compounds that inhibited the growth of Mtb Erdman by 25%–50%. Hypoxia and lysosomal acidification in THP-1 tuberculomas infected with WT Erdman were investigated using hypoxia red and Lyso-ID red reagents. In contrast, autophagy was studied in tuberculomas of THP-1 RFP-GFP-LC3 reporter cells infected with WT Erdman. Blue arrows indicate aggregates of viable cells stained with hypoxia red, clearly distinct from unstained Mtb permissive lesions (white) in granulomatous areas or the core. Yellow arrows indicate aggregates of cells lacking autophagosome maturation (yellow-green spots) and lysosomal acidification (no Lyso-ID red staining).

[0052] Figure 29 This study demonstrates how HDT compounds can be categorized into functional clusters based on their immune mechanisms and therapeutic efficacy within a 3D biological platform. Compounds belonging to cluster 1 (cytotoxic "preferred precursors"), cluster 2 (non-cytotoxic "preferred precursors"), cluster 3 (potential cytotoxic "preferred precursors"), cluster 4 (non-cytotoxic potential "preferred precursors"), cluster 5 (cytotoxic with insignificant effects), cluster 6 (non-cytotoxic with insignificant effects), and cluster 7 (insignificant effects) are listed. Key innate immune mechanisms induced by representative compounds, such as hypoxia, autophagy, and lysosomal acidification, are also illustrated.

[0053] Figure 30A and Figure 30BThis study demonstrates that AT9283 (a small molecule and aurora kinase inhibitor) significantly inhibited bacterial load and granulomatous lesions in the lungs of mice infected with Mtb Erdman aerosol. Treatment began seven days after Mtb aerosol inoculation, with mice receiving either AT9283 (test drug) at a dose of 35 mg / kg in 150 µl of sterile water or rifampin (positive control antibiotic) daily, five days a week for three weeks. Thus, mice received a total of 16 doses of either AT9283 or rifampin. Initially, the drug was dissolved in DMSO at 200 mg / ml to prepare a cryopreservation stock, and fresh dilutions in distilled water were prepared daily for oral administration via tube feeding. A group of mice received DMSO diluted in sterile water as a negative control. On day 28, following aerosol infection, mice were euthanized, and a homogenate of the left lung was plated for CFU. The right middle lobe of the lung was fixed with paraformaldehyde, embedded in paraffin, and the tissue sections were stained with hematoxylin and eosin (H and E) as well as immunostaining against Mtb bacteria. Figure 30A This study shows a reduction in Mtb Erdman CFU load in the lungs of mice treated with AT9283 or rifampin compared to untreated and DMSO controls. Data from two independent experiments are shown. *p < 0.05, **p < 0.01 compared to DMSO controls, by Kruskal-Wallis and Dunn post-hoc tests. Error bars represent standard deviations, and the horizontal line indicates the geometric mean. Figure 30B The reduced granulomatous lesion burden in tissue sections of the right middle lobe of mice treated with AT9283 and rifampin is shown compared to the DMSO control. Blue stars indicate granulomatous lesions.

[0054] Figures 31A-31D A workflow for evaluating the therapeutic efficacy of selected HDT compounds in reducing bacterial load and tuberculous granulomatous lesions in C3Heb / Fej mice is shown. Figure 31A A flowchart illustrating the workflow for evaluating the therapeutic efficacy of selected HDT compounds is shown. Figure 31B The Glas-Col inhalation exposure system is illustrated. Female, 6–8 week old C3Heb / Fej mice were aerosol-infected with 10–15 Mtb Erdman CFU using the Glas-Col inhalation exposure chamber at the ABSL-3 facility. Seven days post-infection, mice in each group received a potential HDT compound (35 mg / kg / day), rifampin (10 mg / kg / day), or drug-free or drug-carrier DMSO (< 5% v / v) five days a week for three weeks. Mice were also administered oral via tube feeding daily with a fresh drug dilution prepared from a frozen stock solution of the drug (200 mg compound / ml dissolved in DMSO) in distilled water, and received 16 doses. Figure 31C Screening for HDT compounds is shown (n = at least 5-6 mice / group). Mice were euthanized on day 28 post-infection, and left lung and spleen homogenates were plated on Middlebrook 7H10 agar plates. Mtb CFU were counted at 4 and 8 weeks post-plating and incubation. Paraformaldehyde-fixed middle or lower right lung lobe sections were stained with hematoxylin and eosin, as well as immunostaining against Mtb bacteria. Serum was separated from the blood to determine the circulating drug concentrations achieved. Figure 31D The bacterial load determination in organ homogenates from control mice infected with Mtb Erdman but not treated with drugs is shown. Total lung (right and left lung) homogenates from plated mice euthanized 24 h after aerosol infection revealed an average of 12 CFU deposited in the lungs of a single mouse. The Mtb Erdman load in the left and right lungs of mice euthanized 4 weeks after aerosol infection was comparable (approximately 10 CFU). 7 CFU confirmed that both lungs were equally infected after low-dose mycobacterial infection and had similar levels of bacterial load at the peak of infection.

[0055] Figures 32A-32B This study demonstrates the validation of the “sprout” HDT compounds screened and identified from the 3D tuberculoma bioplatform in reducing bacterial load in a C3Heb / FeJ mouse model infected with Mtb aerosols. Figure 32A This study demonstrates a reduction in Mtb Erdman bacterial load in a 3D tuberculoma bioplatform following treatment with a single selected potential HDT compound. 3D tuberculomas infected with Mtb Erdman (tdTomato) were treated with a 20 µM dose on day 6, and fluorescence intensity (FI) was measured 6 days post-treatment. Results are expressed as normalized bacterial load (%) compared to a control without treatment (i.e., culture medium only). Data were obtained from two independent experiments (n = 6 tuberculomas / compound, 36 tuberculomas / DMSO, 72 tuberculomas / no-drug control (not shown)). Solid circles represent bacterial load in individual 3D tuberculomas. *p < 0.05 and ****p < 0.0001, ns, were not significant compared to the DMSO control by one-way ANOVA and Holm-Hidak multiple comparison test. Figure 32BThis illustrates a reduction in MtbErdman (WT) bacterial load in the left lung of aerosol-infected C3Heb / Fej mice after treatment with a single potential HDT compound. The workflow for evaluating the therapeutic efficacy of the selected HDT compound is described in Figure 31 above. Solid circles represent the bacterial load in the left lung of a single mouse. Mice treated with the antibiotic rifampin were used as a positive control. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to the DMSO control were determined by the Kruskal-Wallis & Dunn post-hoc test.

[0056] Figure 33 This study demonstrates the regression of tuberculous granulomatous burden in the lungs of C3Heb / Fej mice infected with Mycobacterium tuberculosis after treatment with selected HDT compounds. The number and size of tuberculous granulomatous lesions in the lungs of mice infected with Mtb Erdman and treated with HDT compounds alone were reduced compared to control mice treated with DMSO. Middle or lower right lung lobes (n = 5–7 lobes / group) were randomly collected from individual mice in each treatment or control group and fixed in 4% paraformaldehyde in PBS, followed by fixation in 80% ethanol. The tissues were processed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin or anti-mycobacterial antibodies. Representative lung lobe images (n = 5–6 mice / group) are shown. Lung lobes collected from untreated uninfected mice, Mtb-infected mice treated with DMSO, and Mtb-infected mice treated with rifampin were used as negative and positive controls, respectively. Detailed Implementation

[0057] The inventors have developed an in vitro bioplatform using mycobacterial species and human immune cells that can be used in BSL-2 or BSL-3 level laboratories to screen for novel anti-TB patterns. This in vitro bioplatform comprises a 3D co-culture of mycobacteria within spheroids (similar to human tuberculomas with granulomatous aggregation). Compared to conventional methods, the bioplatform offers several unexpected advantages, including reduced animal use, cost, and time, as well as increased throughput and efficiency in screening potential host-directed therapy (HDT) compounds, antimicrobial agents (including antibiotics), and biologics within the tuberculoma microenvironment. Furthermore, the BSL-2 platform reduces safety concerns for researchers. To achieve this, the inventors implemented a 3D cell culture method.

[0058] Compared to traditional 2D cell cultures, 3D cell cultures offer numerous advantages: they allow for physiological cell-to-cell contact, cell-extracellular matrix (ECM) interactions, and a diffusion gradient that increases carbon dioxide and waste towards the center of the 3D spheroid while decreasing oxygen and nutrients. All these features are absent in traditional 2D cultures. Since human TB granulomas are primarily organized 3D aggregates of infected macrophages in the lungs, the core of this research was to answer the question of whether it is possible to develop 3D spheroids using human immune cells infected with a BSL-2 organism that expresses fluorescent proteins to aid in the study of pathogen and host cell growth dynamics via in situ imaging.

[0059] The 3D cell cultures of human immune cells and mycobacterial species disclosed in this paper form “nodules or spheroids” within 3 days of infection. In 2D cell cultures, researchers observed only a monolayer of infected immune cells. Researchers observed well-organized, dense granulomatous lesions of green or red color expressed by mycobacterial strains expressing green or red fluorescent proteins (i.e., GFP or tdTomato) in the bio-platform 10 to 12 days after immune cell infection. In 2D cell cultures, they observed that infected cells formed only loose aggregates, and that virulent but unattenuated mycobacterial strains could form organized granulomatous lesions. Similar to human TB granulomas, the inventors also observed increased hypoxia, necrosis, and cavity formation in the 3D bio-platform. They also observed that the increased cell death in the center of infected 3D spheroids compared to uninfected control spheroids was due to necrosis rather than apoptosis. Therefore, key features of human TB granulomas were also observed in 3D in vitro tuberculomas in the bio-platform. Other key features of human granulomas generated in biological platforms include epithelioid macrophage transformation, vascularization and upregulation of angiogenesis markers, collagen secretion, biofilm formation, matrix metalloproteinase (MMP) activity, and acidosis.

[0060] The 3D bio-platform disclosed in this paper can be used to screen potential HDTs, biologics, therapeutic vaccines, antimicrobial agents, and antibiotics. Results from the tests conducted indicate that the 3D bio-platform can indeed be used to screen host- and pathogen-targeted therapeutics, considering the significantly reduced bacterial load and regression of granulomatous lesions in the 3D spheroids. Available 2D and 3D granuloma models lack the ability to study the effects of therapeutics on the regression of granulomatous lesions and cavitation transformation because discrete, well-organized lesions and cavities are not formed in the available models due to their small size.

[0061] Screening a range of FDA-approved drugs on a 3D bioplatform showed that 9% of the drugs reduced bacterial load by more than 75%, 12% reduced bacterial load by 25%-75%, 62% had no significant effect on bacterial load, but 8% increased bacterial load by more than 25%. FDA-approved drugs that significantly reduce bacterial load and granulomatous lesions may be repurposed for the treatment of tuberculosis (TB). Compared to current methods, this bioplatform has the potential to screen HDT compounds and other small molecules at a much lower cost, with higher efficiency, and faster speed.

[0062] Potential uses of the biological platform beyond HDT could include developing treatments for drug-sensitive and drug-resistant Mtb, evaluating vaccine efficacy in in vitro clinical trials using immune cells from vaccinated volunteers, studying immune protection mechanisms, studying TB co-infections and comorbidities in vitro, and potentially serving as a platform for a wide range of granulomatous diseases and other human diseases in which intercellular and cell-matrix interactions are important. 1. Definition.

[0063] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless the context clearly specifies otherwise, the singular forms “a”, “an”, and “the” as used in this specification and the appended claims include plural indicators.

[0064] In the description of the ranges of numbers in this article, each intermediate number with the same precision is explicitly considered. For example, for the range of 6–9, the numbers 7 and 8 are considered in addition to 6 and 9, and for the range of 6.0–7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly considered. 2. Three-dimensional biological platform

[0065] This document provides a 3D biological platform. The 3D biological platform may include a container that may include at least one well suitable for culturing human cells. The well may contain immune cells and mycobacteria. In one example, the well contains a co-culture of mycobacteria within a spheroid containing immune cells and mycobacteria. In another example, the well contains a tuberculoma with granulomatous lesions. The immune cells may be human immune cells, which may include one or more of monocytes and macrophages. The human immune cells may include THP-1 cells, U937 tissue cells, or CD14 cells. +Monocytes. Human immune cells may include peripheral blood mononuclear cells (PBMCs), purified immune cell subsets derived from PBMCs, BAL, or biopsies of myeloid and lymphoid lines. The 3D bioplatform may also include supplementary cells, which may include one or more of epithelial cells, endothelial cells, and fibroblasts, derived from human primary cells or cell lines. Immune cells can be obtained from healthy individuals. These immune cells may be obtained from individuals infected with Mtb, suffering from TB, receiving TB treatment, or vaccinated against TB. Immune cells may be obtained from individuals with comorbidities (e.g., diabetes) or co-infected individuals (e.g., HIV).

[0066] Mycobacteria may include *Mycobacterium marineum* (also referred to herein as Mm), *Mycobacterium tuberculosis* (also referred to herein as Mtb), or Mtb complex species. Mycobacteria may express fluorescent markers, which may be green fluorescent protein (GFP) or red fluorescent protein (RFP). RFP may include tdTomato. In one example, *Mycobacterium marineum* is strain 1218 or strain M. Mm 1218 may express GFP, and Mm strain M may express RFP (e.g., tdTomato), or vice versa, or other fluorescent proteins. Mycobacteria may include Mtb, which may include one or more of strains H37Rv, Erdman, CDC1551, Beijing F2, and clinical isolates of drug-sensitive or drug-resistant Mtb. *Mycobacterium tuberculosis* may express luminescent or fluorescent markers, which may be one or more of GFP, RFP, far-red fluorescent protein, and another fluorescent protein.

[0067] Mycobacteria can be combined with immune cells in amounts of colony-forming units (CFU). In one example, the mycobacteria include Mycobacterium marinum, and the amount used for infection is approximately 600-1200 CFU. The multiple of infection (MOI) can be 10 per 10^10 CFU. 5The number of live immune cells is approximately 0.006–0.012, as measured in CFU. This amount can be approximately 800 CFU (or approximately 0.008 MOI). In another example, the mycobacteria include Mycobacterium tuberculosis H37Rv, and the amount used for infection is approximately 1500–5000 CFU (or approximately 0.015–0.05 MOI). The amount used for infection can be approximately 2500 CFU (or approximately 0.025 MOI). In yet another example, the mycobacteria include Mycobacterium tuberculosis Erdman or Beijing F2, and the MOI is approximately 0.012–0.05. In one example, the mycobacteria include Mycobacterium tuberculosis Erdman, and the MOI is approximately 0.05. The ideal MOI can vary depending on the mycobacterial species or strain used in 3D co-culture and can be experimentally determined by monitoring the kinetics of bacterial growth and granulomatous lesion formation within the 3D spheroid structure and by identifying the Z' factor in high-throughput screening assays using a biological platform.

[0068] The 3D bioplatform container may comprise a microplate, which may be a 3D spherical microplate. The microplate may include at least one pore. The pore may have a U-shaped bottom. The pore may include one or more walls comprising polystyrene (which may be virgin polystyrene). The pore may be optically transparent. The pore may include a coating, which may contain a hydrogel. The hydrogel may be hydrophilic, neutrally charged, and bioinert. The hydrogel may be covalently bonded to at least one surface of the pore. The hydrogel may comprise one or more of naturally derived, synthetically derived, and hybrid materials. The hydrogel may comprise one or more of collagen, fibroin, and alginate. The hydrogel may be synthesized using polyacrylamide and polyethylene glycol. The hydrogel may comprise one or more hybrid materials, which may be one or more of hyaluronic acid, peptides, and polymers. The hydrogel may comprise a perfluorinated polymer, olefin, or a combination thereof. The perfluorinated polymer may include poly4-methylpentene. The hydrogel may be a CORNING® PURAMATRIX™ peptide hydrogel with a synthetic matrix. PURAMATRIX™ peptide hydrogels can contain growth factors, extracellular matrix proteins, and / or other bioactive molecules required for optimal cell culture. CORNING PURAMATRIX peptide hydrogels can contain standard amino acids (1% w / v) and 99% water. Under physiological conditions, the peptide components can self-assemble into a 3D hydrogel with a nanoscale fibrous structure. In one example, the microplate is the CORNING® ultra-low adsorption spherical microplate. In another example, the microplate is the S-BIO PRIMESURFACE® 3D culture spherical plate. The microplate may lack a V-shaped bottom, an M-shaped bottom, or a flat bottom, and may also lack microcavities.

[0069] The wells may contain cell culture medium. Cell culture medium may include Roswell Park Memorial Institute medium (RPMI 1640). RPMI 1640 may contain the following components. Table 1

[0070] The cell culture medium may further contain one or more of L-glutamine, heat-inactivated fetal bovine serum (FBS), sodium pyruvate, and HEPES buffer, and may contain all of the aforementioned substances. The concentration of L-glutamine may be approximately 1-2 mM. The concentration of FBS may be approximately 9.63%-10% (v / v). The concentration of sodium pyruvate may be 0.87%-1.0% (v / v). The concentration of HEPES buffer may be approximately 0.87%-1.0% (v / v). The cell culture medium may exclude mitogens or all mitogens. In one example, the excluded mitogen is PMA.

[0071] Each well may contain a cryopreservative. The cryopreservative may contain cell culture medium and dimethyl sulfoxide (DMSO). The concentration of DMSO may be approximately 5% (v / v). Alternatively, the cryopreservative may contain heat-inactivated FBS and DMSO. The concentration of DMSO may be approximately 5% (v / v).

[0072] In one example, one or more wells of the 3D bioplatform may additionally contain extracellular matrix (ECM). The ECM solution mixture may contain human fibronectin and collagen, which may be type I human collagen. The ECM solution mixture may be added to a co-culture of mycobacteria in spheroids. The added ECM solution mixture may be 50 µl to 200 µl of co-culture cell culture medium per well. The human type I collagen may be derived from VITROCOL® and may contain approximately 97% type I human collagen, with the remainder being type III human collagen. The final concentration of collagen may be approximately 0.048 mg / ml or 0.048%. The final concentration of fibronectin may be approximately 0.0008 mg / ml (i.e., 0.8 µg / ml) or 0.00008%.

[0073] A microplate may include a body. The microplate body may be opaque and may shield each well from crosstalk between wells.

[0074] The 3D biological platform may contain one or more additional cell types, including A549 human lung epithelial cells, HUVEC-1 human fetal endothelial cells, HULEC human lung endothelial cells, and MRC-5 human lung fibroblasts.

[0075] The 3D biological platform can include viruses and human immune cells as alternatives to or supplements to mycobacteria. The virus can be one or more of human immunodeficiency virus (HIV), influenza virus, and coronavirus. The coronavirus can be severe acute respiratory syndrome (SARS)-CoV-2. 3. Methods for manufacturing 3D biological platforms

[0076] This document provides a method for preparing the 3D biological platform described herein. The method may include co-culturing immune cells and mycobacteria. Prior to co-culturing, the immune cells may be suspended in a 3D cell culture medium. The 3D cell culture medium may include RPMI 1640 medium as described herein, supplemented with one or more of L-glutamine, FBS, sodium pyruvate, and HEPES buffer as described herein. The 3D cell culture medium may contain antibiotics during growth, which may be one or more of penicillin and streptomycin, but may not contain antibiotics during co-culturing with mycobacteria. The immune cells may have a threshold viability of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Cell viability can be measured using a trypan blue rejection assay. In one example, the optimal threshold viability is at least 95%. The final concentration of immune cells may be approximately 1 × 10⁻⁶. 6 Live cells / mL. Immune cells can be washed 1, 2, 3, 4 or more times to remove antibiotics, up to 4 times. Washing may include centrifuging the immune cells and resuspending them in cell culture medium. The washed immune cells may have threshold viability.

[0077] If the immune cells are PBMCs, they can contain CD14. + Blood mononuclear cells (PBMCs) can then be purified and enriched into a subset of PBMCs, which are then combined with mycobacteria, as described below. In one example, PBMCs are isolated from human blood and purified by positive selection using a magnetically activated cell sorting (MACS) method. The MACS method may involve contacting a cell suspension containing PBMCs with magnetic nanoparticles. The nanoparticles may contain anti-CD14 antibodies. CD14 can be removed by running the cell suspension on a column placed in a magnetic field. + Monocytes (bound to nanoparticles) from a group containing lymphocytes and CD14 - The nanoparticles were isolated from the lymphocyte-rich cellular fraction of monocytes. A magnetic field can trap the nanoparticles and allow the lymphocyte-rich fraction to pass through. CD14 can then be purified by removing the washed column from the magnetic field. +Monocytes. In another example, if the immune cells are a subset purified from PBMCs, the mycobacteria may contain Mtb. The Mtb may be MtbErdman and may be combined with PBMCs at approximately 5000 cfu or approximately 0.05 MOI. In one example, a lymphocyte-rich cellular fraction purified from autologous PBMCs may be added to the CD14-containing... + Blood mononuclear cells and Mtb in mycobacteria within spheroids, and the resulting 3D cell cultures can be used in the screening methods disclosed herein.

[0078] The method may further include adding mycobacteria to immune cells in a cell culture medium. Mycobacteria may be added in the amounts or MOIs disclosed herein. The mycobacteria may be suspended in a 3D cell culture medium. Prior to suspension, the recombinant mycobacteria may be grown in a bacterial growth medium containing an agent that exerts selective pressure on the plasmid expressing the fluorescent marker. The agent may be an antibiotic, such as hygromycin or kanamycin. The bacterial growth medium may be Middlebrook 7H9 broth, which may contain 7H9 broth and Tween-80 (at a concentration of 0.05% (v / v)). The bacterial growth medium may also have a pH of 6.8-7. The bacterial growth medium may also be supplemented with ADC (at a concentration of 10% (v / v)) and glycerol (at a concentration of 0.4% (v / v)). The ADC may contain 2.5 g bovine albumin fraction V, 1 g dextran, and 0.0015 g catalase / 50 mL ADC. After suspension in 3D cell culture medium, the suspension can be passed through a 25-27G needle 10-15 times to produce a single bacterial cell suspension. The mycobacterial suspension can be diluted with 3D cell culture medium to the amounts or MOIs disclosed herein.

[0079] An immune cell suspension can be added to one or more wells of the container disclosed herein. Approximately 10 5Live cells are added to each well. The volume of the cell suspension can be as low as 100 µl. Mycobacterial suspensions can be added to one or more wells of the container, either in the amounts disclosed herein or at MOI. The volume of the mycobacterial suspension can be as low as 100 µl. The immune cell suspension and the mycobacterial suspension can be mixed after being added to the wells, and sterile 3D cell culture medium or cell culture-grade water can be added to the outer wells to avoid drying or boundary effects. The container can be incubated at 37ºC with 5% CO2 and 100% humidity. Incubation can be sustained for a sufficient time to form mycobacterial structures within the spheroids. Incubation can be between 6 and 14 days to allow for the formation of tuberculoma-like features in the mycobacterial co-culture within the spheroids. If the intention is to incubate the mycobacterial culture within the spheroids for more than 6 days, a minimum volume of 50 µl of fresh RPMI medium should be added weekly. The CFU of each well can be measured by spreading a dilution of the mycobacterial inoculum on an agar plate (which can be a Middlebrook 7H10 agar plate) at 30ºC for 12-14 days (if the mycobacterium is Mm) or at 37ºC for 3-4 weeks (if the mycobacterium is Mtb).

[0080] In one example, ECM is added to mycobacteria within spheroids. The ECM can be in a solution as described herein, and can be added approximately 3 days after co-culturing the immune cells and mycobacteria.

[0081] A 3D biological platform containing mycobacteria encapsulated in spheroids can be frozen using a cryopreservative-containing cryoprotectant. In one example, the microplate can be frozen at -80ºC within 30 minutes of co-culturing the mycobacteria and immune cells as described herein. If the aim is to freeze the microplate for more than 72 h, it can be placed at -160ºC to -196ºC, which may be in liquid nitrogen. In another example, after combining the immune cells and mycobacteria, the co-culture can be incubated at 37ºC with 5% CO2 and 100% humidity for 16–72 h, the 3D cell culture medium can be removed and replaced with a cryopreservative-containing cryoprotectant as described herein, and the microplate can then be frozen as described herein. As described herein, the frozen 3D biological platform can be thawed, the cryoprotectant removed, the cryopreservative-containing medium replaced at least three times with fresh 3D cell culture medium, and incubated in fresh 3D cell culture medium. The 3D biological platform can then be used in the screening methods described herein. 4. Screening Method

[0082] This document provides a method for screening candidate molecules that may be capable of treating or reducing mycobacterial infection or one or more pathological features thereof. The method may include contacting or exposing a 3D bioplatform disclosed herein to one or more candidate molecules or serial dilutions thereof. The method may include measuring one or more characteristics of mycobacteria in spheroids formed by a mixture of immune cells and mycobacteria from the 3D bioplatform. The characteristic may be the amount of fluorescence produced by the mycobacteria, which may be fluorescence intensity. The amount of fluorescence may indicate bacterial load. In one example, the characteristic includes loss of granuloma integrity. In another example, the characteristic includes one or more of reduced bacterial growth and granuloma formation. In one example, the characteristic includes a reduction in the number and size of granulomas. In one example, the characteristic includes autolysosome formation or autophagy flux. Changes in one or more characteristics compared to a control may indicate that the molecule is capable of treating or reducing mycobacterial infection or one or more pathological features thereof. In one example, the control is a negative control, which may include only a drug carrier. In another example, the control is a positive control, which may include an agent known to treat or reduce mycobacterial infection or one or more pathological features thereof.

[0083] A reduction in mycobacterial load in the 3D bioplatform reaching a threshold can indicate that a molecule can treat or reduce mycobacterial infection. The threshold can be greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In one example, the threshold is >25%. In another example, the threshold can be a z-score less than -2. In yet another example, the z-score threshold is <-4. The MOI for screening mycobacteria in the 3D bioplatform can be 0.007–0.012, which can be used when the 3D bioplatform contains Mm and THP-1 cells; or 0.012–0.05, which can be used when the 3D bioplatform contains Mtb H37Rv, Erdman, or Beijing F2 and THP-1 cells. In one example, the MOI is 0.025, which can be used when the 3D bioplatform contains Mtb H37Rv. In one example, the MOI is 0.05, which can be used when the 3D bio platform includes Erdman or Beijing F2.

[0084] In another example, the 3D biological platform can be exposed to one or more candidate molecules or serial dilutions thereof, and the quantity and amount of one or more upregulated or downregulated genes or one or more proteins expressed by cells of the 3D biological platform can be measured, as compared to a control. Each of the one or more genes or proteins can be associated with cell activation and differentiation, signal transduction, cell adhesion, granuloma and cavity formation, angiogenesis, hypoxia, cytokines and chemokines, immune responses, autophagy, necrosis, necroptosis, pyroptosis, and other cell death pathways. The control may contain uninfected control human immune cells. The 3D biological platform cells may or may not have been exposed to the ECM. Protein expression levels can be measured from cell lysates or culture supernatants. Gene expression levels can be measured using cell-purified RNA.

[0085] In another example, after contact with candidate molecules, the expression levels of one or more genes on a 3D biological platform can be quantified compared to a control.

[0086] Each candidate molecule can be diluted in 3D cell culture medium and added to mycobacteria in spheroids. Each dilution of the candidate molecule can be added approximately 0–6 days after co-incubation with mycobacteria and immune cells. In one example, molecules are added on day 0. In another example, molecules are added on day 6.

[0087] Approximately 1–14 days after the addition of each candidate molecule, one or more characteristics of mycobacteria within the spheroids can be observed or measured. In one example, one or more characteristics are observed 6 days after the addition of each candidate molecule. In another example, observations are performed on days 7, 9, and 12. Granulomatous lesion formation can be observed using a cell imaging reader or an inverted microscope. The cell imaging reader may be capable of automated microscopy and microplate detection. The cell imaging reader may be a Cytation 5 imager. The fluorescence intensity produced by mycobacteria can be measured using a cell imaging reader. Differences in one or more characteristics of mycobacteria within the spheroids compared to a control can indicate that the candidate molecule has a therapeutic effect against mycobacteria. The control can be a negative control, such as DMSO; or it can be a positive control, such as a molecule known to treat mycobacteria. a. Methods for identifying the in situ mechanisms of action of host-targeted drugs, biologics, and immunotherapeutic agents in 3D tuberculomas (1) Methods for screening candidate molecules for their role in autophagy in 3D tuberculoma

[0088] This article provides a method for screening candidate molecules to induce or inhibit autophagy in 3D tuberculomas. (a) First exemplary method

[0089] The method may include a 3D biological platform comprising a co-culture of THP-1 autophagy reporter cells (which may include THP-1 hLC3-GFP-RFP cells) and wild-type Mtb or Mm (non-fluorescent) mycobacteria. The method may include contacting the 3D biological platform with a candidate molecule on day 6 after co-incubation and imaging (using Cytation 5) on days 7, 9, and 12 after contact with the candidate molecule to determine autophagy flux. The reporter cells may express the human autophagy protein LC3B fused with two fluorescent proteins, GFP and RFP. GFP (acid-sensitive) is quenched or degraded in the acidic environment of autolysosomes, while RFP remains stable. Blockage of the autophagy process results in a higher yellow signal (red plus green colocalization) compared to autophagy induction (red signal) in 3D tuberculomas. Therefore, the red signal indicates autolysosome formation or induction of autophagy, while the yellow signal indicates incomplete autophagy—with autophagosome formation but without fusion with acidic lysosomes. During periods of inhibited autophagy, both yellow and red spots decreased. (b) Second exemplary method

[0090] The method may include a 3D biological platform comprising a co-culture of THP-1 cells (or human monocytes) transfected with the autophagy sensor LC3B-RFP (BacMam 2.0). The 3D biological platform may contain transfected monocytes and WT Mtb or Mm (non-fluorescent). The 3D biological platform may be contacted with the candidate molecule or a serial dilution of the candidate molecule on day 6 post-co-culture. The contacted 3D biological platform may be imaged using Cytation 5 on days 7, 9, and 12 post-contact to determine autophagy flux. A red signal indicates autolysosome formation or induction of autophagy.

[0091] Commercially available BacMam technology is based on insect viruses (baculoviruses) to aid in the efficient delivery and expression of genes in mammalian cells. The baculoviruses have been modified to include expression cassettes for transgene expression in mammalian cells. BacMam 2.0 incorporates elements that significantly improve transduction efficiency and expression levels: pseudocapsid proteins for more efficient cell entry and genetic elements that enhance expression levels (enhanced CMV promoters and prairie dog posttranscriptional regulatory elements). Baculoviruses do not replicate in mammalian cells and therefore possess excellent safety profiles and do not cause cytopathic effects. (c) Third exemplary method

[0092] The method may include a 3D biological platform comprising a co-culture of THP-1 monocytes and Mtb or Mm (non-fluorescent or red fluorescent). The 3D biological platform may be contacted with the candidate molecule or its serial dilutions on day 6 after co-culture. Enzo Life Sciences CYTO-ID may be used. ® An autophagy detection kit is used to detect autophagy. The kit uses a novel dye that selectively labels accumulated autophagosomes to measure autophagosomes and monitor autophagy flux in lysosomal-inhibited live cells. A 488 nm excitable green dye has been optimized by identifying a titratable functional moiety that allows for minimal staining of lysosomes while exhibiting bright fluorescence (green) upon incorporation of pre-autophagosomes, autophagosomes, and autolysosomes (autolysosomes). The kit also includes Hoechst 33342 dye for nuclear staining, an autophagy inducer (rapamycin), and a lysosomal inhibitor (chloroquine). (2) Methods for screening candidate molecules to induce or inhibit inflammasomes and pyroptosis in 3D tuberculomas

[0093] This article provides a method for screening candidate molecules for inducing or inhibiting inflammasomes and pyroptosis in 3D tuberculomas. The method may include using a 3D biological platform comprising a co-culture of THP-1 inflammasome reporter cells (THP-1 LC3-GFP cells) and WT Mtb (non-fluorescent). The 3D biological platform may be contacted with candidate molecules on day 6 post-co-culture, and Cytation 5 imaging may be used on days 7, 9, and 12 post-contact to determine the effects on Mtb-induced inflammasomes, ASC-GFP spot formation, and pyroptosis in 3D tuberculomas. In these reporter cells, Mtb infection or antigen exposure leads to ASC-GFP expression and ASC-spot formation following inflammasome activation. Exposure to the test molecule may increase inflammasome activation, spot formation, and pyroptosis (cell death) or decrease inflammasome activation and pyroptosis, resulting in increased cell survival. Increased inflammasome activation in macrophages is thought to be associated with reduced bacterial growth. (3) Methods for screening candidate molecules for hypoxia induction or inhibition in 3D tuberculomas

[0094] This article provides a method for screening candidate molecules for the effects of hypoxia induction or inhibition in 3D tuberculomas. The method may include the use of a 3D biological platform comprising a co-culture of THP-1 monocytes and WT Mtb or Mm (non-fluorescent). The 3D biological platform may be contacted with the candidate molecule or its serial dilutions on day 6 after co-culture. Hypoxia can be detected using the Enzo Life Sciences ROS-ID Hypoxia / Oxidative Stress Detection Kit. 3D tuberculomas or control spheroids are exposed to pre-titrated hypoxia red fluorescence in 100 µl RPMI-1640 medium on day 12 and cultured in a CO2 incubator for 24 h. After incubation, the RPMI-1640 medium in the wells can be exchanged twice with sterile PBS to remove the hypoxia red probe post-incubation. The stained 3D spheroids can be imaged using the Texas Red Channel in a Cytation-5 cell imager. The red hypoxia detection reagent (probe) is a non-fluorescent or weakly fluorescent aromatic compound containing a nitro (NO2) moiety. Because of the presence of nitroreductase activity in hypoxic cells, the nitro group is converted to hydroxylamine (NHOH) and amino group (NH2) in a series of chemical steps. The original molecule then degrades, releasing a fluorescent probe that stains hypoxic cells in 3D tuberculomas red. The test molecule's induction of hypoxia and HIF-1 in infected macrophages can activate an antimycobacterial response, but increased hypoxia can lead to increased cell death (via necrosis). (4) Methods for screening candidate molecules for lysosomal acidification

[0095] This article provides a method for screening the effects of candidate molecules on lysosomal acidification. The method may include a 3D biological platform comprising a co-culture of THP-1 monocytes and WT Mtb or Mm (non-fluorescent). The 3D biological platform may be contacted with the candidate molecule or its serial dilutions on day 6 after co-culture. Lysosomal acidification on day 12 after contact can be detected using the Enzo LifeSciences Lyso-ID Red Cytotoxicity Kit. Cell culture medium can be aspirated from 3D tuberculomas or control spheroids. They can be washed once by exchanging the culture medium with the assay buffer provided in the kit. 3D tuberculomas and control spheroids can be exposed to 50 µl of a two-color assay reagent for 2 h. The assay reagent can be aspirated, and the 3D tuberculomas or control spheroids can be washed with 100 µl of assay buffer. The spheroids can be suspended in 50 µl of assay buffer and imaged. Red lysosomal staining can be read using a Texas Red filter (excitation 540 nm, emission 680 nm), and blue nuclear counterstaining can be read using a DAPI filter (excitation 340 nm, emission 480 nm). The assay reagent comprises a unique drug-like dye that rapidly dispenses into cells and labels acidic organelles (red), and is suitable for monitoring the accumulation of lysosomes and lysosome-like structures in living cells.

[0096] The dye also fluoresces in the acidic environment at the center of the 3D tuberculoma, resulting from increased cell death (necrosis) and the release of acidic organelles. b. Methods for identifying host cytotoxicity induced by candidate molecules in 3D tuberculomas

[0097] This article provides a method for screening candidate molecules for cytotoxic effects in 3D tuberculomas. (1) Method 1: CytoTox-Glo cytotoxicity assay

[0098] The method may include: using a 3D biological platform containing a co-culture of THP-1 cells, and using a CYTOTOX-GLO assay (G9291, Promega, Mannheim, Germany) to assess cytotoxicity. This assay measures the activity of dead-cell proteases released from cells that have lost membrane integrity. The method may include a 3D biological platform containing a human immune cell-mycobacterial co-culture in a U-shaped clear-bottom 96-well plate (Corning). The 3D biological platform may contain THP-1 monocytes or human CD14 cells. +Combinations of monocytes and other PBMC subsets were co-cultured with Mtb or Mm (fluorescent or non-fluorescent) in 3D. The candidate molecule or its serial dilutions could be contacted with the 3D bioplatform on day 6 post-co-culture. Cytotoxicity could be measured on days 6, 9, 12, and 14. During assays, each 3D sphere (200 µl volume) could be dissociated and resuspended by pipetting, divided into two equal volumes (100 µl each), and transferred to two separate wells (well A and well B) in a new white-background luminescent plate. 50 µl of lysis reagent (digitalis saponin 30 µg / ml) could be added to well A, and 50 µl of RPMI medium could be added to well B. 50 µl of pre-warmed CYTOTOX-GLO reagent (1:4 dilution) could be added to all wells of the plate, and the cells could be incubated with orbital oscillation at 700–900 rpm for 15 min at room temperature. Luminescence could be measured using a BioTek Cytation 5 cell imaging multimodal reader. Control samples included 2-fold serial dilutions (from 5 × 10⁻⁶) of freshly grown THP1 cells. 6 A standard curve was obtained in each plate (starting with cells / well). Using the standard curve obtained from the control wells, the total number of cells could be calculated from well A, and the total number of dead cells could be calculated from well B. The data were analyzed using Microsoft Excel.

[0099] Cytotoxicity = (2) Use of THP-1-RFP cells

[0100] The method may include measuring the cytotoxicity of a candidate molecule or its serial dilutions by determining the effect of the candidate molecule on host cell viability and finding the cytotoxicity 50 (CC50) and effective concentration 50 (EC50) values ​​of the candidate molecule. The method may include a 3D biological platform containing a co-culture of THP-1 cells expressing RFP and WT Mtb or Mm (non-fluorescent). The 3D biological platform may be contacted with the candidate molecule or its serial dilutions on day 6 after co-culture. THP-1 red fluorescence (excitation 588 nm and emission 633 nm) may be measured on days 6, 9, 12, and 14 after contact. Untreated or DMSO-treated 3D tuberculomas may be used as controls. The amount of red fluorescence indicates viable THP-1 cells, and the loss of red fluorescence indicates dead cells. The cytotoxicity (%) induced by the test molecule in 3D tuberculomas relative to untreated or DMSO-treated cells is calculated. 5. Treatment methods

[0101] This document provides a method for treating or reducing mycobacterial infection, which may be tuberculosis. The method may include treating or reducing tuberculomatous lesions. The method may include administering an antimycobacterial agent to a subject in need. This document also provides an antimycobacterial agent for treating or reducing tuberculomatous lesions, or the use of said antimycobacterial agent in the manufacture of a medicament for treating or reducing tuberculomatous lesions. The antimycobacterial agent may include one or more of the following: tumor necrosis factor (TNF) blockers, such as anti-TNF antibodies; CD11a blockers, such as anti-CD11a antibodies; anti-vascular endothelial growth factor (VEGF) blockers, such as anti-VEGF antibodies; integrin α4β7 blockers, such as anti-integrin α4β7 antibodies; CD30 blockers, such as anti-CD30 antibodies; insulin-like growth factor-1 receptor (IGF1R) blockers, such as anti-IGF1R antibodies; IL-6Rα blockers, such as anti-IL-6Rα antibodies. ; IL-1β blockers, such as anti-IL-1β antibodies; IL-1R blockers, such as anti-IL-1R antibodies; compound AT9283; aurinophene; chlormastine fumarate; chlorpromazine hydrochloride; dichlorobenzamil hydrochloride; dovirtinib; doxycycline; H89; lansoprazole; methaxaxone hydrochloride; nizonitrazepam; zuclothiasol dihydrochloride; GW5074; simvastatin; vorinostatin; all-trans retinoic acid; pazopanib; porphyrin IX; fluoxetine hydrochloride; dasatinib; GW 5074; SRT 1720; gefitinib; loperamide; ezetimibe; and active metabolites of the above compounds. In one example, the antimycobacterial agent includes AT9283.

[0102] In one example, an antimycobacterial agent includes an antibody. The antibody may be an anti-CD11a, anti-α4β7 integrin, anti-CD30, anti-IGF1R, or anti-IL-6R antibody. The antibody may be monoclonal and may be humanized. Antimycobacterial agents may include biosimilars of the antibody. In another example, an antimycobacterial agent includes a compound. The compound may include AT9283, tinizonide (the active metabolite of nitrozonide), dasatinib, quinacrine dihydrochloride, all-trans retinoic acid (ATRA), vorinostat, sitagliptin, H89, or lansoprazole. Antibodies, their biosimilars, or compounds can kill mycobacteria and reduce granulomatous lesions by activating host cell mechanisms in 3D tuberculomas. Antimycobacterial agents can be used to treat mycobacterial infections or tuberculosis.

[0103] Antimycobacterial agents may include one or more host-directed molecules or biological agents that act via host cells, as well as one or more antibiotics or other antimicrobial agents that act directly on mycobacteria.

[0104] The present invention has several aspects, which are illustrated by the following non-limiting embodiments. Example 1 Three-dimensional biological platform

[0105] This embodiment demonstrates a method for preparing the 3D biological platform disclosed herein and its use in screening. summary

[0106] Tuberculomas are well-organized, 3D clumps of tuberculous granulomas caused by Mycobacterium tuberculosis infection and are one of the more severe forms of tuberculosis (TB). Several in vitro models mimicking human TB granulomas have been reported to date to decipher the complex host-Mtb biology and discover novel preventative and therapeutic interventions. However, these models lack well-organized granulomatous lesions and classic tuberculoma structures. Furthermore, they are impractical for screening large libraries of compounds due to their low throughput, limited scalability, batch-to-batch variability, and high cost. Here, we describe a co-culture workflow of "Mycobacteria in Spheroids" in standard 96-well plate form to produce a robust 3D cell culture model. This model reproduces the key properties and microenvironment of human tuberculomas and can be scaled up as a high-throughput screening platform for biotechnology. The resulting tuberculoma-like structures contain well-organized, vigorous granulomatous lesions and exhibit solid, necrotic, and cavitary morphologies not previously described in 3D cell culture models of TB. This model can be developed using freshly isolated primary human blood mononuclear cells or mononuclear cell lines with virulent mycobacteria. The platform integrates the entire workflow from generation to imaging of in situ tuberculoma-like structures. It allows for continuous quantification of drug efficacy after a single treatment over a period of days to weeks, as well as observation of lesion size and number regression. A method for employing this workflow as a bio-platform for cryopreservation for potential commercial use is also described. The bio-platform's ease of generation, reproducibility, adaptability, and cryopreservation stability make it ideal for HTS applications and suitable for easy scaling and implementation in tuberculosis and other granulomatous disease discovery projects. background

[0107] Tuberculosis (TB) (an airborne disease caused by Mycobacterium tuberculosis (Mtb)) is one of the leading infectious killers worldwide. In 2021, an estimated 10.6 million people were infected with TB, and 1.6 million died from it (WHO Global Tuberculosis Report, 2022). TB is now projected to kill almost twice as many people daily as COVID-19 (Bagcchi, 2023). The histopathological hallmark of TB is the formation of dynamic, spatially organized, multicellular clusters called granulomas. These macrophage-rich structures serve as microenvironments for Mtb growth and spread, while also providing an environment for infected macrophages to interact with other recruited cells to “contain” and combat the invading pathogen (Ramakrishnan, 2012; Pagan and Ramakrishnan, 2018; P. Elkington et al., 2022). One of the more serious clinical manifestations of TB is the formation of tuberculomas, which aggregate tuberculous granulomas into well-defined masses, most commonly found in the lungs and brain, resembling cancerous tumors in these organs (Culver et al., 1950; Moyes, 1951; Sochocky, 1958; Nicols et al., 2005; Monteiro et al., 2013). Tuberculomas exhibiting severe morphological forms are present in approximately 5%–10% of patients with pulmonary TB (Lee et al., 2004; Wetscherek et al., 2022). In patients with pulmonary TB, granulomas are highly polymorphic and exhibit a range of structures, including solid, hypoxic, necrotic, and cavitary transformations, and display complex morphologies in lung sections (Cadena et al., 2017; Wells et al., 2021; Sawyer et al., 2023). Although spherical granulomatous nodules with a diameter of 2–5 mm are common in the lungs, the size of tuberculoma structures varies from a few mm to >10 cm (Lee et al., 2004; Wells et al., 2021). Cavitotic transformation of tuberculomas increases the risk of person-to-person transmission. Furthermore, it is associated with poor treatment outcomes, recurrence, and potential drug resistance in lung TB (Urbanowski et al., 2020).

[0108] The recent Covid-19 pandemic has dramatically disrupted and reversed the positive trend of sustained decline in global TB incidence and mortality over the past decade. It continues to negatively impact TB diagnosis and care. This setback in TB control efforts is projected to lead to a long-term increase in TB-related deaths and delay the achievement of the UN Sustainable Development Goal of ending TB endemicity by 2030 (WHO Global Tuberculosis Report, 2022) (Pai et al., 2022; Bagcchi, 2023). To achieve TB elimination, there is an urgent need for improved and shorter treatment regimens for both drug-resistant and drug-sensitive forms of TB. Such regimens may stem from a better understanding of the efficacy of novel therapeutics across granulomatous forms and microenvironments. A growing body of preclinical and clinical studies highlights the importance of designing novel pathogen-targeted and host-directed therapies (HDTs) that are readily permeable and act within the granulomatous environment (Prideaux et al., 2015; Strydom et al., 2019; Larkins-Ford et al., 2021; Dartois and Rubin, 2022; Wallis et al., 2022). Animal models and in vitro cell culture systems can be used to identify therapeutics that modulate host-Mtb interactions in granulomas. Several 2D and 3D in vitro cell culture models using primary human cells infected with Mtb to mimic neonatal TB granulomas have been described in recent years (Puissegur et al., 2004; Birkness et al., 2007; Guirado et al., 2015; Kapoor et al., 2013; Tezera et al., 2017a; Berry et al., 2020; Kotze et al., 2021). While helpful in screening a limited number of compounds and probing Mtb-host interactions (Bielecka et al., 2017; Tezera et al., 2017b), these models suffer from low throughput, limited plasticity, and limited scalability (P. Elkington et al., 2019; Arbues et al., 2020). They form small granulomatous aggregates of macrophages but lack organized granulomatous lesions and the associated tuberculoma size, structure, and form.

[0109] Therefore, the physiological gradients of nutrients, oxygen, pH, and associated microenvironments present in heterogeneous TB lesions are either absent or not fully reproduced in these models. Key features such as hypoxia and Mtb dormancy observed in some of these 3D models with small granulomas are primarily due to macrophages encapsulated in microspheres and embedded in the extracellular matrix (ECM) (rather than the granuloma structure itself) (Colom et al., 2014; Figueiredo et al., 2018; Arbues et al., 2021). The lack of a sustained influx of immune cells in these models makes maintaining dynamic structures and extending experiments over long periods challenging. Using animal models for this screening is expensive, time-consuming, and limits the number of compounds that can be screened in high-level protective facilities. A high-throughput screening (HTS) compatible and widely applicable biological platform that reproduces the key features and microenvironment of TB lesions and allows for continuous multi-parameter readouts of host and pathogen physiology and spatiotemporal presence is highly desirable.

[0110] The protocol described here provides a simple workflow (Figure 1) that uses human monocytes and a virulent mycobacterial strain expressing bright red fluorescent protein (tdTomato) to develop an HTS-compatible bioplatform for evaluating drug efficacy in 3D cell culture microplates based on fluorescence intensity and imaging. The “mycobacteria in spheroids” co-culture generated in the microplates consistently mimics the structure and size of 3D human tuberculomas. Since macrophages are fundamental to the formation of the core scaffold, which determines the host immune response and therapeutic accessibility in tuberculous granulomas (Cronan, 2022), we employed human THP-1 monocytes or peripheral blood mononuclear cells (PBMCs) as the source of macrophages. The use of freshly cultured THP-1 monocytes or purified CD14 from PBMCs is described. + Three different versions of biological platform methods for monocytes ( Figures 1A-1C Surprisingly, in 3D co-culture, individual human THP-1 mononuclear cells with self-renewal and recruitment capabilities could generate structurally organized granulomatous lesions in the absence of other myeloid, lymphoid, and non-hematopoietic cells.

[0111] Furthermore, this 3D spheroid co-culture produced a range of morphological forms mimicking solid, necrotic, and hollow tuberculomas that could not be described by existing in vitro 3D granuloma models. Other advantages of this platform include ease of development, increased throughput, scalability, adaptability, and in situ real-time monitoring of bacterial load and lesions. Additionally, we successfully used the risk group 2 pathogen *Mycobacterium marineum* as an alternative to *Mtb* to develop this bioplatform in a BSL-2 laboratory, resulting in a bioplatform comparable to those created using *Mtb*. *Mtb* can cause granulomatous skin infections in humans and generate human TB-like disease with macrophage epithelialization and tuberculous granulomas in zebrafish (Cosma et al., 2003; Cronan et al., 2021). The *Mtb*-zebrafish infection model has emerged as a useful in vivo platform for studying the dynamics of tuberculous granuloma formation and host-pathogen interactions (Davis et al., 2002; Cronan, 2022). Using this workflow with Mm can significantly reduce the time required for extensive screening in BSL-3 laboratories, while potentially reducing safety issues and costs.

[0112] Because the successful preservation, maintenance, and long-term storage of the screening biological platform are highly desirable, we adapted our workflow to develop a cryo-stable version for potential commercialization, which can be frozen for future use and thawed as needed. Figure 1D The described biological platform and technology have the potential to minimize the amount of animal testing in high-security facilities by improving screening capabilities and efficiency while saving time and resources. Although the system was developed for Mtb, we have provided a foundation to allow others to modify this system for other granulomatous inflammatory and infectious diseases, including comorbidities and co-infections such as Mtb infection with HIV, influenza, or SARS coronavirus 2. The biological protocol described herein advances the technology of “human in vitro 3D granuloma model”. Materials and reagents

[0113] RPMI 1640 medium (Gibco) containing L-glutamine and with or without phenol red. TM (Catalogue numbers: 11875-093 and 11835-030)

[0114] Penicillin-streptomycin solution, 10,000 units / ml for each antibiotic (Gibco) TM (Catalog number: 15140-122)

[0115] Sodium pyruvate solution, 100 mM (Gibco) TM (Catalog number: 11360-070)

[0116] HEPES buffer, 1M (Gibco) TM (Catalog number: 15630-080)

[0117] Fetal bovine serum (FBS), tested for endotoxin levels and heat-inactivated, of U.S. origin (AtlasBiologicals, catalog number: F-0500-A).

[0118] Nalgene® Rapid-Flow™ disposable vacuum filter unit, sterile (500 ml, with 0.2 µm membrane) (e.g., Thermo Fisher Scientific, catalog number: 5660020)

[0119] Reagent storage containers, sterile, single-use (e.g., Aquafill, catalog number: S-5501080).

[0120] Micropipette tips with filters (various volumes), sterile, disposable (e.g., Rainin) TM (Catalogue numbers: 30389257, 30389272, 30389276 and 30389274)

[0121] Serological pipettes (various volumes) (e.g., Pyrex, catalog numbers: 7077-5N and 7077-10N)

[0122] Cell culture flask, 75 cm 2 150 cm 2 and 175 cm 2 Aseptic (Corning) TM (Catalog numbers: 430720U, 431465 and 431080)

[0123] Falcon TM Conical centrifuge tubes, 15 and 50 ml, sterile (Corning) TM Falcon® (catalog numbers: 352097 and 352098)

[0124] Microcentrifuge tubes (various volumes, sterile) (e.g., GreenTree Scientific, catalog number: T5040G, and LabSource, catalog number: T56-950)

[0125] Test tube racks or supports (e.g., Fisher Scientific, catalog numbers: 21-200-285, 03-448-17, 21-402-18).

[0126] Human THP-1 cells (American Type Culture Collection, catalog number: TIB-202)

[0127] Mtb strain H37Rv or Erdman expressing the deep red fluorescent protein tdTomato, Mm strain M expressing tdTomato, and Mm strain 1218 expressing the green fluorescent protein (GFP)

[0128] Note: Co-culture requires pathogenic mycobacteria (risk group 2 or 3). Attenuated Mycobacterium bovis BCG strains lack the genomic virulence locus RD1 and are unsuitable.

[0129] Sterile syringes equipped with needles (25 or 27G) (e.g., Becton Dickinson (BD), catalog number: 309626).

[0130] Cell culture grade water, sterile (Corning) TM (Catalog number: 25-055-CM)

[0131] Duchenne phosphate-buffered saline (PBS), calcium and magnesium-free, sterile, pH 7.4 (Gibco) TM (Catalogue number: 14190-136)

[0132] 3D Cell Culture Plate (Corning® Spherical Microplate, Catalog No.: 4515)

[0133] We tested microplates from several different vendors. The Corning® Ultra-Low Adsorption (ULA) spherical microplate was optimal for generating the 3D tuberculoma structures described herein. This 96-well microplate has an opaque black body that shields against inter-well crosstalk from each optically transparent microwell. The round-bottomed microwells have a hydrophilic, bioinert surface. They are coated with a covalently bonded, nonionic, neutrally charged hydrogel that minimizes activation and cell adhesion to the pore surface, enabling uniform and reproducible formation of 3D tuberculoma-like structures. Another useful alternative is the S-BIO PrimeSurface® 3D Culture Spherical Plate (S-BIO, catalog number: MS-9096UZ).

[0134] Trypan blue staining solution (e.g., Invitrogen) TM (Catalog number: T10282)

[0135] Countess™ Cell Counting Chamber Slides (Invitrogen) TM(Catalogue No.: C-10283) or Neubauer cell counting chamber (e.g., Millipore Sigma, Bright-Line blood cell counter, catalogue No.: Z359629)

[0136] Mycobacterial culture flasks (e.g., 490 cm⁻¹) 2 Aseptic roller bottle, Corning®, catalog number: 430195

[0137] Mycobacterium liquid growth medium, Middlebrook 7H9 culture medium with supplements (e.g., internal preparation, see formulation. Middlebrook 7H9 culture medium dehydrated substrate, BD, catalog number: 271310; Tween 80, Fisher, catalog number: BP338-500; Middlebrook bovine albumin fraction V, dextrose, catalase (ADC) enrichment, BD, catalog number: 212352; and glycerol, Sigma-Aldrich, catalog number: G7893)

[0138] Mycobacterial culture plates (e.g., internally prepared Middlebrook 7H10 agar plates, antibiotic-free and supplemented with oleic acid, albumin, dextrose, and catalase (OADC) enriched at 10% (vol / vol) and glycerol at 0.5% (vol / vol). Middlebrook 7H10 agar, BD Difco TM Catalog number: 262710; Middlebrook OADC, BD, catalog number 212351; and glycerol, Sigma-Aldrich, catalog number: G7893)

[0139] Bacterial cell applicator (e.g., Fisher Scientific, catalog number: 14-665-230)

[0140] Triton X-100 (e.g., Sigma-Aldrich, catalog number: X-100)

[0141] In BD Vacutainer ® Cell preparation tube (CPT) TM Human PBMCs (collected from whole blood from healthy tuberculin skin test negative donors) (Catalogue No.: 362761).

[0142] VitroCol Type 1 Human Collagen Solution (Advanced BioMatrix, Catalog No.: 5007-20ml)

[0143] Fibronectin from human plasma, 0.1% solution, 1 mg / ml (Sigma-Aldrich, catalog number: F0895)

[0144] 0.1 M sodium hydroxide solution (NaOH) for cell culture (Advanced Biomatrix, catalog number: 5078)

[0145] Calcium and magnesium-free phosphate-buffered saline (PBS) 10X (Advanced BioMatrix, catalog number: 5076-B)

[0146] Red blood cell (RBC) lysis buffer, 1X eBioscience TM (Invitrogen, catalog number: 00-4333-57)

[0147] Ethylenediaminetetraacetic acid (EDTA) solution, 0.5 M, sterile (Amresco, catalog number: C-177)

[0148] Magnetic-Assisted Cell Sorting (MACS) LS Column (Miltenyi Biotec, Catalog No.: 130-042-401)

[0149] MACS ® CD14 microbeads, human CD14 + Mononuclear cell isolation reagent (Miltenyi Biotec, catalog number: 130-050-201)

[0150] Cell scrapers (e.g., Costar) TM (Catalog number: 3010)

[0151] Petri dishes (e.g., Falcon) TM (Table of Contents: 351029)

[0152] Dimethyl sulfoxide (DMSO), cell culture grade and endotoxin level tested (Sigma-Aldrich, catalog numbers: D2650-5 × 10ML and D5879).

[0153] Test block 96 wells, 2 ml capacity, sterile (Corning) TM (Table of Contents: 3960)

[0154] Leibovitz L-15 medium (Gibco) TM (Catalog number: 11415-064)

[0155] Polyvinylpyrrolidone (PVP) (MP Biomedicals, catalog number: 102786)

[0156] Cell growth medium (complete RPMI-1640 medium containing antibiotics) (see formulation)

[0157] 3D cell culture medium (antibiotic-free complete RPMI-1640 medium) (see formulation)

[0158] Middlebrook 7H9 culture medium (see formula)

[0159] Extracellular matrix (ECM) solution (see formulation)

[0160] PBMC washing buffer (see formulation)

[0161] MACS buffer (see recipe)

[0162] 3D cell culture cryopreservation medium (see formulation) equipment

[0163] Class II A2 Biosafety Cabinet (BSC) (e.g., Nuaire, model: NU-543-600)

[0164] Micropipettes (various volumes) (e.g., Rainin) TM Catalog numbers: Pipet-Lite LTS, Pipettes L-20XLS, L-200XLS, L-1000XLS, L-5000XLS and L8-200XLS

[0165] Novaspec II spectrophotometer (Amersham Pharmacia Biotech) or equivalent

[0166] Aerosolve ® can or equivalent

[0167] Cell culture incubator, set to 37ºC, 5% CO2, and 100% humidity (Forma Scientific, model: 3110).

[0168] Microcentrifuge for microtubes (Eppendorf, model: 5430 R)

[0169] Centrifuge for 15 or 30 ml conical tubes (Eppendorf, model: 5810 R)

[0170] Cell counting equipment (Thermo Fisher Scientific, model: AMQAX1600 Countess II or AMQAX2000 Countess III)

[0171] pH meter or pH test strips (e.g., Fisher brand, catalog number: 13-640-510)

[0172] Cytation 5 microplate fluorescence reader and cell imager or an equivalent device with an optional CO2 source (i.e., CO2 controller) (Agilent / BioTek, model: CYT5MPV with GEN5PRIME and GEN5SPOT).

[0173] Note: Fluorescence readers and cell imagers other than Cytation 5 can be used.

[0174] MidiMACS TM The separator (Miltenyi Biotec, catalog number: 130-042-302) and the MACS multi-stent (Miltenyi Biotec, catalog number: 130-042-303)

[0175] Water bath (Precision 180 series)

[0176] Refrigerator, set to 2ºC to 8ºC

[0177] Freezer, set to -80ºC

[0178] Liquid nitrogen storage for cell culture (temperature -196ºC)

[0179] autoclave software

[0180] Gen5 V3.08 (Agilent / BioTek) with a spot counting add-on module

[0181] The Gen5 Image+ software controls the operation of the Cytation 5 for microplate reading and automated digital microscopy based on photomultiplier tubes (PMTs).

[0182] GraphPad Prism V9.3 (GraphPad by Dotmatics) program Workflow of a 3D tuberculoma bioplatform using freshly cultured THP-1 monocytes

[0183] This optimized workflow describes the steps for developing a tuberculoma bioplatform in 96-well 3D cell culture microplates using a simple co-culture of THP-1 mononuclear cells and fluorescently pathogenic mycobacteria for high-content screening of potential therapeutic agents. All work involving the handling of virulent Mtb strains (risk group 3 organisms) should be performed in a BSL-3 laboratory under a Class II BSC while wearing appropriate personal protective equipment (PPE). If the target organism is Mm (risk group 2), experiments are performed in a BSL-2 laboratory under a Class II BSC while wearing appropriate PPE. A typical workflow for screening test compounds, small molecules, or biologics spans 14 days. Depending on the experimental objectives, the workflow can be extended to more than two weeks if the microwells are replenished with fresh medium and THP-1 cells. Using the Mm 1218 strain and carefully exchanging 50% of the medium in the microwells with fresh medium every seven days, we successfully cultured a "mycobacteria in spheroids" model for up to 50 days. Day 1. Production of 3D co-cultures of "mycobacteria in spheroids".

[0184] Prepare a THP-1 cell suspension for 3D cell culture.

[0185] Divide the 3D cell culture medium equally into 50 ml conical tubes (see Formula 1).

[0186] Note: Please check the sterility of the culture medium before use.

[0187] The 3D cell culture medium was preheated at 37ºC for 1 h in a cell culture incubator.

[0188] THP-1 cells cultured in antibiotic-containing growth medium (see Formulation 2) were counted and their viability was assessed using the trypan blue staining method.

[0189] Cell viability must be ≥ 95%, and a final concentration of 1 × 10⁻⁶ is required. 6 viable cells / ml. Use completely dissolved trypan blue dye to avoid interference with automated cell counting caused by dye precipitation. We use an automated cell counter, Countess II or III, for cell counting. Cells can be counted from two or more 150cm cells cultured in the same batch. 2 Cell culture flasks are merged and subjected to cell counting and viability assessment. For extensive experiments requiring >4 microplates, THP-1 cells will need to be cultured in two or more flasks.

[0190] Centrifuge the cells at 200-250 xg in a 50 ml conical tube at room temperature (15ºC-20ºC) for 6 min, and discard the supernatant without disturbing the cell pellet.

[0191] Resuspend the cell pellet in 15 ml of preheated (37ºC) 3D cell culture medium (see Formula 1) and gently mix using a pipette. Centrifuge the cells again at 200–250 x g at room temperature for 6 min and discard the supernatant.

[0192] Repeat this washing step twice more.

[0193] After the third wash, the cell pellet was resuspended in 15 ml of preheated (37ºC) 3D cell culture medium, gently mixed with a pipette, and the cell suspension was allowed to stand at room temperature for 30 min.

[0194] This step is incorporated to exchange and remove antibiotics via endocytosis of THP-1 cells during monolayer culture in an antibiotic-containing growth medium.

[0195] Centrifuge the cells at 200–250 xg at room temperature for 6 min and discard the supernatant. Resuspend the cell pellet in 10 ml of preheated (37°C) 3D cell culture medium and mix gently with a pipette.

[0196] The final cell count and cell viability were assessed using the trypan blue rejection method.

[0197] The cell concentration was adjusted to 1 × 10 using 3D cell culture medium. 6 live cells / ml.

[0198] In the microplate format tested, each well required precisely 6 ml of THP-1 cell suspension. Cells were not dispensed into the wells at the periphery of the plate to avoid the “edge effect.” The outer wells were filled with sterile 3D cell culture medium or cell culture-grade water to mitigate evaporation.

[0199] Preparation of Mtb H37Rv(tdTomato) or Mm M(tdTomato) suspensions for 3D cell culture

[0200] For this workflow, we tested Mtb (H37Rv, Erdman, Beijing F2, and CDC 1551) and Mm (M) strains expressing the red fluorescent protein tdTomato. The fluorescent protein tdTomato was cloned into the constitutively expressed mycobacterial pmsp12 vector using the pTEC27 plasmid (Takaki et al., 2013), and see also Example 2 described below. This red fluorescence provided optimal brightness and low background autofluorescence in spheroid co-cultures. To prepare frozen mycobacterial stock solutions, the fluorescent Mtb and Mm strains were grown in Middlebrook 7H9 medium (see Formulation 3) supplemented with appropriate antibiotics (50 µg / ml hygromycin for tdTomato-expressing strains; or 25 µg / ml kanamycin for GFP-expressing strains). We inoculated 5 ml of 7H9 culture medium into 30 ml 25 × 150 mm glass culture tubes with 100–150 µl of a strain stock stored at -80°C, and incubated at 30°C (Mm) or 37°C (Mtb) for 5–10 days. In experiments comparing multiple strains, stock solutions were prepared simultaneously in batches using the same growth medium. At 600 nm (OD) 600 The optical density of the inoculum was measured at ( ). When the OD of the inoculum... 600 When the concentration reaches 0.6-1.0, add 5 ml of inoculum to a 490 cm² area. 2 Add 150 ml of 7H9 culture medium to a sterile culture flask. Incubate the culture at 30ºC (Mm) or 37ºC (Mtb) for 10–12 days, manually shaking once daily until OD (dose retardation). 600 Achieve a concentration of 1.0–1.5, as measured by a Novapec II spectrophotometer. Accumulate the culture by centrifugation at 4000 xg for 30 min at room temperature. Remove the supernatant by decantation or pipetting, and resuspend the precipitate in 10 ml of fresh 7H9 culture medium (see Formulation 3), aliquoting 1.0 ml / frozen flask and storing at -80ºC. Prepare stock solutions of the recombinant fluorescent strain using 7H9 culture medium supplemented with appropriate antibiotics. Thaw aliquots seven days after freezing and further aliquot in 100 µl of working stock solution. Determine the viability count on antibiotic-free 7H10 agar by incubation at 30ºC for 12–15 days (Mm) or at 37ºC for 28–30 days (Mtb). Frozen stock solutions can be stored for one year after preparation; after one year, new stock solutions must be prepared using the original strain preservation.

[0201] In AEROSOLVE ®Cryopreservation vials containing Mtb H37Rv (tdTomato) or Mm M (tdTomato) cryopreservation stock solution in the (transport) tank are transported from the storage freezer to the BSC. The cryopreservation vials are then placed in the test tube rack in the BSC.

[0202] Transfer 900 µl of 3D cell culture medium to a microtube containing 100 µl of mycobacterial working stock solution and mix thoroughly.

[0203] Centrifuge at 5000 xg for 30 min at 8ºC to 10ºC. When finished, carefully discard the supernatant using a pipette without disturbing the mycobacterial precipitate.

[0204] Repeat this washing step using 1 ml of fresh 3D cell culture medium and resuspend the precipitate in 1 ml of preheated (37ºC) 3D cell culture medium.

[0205] To prepare a single-bacterial cell suspension, pass the mycobacterial suspension through a 25-27G needle attached to a 1 ml syringe 10 to 20 times.

[0206] Perform this step immediately before infecting THP-1 cells to avoid bacterial aggregation. Note: Do not cap the needle on the syringe. Carefully place the syringe with the needle into the sharps disposal container.

[0207] Transfer the required amount of mycobacterial single-cell suspension to a sterile reservoir. Dilute the mycobacterial suspension with a volume of preheated 3D cell culture medium for THP-1 cell infection in microplates, the volume being required to achieve the desired multiple of infection (MOI) and to prepare the required volume of bacterial inoculum.

[0208] Our experiments are routinely performed using five-well microplates, and we typically prepare 35 ml of THP-1 cell suspension and mycobacterial suspension (inoculum) in sterile reservoirs. During transfer to the microwells, we frequently mix the inoculum by up-and-down pipetting to maintain a homogeneous single-cell suspension and prevent mycobacteria from settling and agglomerating in the reservoir. In preliminary experiments, we determined the optimal range of bacterial numbers required to infect monocytes in 3D co-culture that does not lead to overcolonization in the 3D spheroids formed within 2–3 weeks of incubation (e.g., using fluorescent Mm strains, see [link]). Figures 2A-2DMOI was calculated as input CFU / input monocytes per well. We did not wash monocytes after infection and avoided using aminoglycoside antibiotics to kill extracellular mycobacteria (if any). Pinocytic aminoglycosides can reach macrophage phagosomes and significantly promote the antimicrobial activity of cells (Vogt and Nathan, 2011). We determined the optimal MOI for using MmM(tdTomato) to be 0.008 (i.e., 800 CFU / 10). 5 The optimal MOI range is 0.006–0.012. Our optimized MOI for Mtb H37Rv (tdTomato) is 0.02 (MOI range 0.015–0.025). The ideal MOI varies depending on the mycobacterial species or strain used in 3D co-culture and can be determined experimentally by monitoring the kinetics of bacterial growth and granulomatous lesion formation within the 3D spheroid structure and by determining the Z' factor in HTS assays performed using a biological platform (see Data Analysis section below). A low physiological MOI ensures complete aggregation of bacteria by accumulating THP-1 cells during 3D spheroid formation and avoids unwanted colonization outside the spheroid structure within two weeks without the need for the use of aminoglycoside antibiotics, which are believed to kill only extracellular bacteria.

[0209] Preparation of 3D microplates for 3D cell culture

[0210] The label includes the plate number, date, experiment name, and the name of the person performing the experiment. ® 3D spherical microplate. Marked outer boundary wells. See the Data Analysis section for a schematic diagram of the plates used in compound screening assays.

[0211] Add 100 µl of THP-1 cell suspension (1 × 10⁻⁶) 5 (One live cell) is added to each micropore except for the peripheral ones.

[0212] Use the six micropipette tips mounted on the multichannel pipette. When dispensing cell suspensions, tilt the micropipette tips against the well walls. Therefore, avoid allowing the micropipette tips to touch the bottom of the wells.

[0213] Add 100 µl of mycobacterial suspension to each microwell except for the peripheral ones.

[0214] Control wells containing THP-1 cells can remain free of mycobacterial infection, depending on the experimental objectives. Add 100 µl of 3D cell culture medium to these wells instead of the bacterial suspension.

[0215] Mix thoroughly up and down at least three times without touching the bottom of the micropores to obtain a homogeneous suspension of THP-1 and mycobacteria. Avoid bubble formation during mixing.

[0216] Use a multichannel micropipette to fill the peripheral microwells with 250-300 µl of sterile 3D cell culture medium or cell culture grade water.

[0217] The microplates were placed in a cell culture incubator at 37ºC, 5% CO2, and 100% humidity to continue the co-culture experiment and to generate 3D “mycobacteria in spheroids” structures in the ULA round-bottom microwells.

[0218] The actual CFU count in 100 µl of mycobacterial suspension was determined by platenting the dilution onto Middlebrook 7H10 agar plates. The agar plates were incubated at 30ºC (for Mm M(tdTomato)) for 12–14 days and at 37ºC (for MtbH37Rv(tdTomato)) for 3–4 weeks.

[0219] Clean the BSC with a tuberculosis-killing disinfectant (e.g., Cavicide).

[0220] Fill in the experiment worksheet. Day 6. Testing the addition of compounds, small molecules, or other therapeutic agents.

[0221] Drug diluents for screening were prepared and added in a 3D bio-platform.

[0222] A dilution of the test drug was prepared in preheated (37ºC) 3D cell culture medium.

[0223] Optimal drug concentrations can be evaluated experimentally. We routinely use a standard concentration of 20 µM drug / microwell for screening experiments. Additionally, we typically test six different concentrations of selected drugs ranging from 20 µM to 0.625 µM (using 2-fold serial dilutions). We perform drug dilutions in 96-well, 2 ml volume, sterile assay blocks. For screening large compound libraries, stock compound solutions (10 mM) in DMSO are obtained from commercial suppliers, aliquoted, and stored at -20ºC or the recommended temperature. To obtain the final concentration of 20 µM test compound / microwell, 10 µl of the stock compound (10 mM) is diluted in 990 µl of preheated (37ºC) 3D cell culture medium, and 50 µl of this diluted compound solution is added to microwells containing spheroids in 200 µl of 3D cell culture medium.

[0224] Use the manual mode or inverted microscope (optional) in the Cytation 5 imager to observe granulomatous lesion formation in a 3D co-culture of "mycobacteria in spheroids".

[0225] In 3D spheroids infected with optimized low MOI Mm M and Mtb H37Rv, vigorous granulomatous lesions began to develop between day 5 and day 6 (and for Mm 1218 between day 8 and day 10). The granulomatous lesions grew and became structurally organized over time.

[0226] The diluted drug in 50 µl volumes is slowly and carefully added to the microwells without disturbing the 3D spheres. At least three technical replicates of the drug are retained for each test, and appropriate positive and negative control wells are included in each microplate.

[0227] Following drug treatment, the final volume of culture medium in the microwells will be approximately 250 µl. The negative control wells will receive 50 µl of 3D cell culture medium. The pipette tip should be tilted against the microwell wall while slowly dispensing the drug solution to avoid disturbing any 3D spheroids and granulomatous lesions that may form. For more information on the positive and negative controls maintained in our drug screening assays, please refer to the Data Analysis section. Day 12. Fluorescence intensity reading and imaging used to assess the efficacy of anti-tuberculosis drugs.

[0228] 1. Read the fluorescence intensity of the microplate as a measure of bacterial load.

[0229] Place the capped microplate (without a base plate support) into the Cytation 5 multimode plate reader, which is pre-set to 30ºC or 37ºC and 5% CO2.

[0230] Cytation 5 combines automated digital wide-field microscopy with routine multimodal microplate detection to provide phenotypic cell information and well-based quantitative data. In the Gen5 software, ensure the appropriate plate type (e.g., Corning ULA round well bottom) is selected and the bottom height of the vessel is defined. Fluorescence intensity in co-cultures can be measured in the absence of CO2 using the Cytation 5 reader. For Mm co-cultures, we performed in situ fluorescence readings and imaging at 30ºC without a CO2 source and controller. We tested automatic gain and several fixed gain readings in microplates with 3D spheroids and found that automatic gain readings were suitable for compound screening assays. For fluorescence intensity readings in the generated 3D spheroids, “top” readings of the microplate were better than “bottom” readings. Microplate fluorometers can be a low-cost alternative to multimodal fluorescence readers. The optimal time for reading fluorescence intensity or imaging after drug treatment should be determined experimentally.

[0231] The fluorescence intensity of tdTomato was read using a developed, validated fluorescence intensity reading scheme stored on a computer connected to Cytation 5. Table 2 Fluorescence intensity reading parameters.

[0232] To read the fluorescence intensity and growth of mycobacteria expressing tdTomato in Corning 96-well 3D cell culture plates, the following four steps were followed.

[0233] i. Open the Gen5 software. Next, select the "Experiments" tab on the left panel of the "Task Manager" screen.

[0234] ii. On the right side of the screen, select "Create using an existing scheme" and choose the pre-developed and saved tdTomato fluorescence intensity reading scheme. The Task Manager screen will close. The parameters of the tdTomato fluorescence reading scheme are listed in Table 2.

[0235] iii. On the top panel of the Gen5 software, click the green "Read New" button. The software will ask you to save the experiment name and other details.

[0236] iv. After saving the experimental file, the software will continue to read the fluorescence intensity in the pre-selected microwells of the plate.

[0237] The fluorescence intensity data was exported as an Excel file generated by Gen5 and saved on the computer for data analysis.

[0238] 2. Automated plate imaging using Cytation 5

[0239] Place the capped microplate in Cytation 5, set to 30ºC or 37ºC and 5% CO2.

[0240] Images were captured using the optimized scheme and the parameters listed in Table 3.

[0241] Co-culture imaging of mycobacteria within spheroids was performed to produce high-quality images mimicking the structure of 3D tuberculomas and to evaluate the efficacy of drugs in reducing the growth of fluorescent mycobacteria and the regression of granulomatous lesions. The entire pore was imaged using a 2.5x objective and a 2 × 2 image montage. Bright-field imaging was used to capture images of the total spheroids, and the red fluorescence channel (Texas Red) was used to image mycobacteria expressing tdTomato. Table 3 Parameters for Automated Fluorescence Imaging

[0242] To image 3D cell culture microplates, follow these steps.

[0243] i. Open the Gen5 software. In the Task Manager window, select the "Experiment" option.

[0244] ii. On the right side of the screen, select "Create using an existing scheme" and choose a pre-developed 3D spherical imaging scheme. The "Task Manager" screen will close. The complete parameters for the imaging and analysis scheme are listed in Tables 3-7.

[0245] iii. Click the "Read New" button on the top panel of the software. The software will ask you to save the experiment name and details in a specific location (directory) on your computer.

[0246] iv. After saving the experimental file, the software will automatically begin imaging the pre-selected microwells in the plate.

[0247] We used montage imaging, image stitching, and Z-stacking in the experimental modes of Gen5 to capture 3D tuberculoma images. To capture the entire 3D tuberculoma structure (diameter > 2000 µM) spanning beyond the objective's field of view, we used montage imaging mode to capture four image segments (patches) within the micropore. The individual patches in the montage were stitched together to produce a complete single image. The montage was captured using an "automatic stitching" method. The 3D tuberculoma structure exists within a series of Z-planes, and multiple images (slices) must be acquired by moving the objective in the z-axis (or focal) plane. Therefore, we used the Z-stacking imaging procedure within Gen5 by selecting "Image Z-stacking." Multiple automatic image slices were captured below and above the focal plane to ensure that the 3D tuberculoma, cells, and granulomatous lesions were imaged at the appropriate Z-height. After capturing the Z-stacking images, the Z-stacking images were projected using a "Z-projection" step and a focus stacking algorithm to create the final composite image. Gen5 Image+ software is required for image stitching and Z-projection. Using the parameters described, each well requires approximately 2 minutes to complete the imaging process.

[0248] To stitch individual tiles together to produce a single stitched image, use the parameters listed in Table 4. Table 4 Image stitching parameters

[0249] For Z-stacking of individual slices to produce a stacked image, use the parameters listed in Table 5. Table 5 Z-stack parameters for 3D imaging

[0250] 3. Cellular analysis of 3D tuberculoma images in Gen5

[0251] Cellular analysis was performed on the projected images using the parameters listed in Tables 6 and 7.

[0252] We used cell analysis to count the diameter and size of 3D tuberculomas, the number of granulomatous lesions they contain, and the area of ​​the tuberculoma structure affected by bacterial growth and lesions. Table 6. Parameters for 3D spheroid cell analysis [for spheroid analysis] Table 73D Spheroid Cell Analysis Parameters [For Lesion Analysis] Day 13-14. Further fluorescence intensity readings were performed on the 3D cultured microplates.

[0253] For relatively slow-growing virulent Mtb strains, such as Mtb H37Rv or Erdman (tdTomato), we performed additional fluorescence intensity readings on the co-cultures on day 14. We routinely use five-well plates for drug screening experiments, but in some experiments we used up to 12 wells. For experiments using more than five wells, imaging of the remaining plates can be continued on the second day.

[0254] Use the parameters described above to read the fluorescence intensity of tdTomato.

[0255] The above workflow uses 3D co-culture to generate solid tuberculoma-like structures in each microwell. Figure 3A These structures, mimicking 3D tuberculomas, contain well-tissued granulomatous lesions and produce key properties including hypoxia, necrosis, and acidosis. Figure 3B The resulting 3D tuberculoma structures exhibited high homogeneity in size distribution, bacterial growth, and contained granulomatous lesions, as described in Example 2 below. A 96-well bioplatform can be used as a high-content screening and imaging assay platform for anti-TB drug discovery (see Data Processing and Analysis section). Improved workflow of a 3D tuberculoma bioplatform incorporating human extracellular matrix

[0256] This workflow details the steps required to develop a bioplatform using the co-culture of THP-1 cells and pathogenic mycobacterial strains in the presence of physiologically relevant human ECM components. ECM contributes to the architecture of tuberculous granulomas in the human lung, and its immunopathological destruction leads to cavitation formation in TB patients (PT Elkington et al., 2011; Urbanowski et al., 2020; P. Elkington et al., 2022). Furthermore, collagen-rich ECM is known to modulate macrophage survival in 3D in vitro granuloma models (Tezera et al., 2017a). Nevertheless, no human in vitro granuloma models with cavitation transformation have been described. Advances in 3D in vitro granuloma models producing cavitation features will allow for the investigation of drivers of cavitation and pharmacological interventions to identify HDT drugs that can prevent or treat Mtb-induced tissue destruction and immunopathology. The 3D tuberculoma model described here produces cavitation-like features within a spherical structure. Day 1. Production of 3D co-cultures of "mycobacteria in spheroids".

[0257] Prepare the THP-1 cell suspension as described in Workflow A above.

[0258] Prepare the mycobacterial suspension as described in Workflow A above.

[0259] We used the GFP-expressing Mm 1218 strain to infect freshly grown THP-1 cells. This strain allowed for relatively long durations of 3D co-culture when 50% of the medium in the microwells was replaced with fresh medium every seven days. We used a low MOI of 0.005 (optimal range 0.005 to 0.008) to develop structures mimicking tuberculomas with cavitary features. When using different mycobacterial species or strains, it is recommended to optimize the MOI and the amount of added extracellular matrix. We have not yet investigated the ability of other Mm or Mtb strains in our collection to produce this feature in 3D cell culture.

[0260] Prepare 3D spherical microplates for 3D cell co-culture as described in Workflow A above. Day 3. Incorporate ECM solution into 3D co-culture of "mycobacteria in spheroids".

[0261] Preparation of ECM solution

[0262] Add the required amount of human collagen solution (3 mg / ml) to a 15 ml sterile conical tube.

[0263] As described in Formula 4, an ECM solution was prepared using a solution of human collagen and fibronectin.

[0264] We used purified human collagen solution VITROCOL ® VITROCOL collagen is naturally secreted from human fibroblasts in vitro. Use the processed and purified form provided by the supplier. VITROCOL is approximately 97% type I human collagen, with the remainder comprising type III collagen. Storing VITROCOL collagen at 2ºC–8ºC is essential. Do not freeze. Gentle but thorough mixing and careful pH monitoring are crucial when preparing collagen mixtures. Keep the mixture in a refrigerator or on ice at 4ºC to prevent gelation. The mixture can be prepared one day in advance and stored overnight in the refrigerator at 2ºC–8ºC to allow for uniform pH adjustment of the solution. Add the fibronectin solution at the end of the mixing.

[0265] Add ECM solution

[0266] Add 30-50 µl of ECM solution to each microwell containing the 3D co-culture.

[0267] ECM solution was slowly dispensed by tilting the micropipette tip against the sidewall of the microwell. Control microwells without added ECM were kept, depending on the experimental purpose. We have investigated the incorporation of different volumes of ECM solution ranging from 5 to 50 µl into 3D co-cultures. The volume of ECM used for incorporation and the MOI of Mm 1218 used in the co-culture affected the formation of cavitary features in the 3D tuberculoma structures. 30–50 µl of ECM incorporation resulted in the formation of cavitary features observed in this study. Figure 3C Very low MOI (50 CFU) or smaller ECM incorporation (< 25 µl) does not lead to cavity formation.

[0268] The microplate was returned to a CO2 incubator set at 37ºC, 5% CO2, and 100% humidity, and the co-culture of spheroids was continued by incubating the microplate. Add fresh culture medium on days 7 and 14.

[0269] Add fresh 3D cell culture medium (as described above).

[0270] Carefully replace 50% of the cell culture medium in the microwells with fresh, preheated (37ºC) culture medium.

[0271] For co-cultures that have grown for more than 2 weeks, replace with fresh culture medium.

[0272] Observe the growth of the spheroid co-culture using a Cytation 5 imager or an inverted microscope (optional).

[0273] Co-culture was continued by incubating the microplate in a CO2 incubator. Days 16 and 21. Imaging and fluorescence intensity readings of the 3D co-cultured microplates.

[0274] Place the capped microplate in a Cytation 5 container set to 30ºC or 37ºC with or without 5% CO2.

[0275] Fluorescence intensity was read using the optimized parameters described in Table 1 above, except that the excitation and emission wavelengths of GFP were 488 nm and 510 nm, respectively.

[0276] Images were captured using the optimized scheme and parameters for imaging with bright-field and fluorescent filters as described in Table 2 above, except that a GFP filter was used instead of Texas Red.

[0277] The above-mentioned 3D montage imaging, stitching, and Z-stack parameters (Tables 3 and 4) are used for 3D image processing.

[0278] After the images are captured, the focus stacking algorithm described in Workflow A above is used to perform Z-projection of the Z-stacked images to create the final composite image.

[0279] Cell analysis of 3D projection images was performed using the parameters described in Table 5 above. Workflow of a 3D biological platform using purified human PBMC subpopulations

[0280] This optimized workflow details the use of purified human CD14 in 3D cell culture microplates. + The sequence of steps required for developing a biological platform using co-cultures of blood monocytes and the toxic Mtb Erdman (tdTomato). Profiling analysis of granulomas in animal models infected with Mtb and in humans revealed that macrophages constitute a significant portion (>40%–50%) of the granuloma cells. However, cellular composition can vary with early and late granulomas and granuloma morphology (Gideon et al., 2022). Primary monocytes differentiated into macrophages constitute a small fraction of the cells in human PBMC samples, approximately 10%. Due to the insufficient number of monocytes and macrophages in PBMC samples to continue recruiting and maintaining 3D in vitro granulomas, the entire PBMC results in the generation of small cell aggregates (rather than well-organized dynamic granulomas). Therefore, CD14 from PBMCs... + The purification and enrichment of monocytes are key steps in this workflow for developing 3D tuberculoma models. In this workflow, primary human CD14 cells are purified using a MACS column. + Monocytes produce a 3D co-culture of "mycobacteria in spheroids," which differentiate into macrophages to mimic the formation of a core scaffold in human tuberculomas. A virulent Mtb strain is required. The culture is carried out on a MACS column using CD14. + The flow-through unbound fraction produced during monocyte purification contained unlabeled CD14 microbead lymphocytes and CD14 from PBMCs. - Cells. This "lymphocyte-rich" fraction was cultured separately for two days, washed, and added to purified autologous CD14 in 96-well microplates. + In 3D spheroids generated by monocytes, lymphocytes are simulated to aggregate around a human tuberculoma with a macrophage-rich center. Granulomatous lesions are generated in co-cultures of mycobacteria within the spheroids, even without the addition of this "lymphocyte-rich" portion. This bioplatform, employing innate and adaptive cell subsets purified from PBMCs, can be used for confirmatory screening of "preferred seedlings" identified from primary screening on the THP-1 platform. Day 1. Production of 3D co-cultures of "mycobacteria in spheroids".

[0281] PBMCs isolated from human whole blood

[0282] PBMCs can be separated from anticoagulated human blood or the "erythrocyte sedimentation rate (ESR) layer" by density gradient centrifugation (e.g., using Ficoll-Paque™). We used BD Vacutainer® cell preparation tubes (CPT). TM This was used for blood collection and PBMC isolation. PBMCs were isolated following the manufacturer's protocol (BD CPT manual VDP40104-05, pages 1-2). We used 200 ml of blood per human donor to isolate PBMCs and purify CD14. + Monocytes. Isolated PBMCs and purified CD14. + A single cell is sufficient to produce a five-well plate.

[0283] Collect blood into BD Vacutainer® tubes. Gently mix the blood by inverting the tubes 5 to 10 times before centrifugation.

[0284] BD VACUTAINER® tubes should be stored at room temperature (18ºC-25ºC) and properly labeled for human donor identification.

[0285] At room temperature, centrifuge tubes containing blood samples at 1500-1800 xg using a horizontal rotor (with the rotor swung away) for 30 min.

[0286] Immediately after centrifugation, carefully aspirate the plasma using a pipette without disturbing the underlying leukocyte layer. Collect the cell layer containing monocytes into a 50 ml conical tube. PBMCs from several CPT tubes can be pooled into one conical tube. Centrifuge the tube at 1200 xg for 15 min. Remove the supernatant without disturbing the cell pellet.

[0287] RBCs were lysed by resuspending the precipitate in 10 ml of 1x RBC lysis buffer for 5 min at room temperature.

[0288] To stop the lysis reaction, add 25 to 30 ml of PBMC wash buffer (see formulation 5) and gently mix the cell suspension using a pipette. Centrifuge the cells at 275–300 xg for 10 min at room temperature. Carefully discard the supernatant.

[0289] The cell pellet was resuspended in 20 ml of PBMC wash buffer. The washing step was repeated three times by centrifugation at 200 xg for 10 min at 15ºC–20ºC.

[0290] These washing steps, performed at low speeds and centrifugation, are necessary for platelet removal.

[0291] The cell pellet was resuspended in PBMC wash buffer. Cell counts and viability were assessed using trypan blue rejection.

[0292] Cell viability should be ≥ 95%. (In CD14) + During the purification of monocytes, dead cells may nonspecifically bind to MACS microbeads.

[0293] Centrifuge the cell suspension at 300 xg for 10 min. Completely remove the supernatant. Adjust the final PBMC concentration to 1 × 10⁻⁶ in 80 µl of MACS buffer (see formulation 6). 7 Live cells were used to purify CD14. + Mononuclear cells.

[0294] Not recommended for use with Ca 2+ or Mg 2+ Use a cold buffer (2ºC–8ºC) to minimize nonspecific cell markers.

[0295] CD14 was purified from PBMCs by positive selection. + Mononuclear cells.

[0296] We purified CD14 from PBMCs using MACS technology and following the manufacturer's protocol. + Mononuclear cells.

[0297] Add 20 µl of human CD14 beads to 80 µl of MACS buffer. 7 In live PBMCs, and by pipetting thoroughly to remove CD14 from the PBMCs. + Mononuclear cells were magnetically labeled. For higher cell numbers, the volumes of all reagents and the total volume were increased accordingly.

[0298] The microbeads were conjugated with a monoclonal anti-human CD14 antibody. Since CD14 lacks a cytoplasmic domain, it is assumed that antibody binding to CD14 will not trigger signal transduction in monocytes. Therefore, it does not affect phagocytosis by *M. tb.*

[0299] Incubate at 2ºC-8ºC for 15 min.

[0300] Through every 10 7 Add 1-2 ml of MACS buffer (Formula 6) to each cell and wash the cells by centrifugation at 300 xg for 10 min. Remove the supernatant completely.

[0301] Up to 1×10 8 Each cell was resuspended in 500 µl of MACS buffer.

[0302] Magnetic separation is performed using LS columns.

[0303] Based on total cells and CD14 + The number of cells determines the appropriate MACS column and MACS separator. For LS columns, the recommended sample size of white blood cells is 1 × 10⁻⁶. 7 Up to 2 × 10 9 10 out of 10 total cells 5 -10 8 A number of labeled cells.

[0304] Place the LS column in the magnetic field of the MidiMACS separator attached to the MACS MultiStand.

[0305] The column was prepared by washing it with 3 ml of MACS buffer (Formula 6). The collected effluent was labeled “wash”.

[0306] The cell suspension was applied to the column.

[0307] Collect unlabeled cells that have passed through the column, wash the column with 3 ml of MACS buffer, and collect the total effluent in a 50 ml conical tube. This unlabeled cell fraction contains lymphocytes and cells other than CD14. + Extracellular mononuclear cells. Perform a washing step by adding 3 ml of MACS buffer three times. Label the tube "Lymphocyte-rich".

[0308] Add fresh buffer only during the washing step if the column reservoir is empty.

[0309] Remove the column from the separator and place it in a new 50 ml conical tube.

[0310] Pipe 5 ml of MACS buffer onto the column. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column. Label this fraction as CD14. + Mononuclear cells.

[0311] CD14 was purified from all isolated PBMCs from the donor. + Monocytes. Containing purified CD14 + Centrifuge the cone-shaped tube containing monocytes or "lymphocyte-rich" portions at 300 xg for 10 min.

[0312] Resuspend the "lymphocyte-rich" fraction in 50 ml of PBMC growth medium (Formula 2). Transfer the cells to a 150 cm⁻¹ container. 2 In cell culture flasks, cells were cultured for two days in a cell culture incubator set to 37ºC, 5% CO2, and 100% humidity.

[0313] Purified CD14 + The monocyte pellet was resuspended in 10 ml of PBMC growth medium (Formula 2) and centrifuged at 300 xg for 10 min. The supernatant was completely removed.

[0314] The purified CD14 was obtained by adding 10–15 ml of preheated (37ºC) 3D cell culture medium (Formula 1) and centrifuging at 300 x g for 10 min. + The mononuclear cells were washed three times. The supernatant was completely removed. The purified mononuclear cells were resuspended in 10 ml of 3D cell culture medium.

[0315] Cell counts and cell viability were assessed using trypan blue rejection.

[0316] CD14 + The concentration of monocytes was adjusted to 2 × 10⁻⁶ using 3D cell culture medium. 6 live cells / ml. Use CD14 + Mononuclear cells produce 3D co-cultures of mycobacteria within spheroids.

[0317] As described above in Workflow A, CD14 is used in a 96-well Corning 3D spherical microplate. + Monocytes and Mtb Erdman (tdTomato) produce a co-culture of "mycobacteria in spheroids".

[0318] Infect 2 × 10 using 5000 CFU of Mtb Erdman. 5 We routinely use live mononuclear cells / microwell (MOI 0.025) to perform our experiments. Day 3. Lymphocytes and other CD14-negative monocytes were added to the co-culture.

[0319] Prepare and add a lymphocyte-rich cell subset to a 3D microplate.

[0320] Collect the “lymphocyte-rich” cell fraction from the cell culture flask into a 50 ml conical tube.

[0321] During the 2-day culture period, gently release any adherent cells attached to the surface of the cell culture flask using a cell scraper.

[0322] Centrifuge the cell suspension at 300 xg for 10 min at room temperature. Remove the supernatant completely. Resuspend the cell pellet in 15 ml of preheated (37ºC) 3D cell culture medium.

[0323] Wash the cells four times with 15 ml of preheated (37ºC) 3D cell culture medium. Centrifuge at 300 xg for 10 min. After the third wash, allow the cell suspension to stand at room temperature for 30 min before the final centrifugation. After the final wash, resuspend the cells in 10 ml of preheated (37ºC) 3D cell culture medium.

[0324] Cell counts and viability were assessed using trypan blue rejection. Cell concentration was then adjusted to 4 × 10⁶ cells / day using 3D cell culture medium. 6 live cells / ml.

[0325] Without disturbing CD14 + In the case of spheroids formed by mononuclear cells, 100 µl of cell suspension (4 × 10⁻⁶) was added. 5 (One live cell) is added to each well. As mentioned above, the peripheral wells in the microplate are not used.

[0326] Adding a "lymphocyte-rich" cell portion helps recruit additional immune cell types, including those involved in adaptive immunity in the spheroid nucleus where monocytes and macrophages are predominant. Day 6. Add test compound or treatment agent.

[0327] Without disturbing the 3D spherical structure, carefully pipette 100 µl of the supernatant culture medium from the microwell. Next, add the test compound to the 50 µl solution described in Workflow A above. Day 12. Fluorescence intensity reading and imaging used to assess the efficacy of anti-tuberculosis drugs.

[0328] As described above in Workflow A for the THP-1 biological platform, fluorescence intensity readings and automated plate imaging are performed.

[0329] The workflow produces a complex, solid 3D tuberculoma-like structure, which incorporates multiple adaptive and immune cell subsets within micropores containing co-cultures. Figure 3D Variation was observed among donors in the number of granulomatous lesions formed. Due to the lack of self-renewal capacity of macrophages derived from primary human blood mononuclear cells, granulomatous lesions may have disintegrated relatively quickly after day 12 for some donors. Day 14. Additional fluorescence intensity readings were performed on microplates containing 3D cultures infected with Mtb.

[0330] As described in Workflow A, an additional fluorescence intensity reading is performed on day 14. Workflow of a cryogenically stable 3D biological platform

[0331] Cryopreservation is one of the most promising methods for long-term storage of cells and tissues. Therefore, we developed a cryo-stable biological platform by in situ freezing of 3D cocultures in microplates at low temperatures. Three different time points (30 min, 16 h, and 72 h post-Mm "M" (tdTomato) infection of THP-1 cells) were tested to determine the optimal time for freezing microplates containing 3D cocultures. We also investigated three different freezing media: 3D cell medium containing 5% DMSO, heat-inactivated FBS containing 5% DMSO, and Leibovitz L-15 medium containing a cryoprotectant (see formulations 7a, b, and c). The best results were obtained in terms of the formation of tissue-bound granulomatous lesions after thawing and resuscitation when the 3D cocultures were frozen within 30 min or 16 h after Mm infection, and cryopreservation was performed using 3D cell medium containing 5% DMSO. Figures 4A-4F However, after longer co-culture periods (i.e., exceeding 72 h), cryopreservation of 3D co-cultures is not optimal as solid 3D structures, intercellular connections, and new damage develop. The optimized workflow described below details the steps required to develop a storage-stable biological platform in 96-well 3D spherical microplates. Day 1. Production and cryopreservation of 3D cell cultures

[0332] Preparation of 3D spherical microplates for co-culture and cryopreservation

[0333] As described above in Workflow A, prepare THP-1 cell suspension and Mm suspension.

[0334] The THP-1 cell pellet was resuspended in frozen medium (see formulation 7a) to prepare 2 × 10⁻⁶ cells / mL. 6 One live cell per ml of cell suspension.

[0335] A greater number of THP-1 cells than in workflow A are required to compensate for the increased cell death during freezing and thawing.

[0336] The Mm M(tdTomato) precipitate was resuspended in frozen medium (see formulation 7a) to prepare a suspension of 800-1000 CFU / ml.

[0337] Add 100 µl of THP-1 cell suspension (1 × 10⁻⁶) 5 Each living cell is distributed into every micropore except for the peripheral ones.

[0338] Add 100 µl of mycobacterial suspension to each microwell except for the peripheral ones.

[0339] Mix thoroughly three times from top to bottom without touching the bottom of the well to obtain a homogeneous suspension of THP-1 cells and mycobacteria.

[0340] Use a multichannel pipette to fill the outer microwells with 250-300 µl of 3D cell culture medium or sterile cell culture-grade water to avoid boundary effects.

[0341] If freezing is performed directly, the microplate should be kept at room temperature for no more than 30 minutes.

[0342] For co-cultures of "mycobacteria in spheroids" intended for freezing after 16 or 72 h, we incubated the plates at 37ºC and 5% CO2 and 100% humidity for 16 or 72 h (see below).

[0343] Within 30 minutes of infection or co-culture, place the microplate in a cryogenic chamber and transfer the 3D microplate to a -80ºC freezer. Incubate the microplate in the -80ºC freezer for 12–72 hours.

[0344] The microplates were transferred to liquid nitrogen at -160ºC to -196ºC for long-term storage. Then, the microplates were transported to a liquid nitrogen storage facility on dry ice.

[0345] For co-cultures of "mycobacteria in spheroids" intended for freezing after 16 or 72 h, we co-cultured them in 3D cell culture medium in microplates as described in Workflow A. After 16 or 72 h, the cell culture medium was carefully removed using a micropipette and replaced with freezing medium. The mycobacterial infection process in the spheroids was significantly accelerated by 72 h as intercellular junctions and adhesions formed. We avoided disturbing the early-formed 3D spheroids when adding freezing medium. Thaw the 3D coculture and culture further as needed.

[0346] Thawing of 3D cell co-cultures

[0347] Microplates on dry ice are transported from liquid nitrogen storage facilities to cell culture laboratories.

[0348] Thaw the frozen co-culture in a CO2 incubator set at 37ºC, 5% CO2, and 100% humidity for 10 to 15 minutes.

[0349] We removed the microplate caps in a sterile incubator to facilitate rapid thawing. Alternatively, the microplates with co-cultures can be carefully thawed on a platform in a water bath set to 37ºC.

[0350] When at least half of the frozen medium has visibly melted, add 50 µl of preheated (37ºC) 3D cell culture medium / microwell.

[0351] After thawing completely at 37ºC for 30-35 minutes, the microplate was centrifuged at 430 xg for 6 minutes.

[0352] Carefully aspirate the supernatant to freeze the culture medium without disturbing the cell pellet.

[0353] Add fresh, preheated (37ºC) culture medium (200 µl / well) using a micropipette, and resuspend the cell pellet by pipetting up and down five times. Centrifuge the microplate at 430–450 xg for 6 min. Perform this washing step three times to completely remove frozen culture medium.

[0354] For co-cultures of "mycobacteria in spheroids" frozen 16 or 72 h post-infection in microplates in which a dense 3D structure (and intercellular connections) had already formed, we did not centrifuge the microplates. A washing step was performed by carefully adding preheated culture medium (37ºC) without disturbing the 3D spheroid structure in the microplates.

[0355] After final washing, the precipitate was resuspended in 200 µl of preheated (37ºC) 3D cell culture medium / microwell.

[0356] Microplates were placed in a CO2 incubator set at 37ºC, 5% CO2, and 100% humidity for further incubation and recovery of 3D cocultures and for the formation of granulomatous lesions. Fresh 3D cell culture medium was added on days 6 and 12 after thawing.

[0357] Add 50 µl of preheated (37ºC) 3D cell culture medium without disturbing the 3D spheres.

[0358] The microplate was returned to the CO2 incubator to continue the growth of the 3D cell co-culture. Days 18 and 21. Fluorescence intensity readings and imaging.

[0359] 1. Perform fluorescence intensity readings as described in Workflow A.

[0360] 2. Perform imaging and lesion counting as described in Workflow A.

[0361] Compared to freshly produced co-culture systems, well-organized lesions develop relatively late in cryopreserved and resuscitated co-cultures of "mycobacteria in spheroids". In this workflow, the optimal time for treatment with the research compound is 12 or 15 days post-resuscitation to investigate drug efficacy. Data Analysis Data processing and analysis

[0362] In previous methods (Sable and Li et al.), the 3D cell culture workflow used human THP-1 cells or primary CD14 cells. + Co-cultures of monocytes and pathogenic mycobacteria. These workflows generate 3D structures resembling solid tuberculomas without the use of ECM embedding, artificial scaffolds, or magnetic levitation. In proof-of-concept experiments, a custom library of known potential HDT drugs was screened using the resulting 96-well solid tuberculoma bioplatform. The efficacy of HDT drugs was measured in terms of bacterial load and inhibition of granulomatous lesions in the 3D spheroid structures. Mycobacterial load was measured by using red fluorescent Mm or Mtb strains expressing tdTomato, or by disrupting the spheroids, lysing cells with Triton X-100 (0.1%) for 10 min, and counting CFUs on Middlebrook 7H10 agar after plating the lysates. The number of granulomatous lesions in the 3D tuberculoma structures was measured by automated imaging and cell analysis of stitched and Z-stacked images. Quality control of granulomatous lesion counting was performed by visually counting the lesions in each 3D tuberculoma by three separate readers and comparing it to the count calculated by Gen5. We determined the homogeneity and reproducibility of the 3D tuberculoma-like structures formed in the 96-well platform in several microplates and different independent experiments.

[0363] Homogeneity and reproducibility of the bioplatform: The diameter and area of ​​3D tuberculoma-like structures generated in different microplates and batches developed by two implementers were measured to determine homogeneity and reproducibility. We also analyzed the bacterial load in the 3D tuberculomas, the number of granulomatous lesions produced, and the percentage of tuberculoma structure area affected by mycobacterial growth and lesions. The resulting 3D structures showed very small intra- and inter-batch variability in size distribution, mycobacterial growth, and granulomas contained in individual tuberculoma structures (Figure 5). Their homogeneity, reproducibility, and ease of development make them ideal for high-throughput screening applications.

[0364] Applications in drug screening assays: The bioplatform allows for continuous quantification of in-situ drug efficacy using a fluorescence plate reader for bacterial load reduction and an automated cell imaging system for granulomatous lesion regression (as described in the workflow above). The efficacy of a test compound in reducing or increasing bacterial load is determined by treating a 3D co-culture with a single test compound in at least three copies of the wells (referred to as test wells). Each microplate contains wells treated with a drug diluent or carrier (i.e., DMSO) and cell culture medium only (negative control) and the antibiotic rifampin or the experimental HDT drug nitrozole (positive control) (see plate plot, Figure 6). A predefined plate plot is used to identify the drugs added to the microwells. First, the average fluorescence intensity readings of the untreated wells (n = 6) are subtracted from the individual readings of the test compound-treated wells (n = 3). Then, the value is divided by the average fluorescence reading of the untreated wells to obtain the normalized percentage reduction in bacterial load in the test compound-treated wells.

[0365] Quality and Applicability of the Assay for High-Throughput Screening: Following treatment with the test compound, the bio-platform assay reliably detected increases or decreases in mycobacterial load, lesion number, and lesion size across a large number of microwells. In the case of rifampin and nitrozonide, when using MmM(tdTomato) with an optimized MOI of 0.008 (range 0.006 to 0.012) in microplates, we obtained consistent results for a reduction in bacterial load with a Z' factor > 0.5, demonstrating the robustness of this assay for high-throughput screening (Figure 6). For further details on additional data processing and analysis aspects when screening potential therapeutic agents, please refer to Example 2 described below.

[0366] Other applications: In addition to drug screening applications, we have demonstrated the utility of the tuberculoma bioplatform in simultaneously assessing the effects of test compounds on host cell viability, the types of cell death induced, and the potential innate immune mechanisms by which test compounds act in the in situ 3D microenvironment (see Example 2 below). We have also demonstrated the utility of this 3D modeling system in studying the effects of biologics and biosimilars on granuloma formation and structural integrity, as well as in deciphering early Mtb-host interactions in solid or hollow tuberculoma environments.

[0367] Statistical analysis

[0368] The normalized reduction (%) of bacterial load in wells treated with the test compound was calculated using the following formula, where F t It measures the fluorescence intensity of the pores treated with the test compound, and the average F... u It is the average fluorescence intensity of untreated tuberculoma pores (n = 6).

[0369] Bacterial load reduction percentage = (F r -Average F u ) / average F u

[0370] Statistical differences between two groups were assessed using the Mann-Whitney test, and those statistical differences between three or more groups were measured using one-way ANOVA and the nonparametric Kruskal-Wallis test followed by Dunn's post-hoc test (GraphPad Prism V9.3). p < 0.05 was considered statistically significant, and *, **, ***, and **** in the figure indicate p < 0.05, < 0.01, < 0.001, and < 0.0001, respectively. The quality of the high-content drug screening assay performed in the 3D tuberculoma bioplatform using co-cultures of THP-1 monocytes and Mm was determined using the Z-statistic. The Z' factor describes the degree of separation between positive and negative controls in the HTS assay without intervention by the tested compound. The Z' factor was calculated as follows, using assays performed in 8–10 different batches of 3D cell culture microplates: Where σ c- It is the standard deviation of the untreated well or the DMSO control well, σ c+ It is the standard deviation of the rifampicin or nitrozanide control wells, in μ. c- It is the average value of the untreated wells or DMSO control wells, and μ c+ This is the average value of the rifampicin or nitrozanide control wells. A Z' value between 0.5 and 1 is considered excellent, a value between 0 and 0.5 is acceptable, and a value less than 0 indicates that the assay is unlikely to be suitable for HTS applications. Outlook

[0371] 3D cell cultures of pathogenic mycobacteria and human immune cells, along with in vitro human TB granuloma models with high content screening or HTS capabilities, constitute informative systems. However, existing systems still require improvement in simplicity, robustness, reproducibility, scalability, high throughput, and efficiency. A simple, high-fidelity biological platform is highly needed. However, the minimum composition of such robust models for studying underlying disease mechanisms and the effectiveness of potential therapeutics without compromising key tuberculoma structures and properties remains a mystery. The use of human monocytes (immortalized THP-1 or primary CD14) is crucial. +The observations from 3D cell cultures of monocytes and pathogenic Mycobacterium tuberculosis (as the smallest basic component of the workflow described herein) are remarkable. This simple 3D co-culture of "mycobacteria in spheroids" resulted in the differentiation of monocytes into epithelioid macrophages and the formation of a series of tuberculoma structures with well-organized granulomatous lesions. It not only validates findings in animal models that the construction of tuberculous granulomas occurs only against the background of innate immunity, without the contribution of adaptive immunity (Davis et al., 2002; Grant et al., 2022), but also provides a range of tuberculoma microenvironments for HTS-compatible platform systems to study host-pathogen interactions and TB treatment findings. This 3D tuberculoma model was developed without artificial scaffolds, matrix encapsulation, or human monocyte collagen embedding. It does not require the incorporation of ECM into the 3D co-culture to form early solid tuberculoma-like structures with hypoxic and necrotic cores. This reduces the complexity of the system and helps maintain the handleability, scalability, and reproducibility of the bioplatform. Furthermore, observations in workflows using ECM suggest that cavitation formation can also occur in an innate immune-only context during lymphocyte entry into the lesion, depending on the amount of ECM deposited and remodeled by innate immune cells, including macrophages and stromal cells in tuberculomas. However, this observation requires further confirmation using an in vivo model of TB cavitation.

[0372] Using primary human cells (such as PBMCs and bronchoalveolar lavage fluid cells) is preferred over ideal cell lines in 3D model systems (P. Elkington et al., 2019), but their scarcity, donor variability, and high procurement costs make them impractical for high-throughput screening systems. Using monocytes derived from human stem cells can circumvent some of these limitations and generate large numbers of macrophages (Hong et al., 2018; Han et al., 2019). However, because differentiated macrophages cannot proliferate and self-renew, they need to be repeatedly incorporated into the biological platform to promote the influx of macrophage aggregation to form and maintain tissueed granulomatous lesions in experiments lasting more than 10 days. Immortalized macrophages or monocyte lines (including THP-1) derived from cancer patients are generally not considered due to concerns that they may carry mutations and create an immunomodulatory microenvironment that could affect Mtb interactions (P. Elkington et al., 2019). However, emerging evidence suggests a striking similarity in the immunomodulatory characteristics and microenvironment of cancerous tumors and TB granulomas (Gern et al., 2021; McCaffrey et al., 2022), which could open new avenues for the treatment of TB using HDT and immunotherapy, both already developed for cancer therapy. Therefore, the large-scale production and uniform infection of self-renewing and recruitable THP-1 mononuclear cells to form well-tissued granulomatous lesions and key tuberculoma properties may represent a simple strategy to improve throughput, reproducibility, and assay robustness in primary screening.

[0373] Our research indicates that simply co-culturing human THP-1 cells or CD14 cells in any 3D cell culture dish demonstrates that... + Mononuclear cells and mycobacterial strains may not lead to well-tissued lesions and the formation of tuberculoma forms. Despite the use of 3D spheroid technology, co-culturing primary macrophages or cell lines with mycobacteria (Mukundan et al., 2021a; Mukundan et al., 2021b; Kotze et al., 2021), recent studies have produced infected 3D spheroid structures with characteristics shared with uninfected control spheroids (such as central hypoxia and necrosis), but without the formation of well-tissued granulomatous lesions or cavitary features. We have found that spatiotemporal factors (including the use of appropriate 3D cell culture dishes, optimal micropore geometry, co-culture surface chemistry, culture conditions, and culture timelines) are critical for mimicking the structure of tuberculomas and the formation of well-tissued granulomatous lesions (see Example 2 below). This invention describes these factors and culture conditions required for in vitro tuberculoma formation, as well as the minimum essential elements (i.e., macrophages and pathogenic mycobacteria).

[0374] As described above, our 3D tuberculoma bioplatform offers several advantages over existing systems. These include ease of development and use, robustness, increased throughput and efficiency, reduced cost, real-time monitoring of bacterial load, granuloma characteristics, and host cell cytotoxicity, and in situ tracking of other properties of bacterial and host cell physiology. However, a limitation of our 3D tuberculoma model and workflow described herein is the absence of relevant helper cells, such as neutrophil subsets and non-hematopoietic cells like fibroblasts, epithelial cells, and endothelial cells, in human tuberculous granulomas. Therefore, our model system cannot investigate the contribution of neutrophils and stromal cells to the formation of necrotizing and caseous tuberculomas, as well as cavitary transformation. To further improve our model, we have successfully attempted to incorporate these cell types into the THP-1 bioplatform in the form of human cell lines to demonstrate the flexibility required by our system and to investigate the effects of adding these cell types on granulomatous tissue formation in preliminary experiments (data not shown).

[0375] Compared to other available 3D in vitro granuloma models, such as those using matrix encapsulation or embedding, bioelectrospraying, and magnetic levitation, the method described herein is simple and requires no specialized instrumentation or complex materials. The biological approach described herein advances human 3D in vitro granuloma technology and provides tools for studying the response of heterogeneous human granulomas to vaccines, immunotherapies, and chemotherapy. In addition to high-throughput screening of potential HDT compounds and antimicrobial agents targeting pathogens, the biological platform also allows for the identification of personalized medicines using engineered primary human cells, such as chimeric antigen receptor (CAR)-macrophages and T cells from patients with refractory mycobacterial infections or granulomatous diseases other than TB.

[0376] formula

[0377] 1.3D cell culture medium

[0378] To prepare 3D cell culture medium, RPMI 1640 containing L-glutamine (2 mM) was supplemented with 9.73%–10% (vol / vol) heat-inactivated FBS, 0.88%–1% (vol / vol) sodium pyruvate solution, and 0.88%–1% (vol / vol) HEPES buffer, and then sterile filtered using a 0.2 µm Nalgene filter assembly.

[0379] Cell growth culture medium

[0380] To prepare complete cell growth medium, RPMI 1640 containing L-glutamine (2 mM) was supplemented with 9.63%–10% (vol / vol) heat-inactivated FBS, 0.87%–1% (vol / vol) sodium pyruvate solution, 0.87%–1% (vol / vol) HEPES buffer, and 1% (vol / vol) penicillin-streptomycin solution and sterilely filtered using a 0.2 µm Nalgene filter assembly.

[0381] Middlebrook 7H9 culture medium

[0382] To prepare 1 liter of Middlebrook 7H9 culture medium, dissolve 4.7 gm of 7H9 culture medium in 896 ml of distilled or deionized water. Add 0.05% (vol / vol) Tween-80 and 0.4% glycerol, mix thoroughly, adjust the pH to 6.8–7.0, and sterilize by filtration using a 0.2 µm filter assembly. Aseptically replenish with 10% (vol / vol) ADC. Store at 2ºC–8ºC until use. Determine the sterility of the replenished 7H9 culture medium by incubating a small volume before use.

[0383] Extracellular matrix (ECM) solution

[0384] Preparation of ECM solution

[0385] Add one part of cooled 10X PBS slowly to eight parts of cooled VitroCol type 1 human collagen solution (3 mg / ml) with gentle swirling.

[0386] Adjust the pH of the mixture to 7.2-7.6 using sterile 0.1M NaOH. Gently mix by pipetting up and down. Carefully monitor pH adjustments using pH test strips or a pH meter.

[0387] Adjust the final volume to 10 parts using sterile cell culture-grade water.

[0388] Keep the temperature of the mixture between 2ºC and 10ºC to prevent gelling.

[0389] Add 20 µl of human fibronectin (0.1%) solution to 5 ml of collagen mixture. Gently mix by pipetting up and down.

[0390] PBMC Wash Buffer

[0391] To prepare the buffer required for washing PBMCs after RBC lysis, add 10% (vol / vol) heat-inactivated FBS and 2 mM EDTA to calcium- and magnesium-free PBS (pH 7.2–7.4). Filter the buffer using a sterile vacuum filter (0.2 µM membrane unit). Keep the buffer cold (2ºC–8ºC).

[0392] MACS magnetic labeling and column elution buffer

[0393] To prepare the MACS buffer, 0.5% (vol / vol) heat-inactivated FBS and 2 mM EDTA were added to calcium- and magnesium-free PBS (pH 7.2–7.4).

[0394] 3D cell culture cryopreservation medium

[0395] 3D cell culture medium containing DMSO

[0396] To prepare the frozen medium, 5% (vol / vol) DMSO was added to the complete RPMI 1640-based 3D cell culture medium. The medium was prepared before use and sterilized by filtration using a 0.2 µm filter.

[0397] FBS containing DMSO

[0398] To prepare the frozen culture medium, add 5% (vol / vol) DMSO to the heat-inactivated FBS. Prepare before use and sterilize by filtration using a 0.2 µm filter.

[0399] L15 medium containing cryoprotectant

[0400] This cryo-medium contains equal volumes of serum cryo-medium “A” 2X and DMSO cryo-medium “D” 2X. First, add 100 µl of cryo-medium “A” to the wells containing the 3D cell culture, and then add 100 µl of cryo-medium “D”. The microplate is then transferred to a -80ºC freezer.

[0401] To prepare 1000 ml of serum-freezing medium "A" 2X, 16 ml of 1X HEPES buffer, 300 ml of heat-inactivated FBS, and 200 ml of PVP-10X stock solution were added to 484 ml of L15 medium, and the medium was sterilized by filtration using a 0.2 µm filter. To prepare the PVP-10X stock solution, 10% PVP (wt / vol) was added to 1X HEPES buffered saline. The 10X HEPES buffered saline stock solution contains 70.7 gm sodium chloride, 17.0 gm glucose (dextrose), 2.0 gm potassium chloride, 19.4 gm sodium dihydrogen phosphate 2H2O, 47.6 gm HEPES buffer, and 0.01 gm phenol red in up to 1000 ml of cell culture-grade water, and is prepared by adding 900 ml of cell culture-grade water to 100 ml of 10X HEPES buffered saline.

[0402] To prepare 1000 ml of DMSO frozen medium "D", 16.02 ml of 1M HEPES buffer and 150.63 ml of DMSO were added to 833.3 ml of L15 medium, and the mixture was sterilized by filtration using a 0.2 µM filter. The shelf life of serum frozen medium "A" and DMSO frozen medium "D" stored in glass vials at -20ºC is one year. References

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[0451] Wetscherek, MTA, Sadler, TJ, Lee, JYJ, Karia, S. andBabar, JL (2022). Active pulmonary tuberculosis: something old, something new, something borrowed, something blue. Insights Imaging 13(1): 3.ncbi.nlm.nih.gov / pubmed / 35001143. Example 2 A high-throughput screening compatible 3D tuberculoma biological platform for the effective identification and characterization of host-targeted therapeutics for tuberculosis. Introduction

[0452] Researchers are using cell culture systems to uncover fundamental principles of life, host-pathogen interactions, and disease intervention. Tuberculosis (TB), an ancient infectious disease caused by the obligate intracellular pathogen Mycobacterium tuberculosis (Mtb), will greatly benefit from in vitro cell culture models that recreate key features of TB lesions in a high-throughput screening (HTS) compatible manner. Despite a wide range of preventative and therapeutic interventions, TB remains one of the leading infectious killers worldwide, causing approximately 1.6 million deaths annually, underscoring the need for improved control measures and tools. An incomplete understanding of host-Mtb interactions and pathogen elimination within the hallmark TB lesion hinders efforts to develop effective control measures. TB exhibits a complex pathological spectrum and diverse clinical presentations in humans. The defining pathological feature of TB is the formation of granulomas (tightly organized, macrophage-rich aggregates in infected tissue, most commonly in the lungs). Pulmonary granulomas begin with macrophages phagocytosing inhaled Mtb bacteria, followed by extravasation from the bloodstream and recruitment of additional leukocytes at the site of infection. In addition to a macrophage-rich scaffold, the granulomatous structure is also shaped by other medullary, lymphoid, and helper cells. While TB granulomas are generated by host immune cells in response to invading pathogens to isolate and control infection, this structure can also promote bacterial growth and spread. Therefore, granulomas are considered central to the host-pathogen battle that determines the outcome of infection.

[0453] TB granulomas are highly dynamic structures and can exhibit heterogeneous morphology, bacterial load, and trajectory even within the lungs of a single individual. Over time, they undergo contraction, expansion, or aggregation with nearby granulomas. In severe cases, granulomas can aggregate into 3D solid masses called “tuberculomas,” resembling solid carcinomas. Over time, this produces hypoxic and necrotic centers, with a vigorous granulomatous response in the surrounding walls containing macrophages at various stages of transformation, and a lymphocyte-rich mantle in the periphery. Pulmonary tuberculomas can become mineralized or form cavitation and breach the airways, releasing and spreading Mtb. Activated T lymphocytes entering the granuloma help control the infection. However, the adaptive response in the lungs of infected human hosts and animal models (particularly within pulmonary granulomas) is delayed relative to other respiratory pathogens. Furthermore, lymphocyte infiltration into the granuloma core and its interaction with infected macrophages are limited by numerous barriers, including epithelioid and foamy transformations of macrophages.

[0454] CD4 + T-helper-1 lymphocyte response is essential for protection; paradoxically, this response is inadequate, and abnormal lymphocyte activity can promote immunopathology. Furthermore, in acute HIV / SIV co-infection, recruited CD4+ cells... + T lymphocytes are rapidly depleted from TB granulomas, leaving macrophages and other phagocytes as the focus of most host-Mtb interactions. Growing evidence suggests that in some hosts, despite having few or no CD4+ cells... + T lymphocytes, but macrophage-rich granulomas can contain Mtb infection. However, Mtb employs multiple immune evasion strategies in both macrophages and granulomas, and often successfully establishes long-term infection. Emerging insights into TB granuloma biology highlight the crucial role of cellular states and interactions within granulomas in shaping infection outcomes. The spatiotemporal dissociation of immune cell subsets and signal transduction in granulomas means that emerging therapeutics must penetrate and exert their effects in macrophage-rich centers. In different granuloma forms, bacteria reside in different microenvironments and physiological states, influencing their susceptibility to antibiotics, making TB drug discovery and development a challenging endeavor.

[0455] A key strategy for achieving the goal of eliminating TB is the development of improved and shorter treatment regimens. Standard antibiotic treatment for TB is lengthy, ranging from 6 to 24 months, depending on whether the disease is drug-sensitive or drug-resistant. This pathogen-targeting approach has the potential drawback of selective microbial resistance. An alternative approach known as host-directed therapy (HDT) has recently gained considerable attention. It involves the therapeutic modulation of the host immune response to enhance immunity or target host cellular processes that the pathogen uses to survive. HDT is a transformative approach that has the potential to shorten treatment time and reduce the likelihood of developing microbial resistance, improve immunopathology, and prevent relapse. Preclinical studies of potential HDTs have made considerable progress over the past decade, and a small number of therapeutics have entered clinical trials. However, the need for a physiologically relevant, high-fidelity in vitro platform for rapidly identifying potential HDTs and predicting their therapeutic efficacy has hindered significant discoveries and translational progress.

[0456] While animal models serve as a preclinical selection tool for understanding the efficacy of potential therapeutics, they fail to reproduce the full spectrum of human tuberculosis (TB). Furthermore, the low throughput and high cost of animal-based screening protocols in high-security facilities pose a significant bottleneck to drug discovery. Various tools and methods have been developed to replace animal testing. However, the complexity of human granulomas means that conventional culture media or monolayer cell culture systems rarely mimic the full spectrum of the granulomatous microenvironment.

[0457] Over the past decade, bioengineered human in vitro granuloma models using primary cells have been widely adopted due to their physiological relevance. Early granuloma models developed using traditional monolayer cultures of peripheral blood mononuclear cells (PBMCs) have produced some features of human granulomas, such as macrophage aggregation and multinucleated giant cell formation. However, existing models lack well-organized granuloma structures, diverse morphologies, and microenvironments. Consequently, some drugs effective against human tuberculomas (TB) granulomas (e.g., pyrazinamide) cannot be identified using these 2D systems. Recently developed 3D cell culture models offer promise for better predicting drug efficacy and providing new insights into early host-Mtb interactions, as they more closely mimic the granuloma microenvironment than 2D systems. However, while these 3D models produce macrophage granuloma aggregation, they fail to generate classic tuberculoma structures and features, such as well-organized lesions and a range of solid, necrotic, and cavitary transformations. Current bioengineered 3D granuloma models using extracellular matrix (ECM), microencapsulation, or magnetic levitation are complex and require specialized instruments for development. Their use in HTS applications is limited by low throughput, limited manufacturability, and limited scalability. Furthermore, releasing live cells from these systems for downstream research requires additional manipulations, such as enzymatic treatments, which can affect their accurate characterization. A novel 3D culture technique for explants from microdissected granulomas in infected animals could overcome these limitations of in vitro models. However, this system is technically demanding, and few granulomas can be obtained from microdissection of infected animals. Since biopsy, leukapheresis, or bronchoalveolar lavage fluid cannot generate sufficient macrophages from a single human donor, existing 3D models using primary cells (although useful in later development) are impractical for primary lead generation work. A 3D model similar to classic TB lesions is needed, which can be scaled up as an HTS-compatible platform to rapidly, efficiently, and cost-effectively test thousands of potential therapeutics.

[0458] Here, we report the development of a robust biological platform that can be used for a wide range of applications, from decoding host-Mtb biology and identifying associated transcriptional changes to in vitro screening of various compounds and biologics. Figure 7AThis physiological system uses 3D co-culture of primary human cells or mononuclear cell lines with virulent mycobacteria to simulate the natural microenvironment. The advantage of this system is the generation of 3D structures that replicate the key features and conditions encountered by Mtb within typical solid, necrotic, or hollow tuberculomas in TB patients, which has not been demonstrated using existing in vitro granuloma models. The system employs a standard 96-well workflow that combines in situ generation, maintenance, fluorescence reading, immunostaining, and high-content imaging of the resulting tuberculoma-like structures. This platform allows for the screening and characterization of immune mechanisms induced by putative HDT compounds that preferentially act on host cells and inhibit pathogen growth within 3D tuberculomas. It also allows for the screening of antimicrobial agents or compounds possessing both pathogen-targeting and host-directed activity. We show that a biosafety level (BSL)-2 alternative pathogen, *Mycobacterium marineum* (instead of Mtb), can be used for pre-screening compounds in this model, saving hundreds of working hours in BSL-3 laboratories while potentially reducing safety concerns. In our proof-of-concept experiments, we used this platform to screen a custom library of potential HDT compounds identified in published studies using 2D cell cultures infected with mycobacteria. We found that selected “preferred seed” compounds significantly reduced granulomatous lesions and bacterial load in 3D in vitro tuberculomas. Although the antibacterial activity of the compounds varied among mycobacterial strains, identified “preferred seed” compounds with different annotation pathway targets consistently reduced bacterial load in 3D tuberculomas, regardless of the Mtb strain used. Pharmacological activation studies of in situ innate defense mechanisms in 3D tuberculomas revealed that several seed compounds induced rapid autophagy flux in macrophages. Most of these drugs are already FDA-approved or have been studied in clinical trials for other indications and are therefore repurposing for TB treatment. Notably, we demonstrated that a “preferred seed” drug with potent aurora kinase inhibitory activity and AT9283, one of the clinically used multi-kinase inhibitors, significantly inhibited Mtb and lesion load in 3D in vitro tuberculomas and mice. The platform described here advances “3D human in vitro granuloma technology” and provides HTS-compatible physiological tools for the discovery of tuberculosis and other granulomatous diseases. result The co-culture of mycobacteria in spheroids established an easy-to-use 3D human tuberculoma-like model.

[0459] To identify host-targeted therapeutic agents and their mechanisms of action in controlling intracellular mycobacterial survival, we sought to develop a simple 3D in vitro granuloma model that could mimic the typical tuberculous lesion and microenvironment in a standard 96-well microplate. However, the minimum composition of such a simple model, capable of allowing for long-term experiments and developing dynamic granulomatous environments to simulate long-term host-pathogen interactions, remains to be determined. 3D cell cultures in spheroid and organoid forms have long been used to simulate the complex microenvironment in human cancers and have found utility in HTS applications. Given the significant overlap between the structure and microenvironment of cancerous tumors and tuberculous granulomas, we generated a self-assembling “mycobacteria within spheroids” co-culture in a 96-well 3D cell culture plate using pathogenic mycobacteria and human macrophages (the centers of granuloma formation). Since increasing the complexity of 3D cell cultures would impair reproducibility, adaptability, and throughput, we omitted ECM embedding in early iterations to reduce batch-to-batch variability. Because macrophages can naturally secrete ECM, we generated a co-culture without scaffold elements or exogenous biological materials. Furthermore, although macrophage survival was prolonged compared to 2D monolayer cultures, incorporation of external ECM into 3D granuloma models reduced the size of formed granuloma aggregates and Mtb proliferation.

[0460] To streamline workflows, improve efficiency, and reduce potential safety issues associated with experiments in our BSL-3 lab, we utilized *Mycobacterium marineum* (Mm), a risk group 2 pathogen and a genetic close relative of *Mtb*, in our BSL-2 lab. As an alternative to *Mtb*, Mm has been extensively studied in zebrafish models to understand the biology of tuberculous granulomas. We first evaluated two distinct Mm strains in 3D co-culture: strain 1218 and “M”. Both Mm 1218 and “M” constitutively express the widely used fluorescent GFP (green) and tdTomato (bright red) to track bacterial growth kinetics, and they were initially isolated from infected fish and human skin lesions, respectively. They have been successfully used to study host-Mm interactions in 2D macrophage cultures and zebrafish models. Since Mm grows well naturally in poikilothermic animals at 30ºC, we first compared the ability of the two strains to grow in vitro at 37ºC. We found that both strains grew well in Middlebrook 7H9 medium at 37ºC, although relatively slower than at 30ºC, but showed robust and comparable growth kinetics over three weeks. Figure 8A However, unlike the 7H9 growth medium, both strains grew very slowly in RPMI-1640 3D cell culture medium, which does not contain macrophages.

[0461] To overcome obstacles in HTS application, particularly the insufficient number of primary macrophages from human donors, we tested the growth of two Mm strains in several human and mouse monocyte-macrophage lines, including THP-1, U937, J774A.1, RAW-264.7, and AMJ2 cells. We used a low multiplicity of infection (MOI) of 0.008 (range 0.005–0.01) and did not wash the co-culture after mixing cells and bacteria in microwells. Because pinocytic aminoglycosides can reach macrophage phagosomes and contribute to the apparent antibacterial activity of macrophages, we avoided using aminoglycoside antibiotics that are believed to kill only extracellular bacteria in cell cultures. The workflow also omits the use of mitogens (e.g., PMA) for macrophage differentiation, which can affect cell signaling and other cellular processes and potentially alter host-pathogen interactions. We used an automated multi-mode plate reader and confocal cell imaging system for in situ fluorescence monitoring, microscopy, and 3D co-culture imaging to study the dynamics of host and bacterial cells over time. Among the selected cell lines, human THP-1 monocytes and U937 tissue cells (tissue macrophages) allowed the most consistent and robust bacterial growth in 3D co-culture. Figure 8B and Figure 8C Conversely, mouse macrophage cell lines J774A.1, RAW-264.7, and AMJ2 allowed for relatively poor growth of the Mm strain over 12 days. Interestingly, cell aggregates resembling well-organized macrophage granulomas supporting mycobacterial growth were observed only in infected 3D cultures of THP-1 cells. No aggregates formed in uninfected control 3D cell cultures.

[0462] Given these exciting findings, we then conducted a detailed study of the spatiotemporal dynamics of THP-1 monocyte and mycobacterial proliferation in 3D co-culture. Real-time visualization and a series of images of post-co-culture events revealed that by day 2, individual spheroids began to form in the micropores. Figure 7B and Figure 7CBecause this co-culture uses a low MOI Mm, the inoculated bacteria completely aggregate by assembling mononuclear cells during 3D spheroid formation, and the bacteria subsequently proliferate primarily within the spheroids. After three days of incubation, dense spheroids form, increasing in size by day 6. Cell aggregates are generated in spheroids infected with strains "M" and 1218, respectively, around days 6 and 8. These cell aggregates grow over time and form large granulomatous aggregates supporting robust bacterial growth by days 12 and 16. Considering the maximum O2 diffusion distance through tissue is approximately 250 µm (beyond which tissue becomes hypoxic), the cell aggregates are generated primarily in oxygen-rich areas. At this stage, the 3D "mycobacteria within spheroids" structure resembles the aggregation of well-tissued, vigorous granulomatous lesions in solid tuberculomas. Figure 7B and Figure 7C ).

[0463] Corresponding to the growth of cell aggregates, once the granulomatous aggregates began to expand, bacterial fluorescence (observed to gradually increase until day 6) significantly increased. Figure 7D This observation is consistent with previously reported results in zebrafish models, suggesting that in 3D tuberculoma-like models, the formed cell aggregates may be true granulomas, and that mycobacterial amplification is consistent with granuloma formation and growth. Plates of THP-1 lysates on 7H10 agar plates and quantitative colony-forming units confirmed this bacterial load increase over three weeks. An advantage of the model developed without ECM embedding is that mature 3D spheroids can be easily dissociated by pipetting, and cells can be released for downstream applications without additional treatment and potential alteration of their established properties. Contrary to the increased bacterial load, THP-1 cell survival in infected Mm-1 3D spheroids was found to gradually decrease more over three weeks compared to uninfected spheroids, as studied by cytotoxicity assays. Figure 7E In summary, these results demonstrate that in vitro models can allow for relatively long-term and quantitative characterization of the growth dynamics of hosts and pathogens within physiologically relevant 3D microenvironments.

[0464] When studying co-cultures of *Mycobacteria in Globules* using the Mm strain, we observed that tdTomato provided a brighter signal and less autofluorescence noise in both fluorescence channels tested, consistent with established observations of less biofluorescence at longer wavelengths in different cell types and tissues. Fluorescence intensity (FI) readings further revealed more background fluorescence in the GFP channel in uninfected control globules compared to the tdTomato channel. Figure 8CTherefore, we selected the Mm strain expressing tdTomato for further FI reading experiments. Importantly, we found a linear relationship between FI and the number of bacteria in 3D spheroids in 96-well plates or liquid medium. This confirms that the fluorescence readings in our model are a reliable indicator of relative bacterial count. Figure 8D Co-culturing THP-1 monocytes and Mm in any 3D cell culture dish does not always produce tuberculoma-like structures with well-organized lesions. It requires the unique spatial characteristics of the 3D cell culture dish. When we screened 96-well plates from several vendors with different microwell surface chemistry and bottom geometry, spheroids with large granulomatous lesions formed only in 3D spheroid microplates with ultra-low adsorption (ULA) surfaces and U-shaped bottoms, but not in microplates with V-shaped bottoms or surfaces with microcavities (Fig. 9). Notably, granuloma aggregates formed in 2D microplates or in 3D microparticles encapsulating Mm-infected monocytes were small and loosely organized. In the tested microplates, well-organized, dense, and relatively stable lesions formed only in CorningULA 3D spheroid plates with U-shaped bottoms and neutrally charged hydrophilic surfaces with covalently bonded inert hydrogel coatings. Therefore, we chose the Corning 3D spherical plate for future experiments.

[0465] Human TB granulomas exhibit diverse trajectories and fates. Therefore, we sought to investigate the spatiotemporal evolution of granulomas in our model. We co-cultured THP-1 cells (THP-1-GFP) with Mm “M”tdTomato or THP-1 cells (THP-1-RFP) with Mm 1218 GFP in 3D. We observed dynamic lesion formation and aggregation with neighboring lesions in the 3D model. Figure 10A Some lesions remained relatively stable, while others progressed and contained high bacterial loads. At day 16 post-infection, viable (fluorescent) monocytes supporting bacterial expansion were observed almost exclusively within granulomatous aggregates, while survival was significantly reduced in the remainder of the spheroids, suggesting that Mycobacterium recruits new viable monocytes or promotes the survival of permissive monocytes in these granulomatous aggregates to benefit them. Mtb strains H37Rv, Erdman, Beijing F2, and CDC 1551 (tdTomato) studied with low MOI (0.01) produced comparable well-tissued lesions. However, live attenuated BCG vaccine strains of Mycobacterium bovis did not form well-tissued lesions, although 0 to 5 loose cell aggregates were observed by day 12. Figure 7FNotably, the virulence locus ESX-1 / RD1 was present in the studied Mm and Mtb strains, but the BCG strains Danish (WT) and Pasteur (GFP) had lost the RD1 locus, explaining their reduced virulence. When using BCG strains with a 10-fold MOI, more cell aggregates formed, but these were miniaturized and loosely organized. Therefore, this model can independently validate established observations from the zebrafish model that Mycobacterium ESX-1 / RD1 is essential for accelerating macrophage recruitment in granuloma formation, and that ΔRD1 infection leads to defects in granuloma formation and expansion. A central feature of granulomas in TB patients is the epithelial-like transformation of macrophages with aggregated, interlocking, and E-cadherin-positive junctions. Therefore, we investigated whether the formed granulomatous aggregates were merely macrophage aggregates or true granulomatous lesions. Similar to tuberculous granulomas, in Mtb Erdman (WT) infected spheroids, primarily in the forming aggregates, we detected the expression of classical epithelial markers and cell adhesion molecules, stained against cadherin and intercellular adhesion molecule-1 (ICAM-1). Figure 7G and Figure 7H Importantly, the cells within the neogranuloma exhibited epithelioid-mesenchymal transition, with higher expression of cell adhesion and junctional markers such as E-cadherin, N-cadherin, ICAM-1, and γ-catenin, confirming that the cell aggregates formed in the model were indeed granulomas. Figure 10B and Figure 10C ).

[0466] We then developed a 3D co-culture using human PBMCs and pathogenic mycobacteria, but only small granulomatous aggregates formed in this model. Figure 7I In contrast, when using primary CD14 purified from PBMCs... + When monocytes were co-cultured with Mtb Erdman (tdTomato), well-tissued granulomas were generated, which were subsequently supplemented with lymphocyte-rich CD14 after 3D spheroid formation. - PBMC section ( Figure 7JHowever, granulomas generated in this model using primary monocytes lacking self-renewal and replenishment capabilities progressed rapidly and disintegrated by day 14. This observation is similar to that in zebrafish granulomas, where accelerated macrophage death due to necrosis is exhibited after macrophage supply in the granuloma decreases below a critical threshold and replenishment diminishes. These results, however, demonstrate that readily feasible 3D co-culture of human monocytes and pathogenic mycobacteria in rationally chosen culture dishes is sufficient to generate tuberculoma-like structures with well-organized granuloma aggregates and validates long-standing observations that tuberculous granulomas form in a background of innate immunity alone. The “mycobacteria in spheroids” co-culture described herein establishes a robust cellular model simulating human tuberculomas, and events in the natural host share many similarities with this model. Key properties and microenvironment of human tuberculomas were generated in 3D models.

[0467] Similar to TB patients and animal models, cells in the granuloma and core of 3D tuberculomas generated using THP-1 monocytes infected with Mm (WT) matured into macrophages, which showed higher expression of CD68, the cytoskeletal marker tubulin, and the angiogenesis marker vascular endothelial growth factor receptor-2 (VEGFR-2). Figure 11 A and Figure 12A Furthermore, compared to control spheroids, cells within infected spheroids exhibited greater expression of the activation or immunomodulatory marker galactagogue-9. Increased type 3 collagen deposition and Mtb-biofilm formation were observed in the core of infected spheroids, as indicated by staining against the biofilm matrix cellulose. Figure 12A and Figure 12B These results indicate that monocyte maturation, differentiation, activation, and immune regulation occur in the 3D model.

[0468] Mtb evades killing by inhibiting macrophage innate defense mechanisms, including phagosome-lysosome fusion, phagosome acidification, autophagy, and inflammasome activation. Therefore, to monitor autophagy induction in 3D in vitro tuberculomas, we generated co-cultures of THP-1-Difluo hLC3 reporter monocytes and Mtb Erdman (WT). These reporter cells expressed human autophagy protein LC3B fused with two fluorescent proteins, GFP and RFP. Since GFP (acid-sensitive) is quenched or degraded in the acidic environment of autolysosomes, while RFP remains stable, blockage of the autophagy process resulted in a higher yellow signal (red plus green colocalization) compared to autophagy induction (red signal). On day 7, we observed more yellow-green spots accumulating in the outer region forming granulomas compared to the spheroid core, indicating that autophagy induction was specifically inhibited or blocked in the Mtb permissive macrophages of granulomas in the model. Figure 11 A and Figure 13A Consistent with these results, we also observed a relatively more acidic environment in the globular core compared to the granuloma-forming region. Since Mtb induces the assembly of apoptosis-associated speckle-like proteins containing CARD domain (ASC)-dependent inflammasomes NLRP3 or AIM2 in infected macrophages, we used a 3D co-culture of Mtb Erdman (tdTomato) and THP-1-ASC-GFP inflammasome reporter monocytes (which stably express genes encoding ASC-GFP fusion proteins to monitor ASC-dependent inflammasome formation). On days 7 and 9, we observed greater ASC-GFP speckle formation and inflammasome activation in these granulomas exhibiting attempts to encapsulate Mtb, particularly in globular scaffold cells and epithelial-like macrophages without significant Mtb growth, compared to granulomas allowing unrestrained Mtb growth. Figure 11 A and Figure 13B These results indicate the functional diversity of granulomatous lesions in the 3D model and suggest autophagy and inflammasome suppression in Mtb permissive granulomas.

[0469] Over time, mature tuberculomas may exhibit hypoxia, necrosis centers, and cavitary morphology. Therefore, we sought to investigate these properties and microenvironments in a 3D model. Using different hypoxic probes that fluoresce in a hypoxic environment, we found greater hypoxia in the center of infected spheroids compared to the periphery of infected WT Mm or Mtb spheroids, and that hypoxia increased more significantly in the center of infected spheroids over time as they matured compared to control spheroids. Figure 11 (A and Figure 14). These results suggest that granulomatous lesions supporting bacterial proliferation preferentially arise in oxygen-rich areas. Although a hypoxic environment is inherently generated in both control and infected spheroids, macrophage transformation with granulomatous aggregation may contribute to increased hypoxia in the form of solid tuberculomas due to the relatively large 3D cellular structure with a diameter > 2000 µM. Inducing hypoxia in Mtb-infected macrophages plays a protective role by stimulating the macrophages' antimicrobial capacity. However, excessive hypoxia and reactive HIF-1 may lead to pathogenic necrosis and matrix destruction. Similarly, we found more cell death (primarily characterized by necrosis rather than apoptosis) in the central region using cell death probes, which increased over time in infected spheroids compared to control spheroids (Figure 14).

[0470] Central necrosis and ECM disruption are considered key pathological drivers of cavitation in human tuberculomas. Although increased matrix metalloproteinase (MMP) activity and matrix remodeling are observed in 3D in vitro granuloma models supplemented with collagen-rich exogenous ECM, cavitation has not been described. Therefore, we next investigated the role of ECM inclusion in a 3D tuberculoma model. We used the Mm 1218 (GFP) strain, which allowed for relatively long co-culture (>50 days) with THP-1 monocytes when using a low MOI (0.005), and 50% of the 3D medium was exchanged weekly with fresh medium. To simulate increased ECM deposition in human tuberculomas, we tested incremental (5 to 50 µl) ECM mixtures composed of human type 1 collagen and fibronectin. The mixture was added on day 3 after co-culture once globular structures had formed. Interestingly, after 21 days of co-culture, a cavity-like feature was generated in infected spheroids with a high amount (≥ 30 µl) of ECM. Despite containing ≥ 30 µl of ECM mixture, cavitation-like features were observed in uninfected spheroids ( Figure 11C and Figure 15 Cavity morphology did not form in infected 3D cultures produced using 1 / 10 of the MOI (which subsequently produced relatively lower bacterial and granuloma loads). These results warrant further in vivo studies and support the hypothesis that enhanced ECM deposition exceeding a threshold limit, along with increased mycobacterial and aggregated granuloma loads, may collectively contribute to cavity formation in tuberculomas. The results demonstrate solid, necrotic, and cavitary transformation in a tuberculoma model.

[0471] Tumor necrosis factor (TNF) has long been known as a key cytokine required to maintain granuloma integrity by limiting bacterial growth and preventing macrophage necrosis. However, excessive TNF in infected macrophages can trigger programmed necrosis. Therefore, we next investigated the effects of the human TNF blocker etanercept (a fusion protein of TNF receptor type II (TNFR2) fused with an Fc fragment) and the human anti-TNF monoclonal antibody (mAb) adalimumab biosimilar on bacterial load and granuloma architecture. Figure 11D). To investigate the high-content screening (HCS) potential of the 3D model in a 96-well format, we simultaneously screened 16 additional human recombinant mAbs or antagonistic protein biosimilars (Figure 16), which are equivalent to those approved for the treatment of cancer, inflammatory disorders, autoimmune diseases, and other conditions. To illustrate the differences in bioavailability, half-life, and pharmacological properties of immunotherapeutic agents, 3D cocultures of THP-1 cells and pathogenic mycobacteria were exposed to these biologics or biosimilars immediately after co-culturing or on day 6 as a single treatment. In the preliminary experiments, we selected high (500 ng) and low (62.5 ng) doses for treatment after stepwise dose adjustments. In the 3D coculture using Mtb Erdman (tdTomato), treatment with a TNF blocker instead of an allotype or polyclonal control antibody on day 10 showed a loss of tissue integrity in the emerging granulomas (blue arrows). Figure 11 D). Clearly comparable bacterial loads in co-cultures treated with TNF inhibitors and control co-cultures (white arrows); Figure 11 D) The treatment dose or the treatment exposure time setting ( Figure 16A Regardless, this loss of integrity occurred. Interestingly, exposure to anti-TNF mAb in spheroids infected with Mm M(tdTomato) resulted in multiple small (miliary) granulomas. Figure 16B ).

[0472] mAb bioanalytes targeting integrin CD11a (a cell surface molecule involved in cell adhesion) instead of CD52 (a molecule with anti-adhesion function) by day 10 resulted in early loss of granulomatous tissue formation and integrity, as well as a reduction in Mtb load. Figure 16A However, in 3D spheroids exposed to TNF antagonists, anti-CD11a mAb, or control antibodies, relatively normal mature granuloma formation with abundant and comparable Mtb loads subsequently progressed to day 14. Figure 16C and Figure 16DThis independently corroborates previous findings using TNF antagonists in animal models. These results could not be reproduced in the ECM-embedded 3D granuloma model, confirming that TNF is required for neogranuloma integrity rather than formation. The finding that integrin CD11a expression on monocytes / macrophages contributes to neogranuloma integrity and early bacterial encapsulation is novel and requires further in vivo studies. Furthermore, exposure to several biosimilars on day 6 (such as those targeting vascular endothelial growth factor (VEGF), integrin α4β7, CD30 (TNFRSF8), insulin-like growth factor-1 receptor (IGF1R), IL-6Rα, IL-1β, or IL-1R) showed a slowing of granulomatous lesion expansion, as evidenced by their smaller size on day 10. Figure 16E In contrast, other biosimilars targeting CTLA-4 and CD52 appeared to contribute to the expansion of granulomas compared to the isotype control mAb. However, at day 14 after exposure on day 6, only the anti-VEGF and anti-α4β7 (500 ng) mAbs significantly inhibited Mtb loading relative to the control, while the anti-CD52 (62.5 ng) mAb increased Mtb loading. Figure 16F These results confirm previous observations published in animal models that targeting the pro-angiogenic molecule VEGF reduces mycobacterial load in granulomas. They also demonstrate that, in the absence of endothelial cells or myeloid cells with adaptive immunity, truncating angiogenesis signaling via VEGF and inhibiting pro-adhesion activity via integrin α4β7 can limit granuloma proliferation and mycobacterial growth, warranting further investigation. However, these results collectively indicate the generation of human tuberculoma-like features and microenvironment in 3D in vitro tuberculomas and indicate the model's HCS potential. Protein biomarker profiling using multiple arrays provides insights into host-pathogen interactions that lead to cavity transition.

[0473] To identify host-pathogen interactions involved in granuloma formation and cavitation in a 3D model, cell lysates and culture supernatants from 3D cell cultures were subjected to Quantibody human Kiloplex proteomics assays (Figure 17). A total of 1,000 key proteins associated with cell activation and differentiation, signal transduction, cell adhesion, angiogenesis, cytokines and chemokines, immune response, apoptosis, and cell death were investigated. The relative fold change (FC) of protein expression in infected 3D co-culture samples in cell lysates or culture supernatants, with or without ECM, compared to uninfected controls, was determined. This study aimed to determine the regulation of protein expression levels and to identify the effect of ECM on changes in the proteomic profile leading to cavitation transformation in our model. Raw values ​​of 1,000 protein markers were determined, and the top-ranked upregulated or downregulated proteins in cell lysates and culture supernatants are presented as a dot plot. Figure 17A and Figure 17B In the figure, the size of the dots represents the relative fold change in protein expression, while red and blue indicate upregulation and downregulation, respectively. Among the top six markers upregulated in cell lysates of 3D co-cultures without ECM addition (… Figure 17A Compared to the control, MCP-2 showed the highest expression level (fold change 250.2). Other top-ranked upregulated proteins in cell lysates included the chemokines MCP-2, MIG, MIP-3α, I-TAC, and GROα. These are previously recognized biomarkers of the early innate immune response following human mycobacterial infection. Another top-ranked upregulated protein, nucleoporin 85 (NUP85), which has not been previously identified during mycobacterial infection, has potential roles in CCR-2-mediated chemotaxis and in influenza and HIV infection. MCP-2 and MIP-3α were also overexpressed in the supernatant. Figure 17B This was also true for IP-10 and MCP-4. Interestingly, albumin and RBP4 were significantly upregulated only in co-culture with ECM. These two proteins have recently been identified as potential serum biomarkers for active TB, indicating their upregulation during late infection. Intermediate upregulated and downregulated proteins (FC: 5-50) are shown in... Figure 17A (cell lysates) and Figure 17B The bottom inset of the supernatant. Most proteins identified using this proteomics approach correlated well with biomarkers identified in other studies using samples from TB patients. The expression of five protein biomarkers identified using this method in a 3D co-culture model was confirmed using ELISPOT. Figure 18ECM plays a crucial role in regulating host-pathogen interactions and granulomatous necrosis, and it has been shown to significantly affect the expression of selected cytokines in our Mm-infected 3D co-cultures compared to uninfected control 3D cultures. The top 25 most affected chemokines were shown in the results of adding ECM to either Mm-infected or control 3D cultures. Figure 17C In the control 3D culture, ECM significantly upregulated CD7 (117), galactagogue-7 (39.6), and transferrin (13.6) levels, and downregulated RBP4 (-17.6) and CD163 (-11.1) levels, as some of these proteins interact with ECM. In the 3D co-culture infected with Mm, ECM significantly upregulated CD7 (189), vWF-A2 (72.6), RBP4 (63.3), and albumin (13.6), and downregulated CEACAM-3 (155.7), DCTN1 (56.1), Flt-3 (41.4), optical proteoglycan (25.5), and PRX2 (21) levels. By comparing infected 3D cultures with control 3D cultures, the most significant changes due to the addition of ECM were the downregulation of CEACAM-3, DCTN1, Fit-3, photoglucan, FABP4, ADAM22, and I-TAC expression, and the upregulation of vWF-A2, RBP4, albumin, and PCK1 in the Mm-infected co-cultures, indicating that their levels are regulated by ECM addition, leading to cavitation. Further research is needed to better understand the role of ECM deposition and the regulation of these proteins in cavitation formation in tuberculomas. RNA sequencing provides insights into transcriptional changes associated with granuloma formation and supports the development of human tuberculoma-like fate in models.

[0474] To identify the transcriptomic dynamics of host-pathogen interactions over time in a 3D model, THP-1 monocytes were co-cultured with Mm at a low MOI. 3D spheroids were collected and subjected to total RNA extraction, cDNA library preparation, and RNA sequencing at days 0 (infection day), 3 (spheroid formation), 6 (initiation of granuloma formation), 9 (formation of well-organized granulomas), and 12 (late window of granuloma formation). Figure 20 Uninfected 3D cultures collected on the corresponding incubation days were prepared using the same procedure.

[0475] We sequenced a total of 36 cDNA libraries. Each library yielded 26.4-94.4 million paired-end reads, averaging 28.1 million reads per sample. Initial quality assessment of the 36 raw FASTQ files confirmed that all libraries met the Phred score > 30 quality criterion. For each sample, an average of 85.1% of reads aligned to unique locations on the reference genome. The total read length, GC%, aligned reads, and mapping quality were determined for each sample. Figure 19A Principal component analysis (PCA) showed that, except for one outlier in the uninfected samples on day 3 (which was included in the downstream analysis), biological replicates of the same samples exhibited good clustering. Samples infected with Mm were located in the right region of the graph, clearly separated from the control samples (left region). Early-infection samples (days 3 and 6) were primarily found at the bottom of the graph, while late-infection samples (days 9 and 12) clustered in the top region. Hierarchical clustering heatmap ( Figure 21A This also demonstrated the clear separation between untreated samples and samples infected with Mm, as well as the good clustering of biological replicates within each sample.

[0476] We used the DESeq2 tool to analyze differentially expressed genes (DEGs) between untreated and infected samples. DEGs were filtered for biological significance using an adjusted p-value < 0.05 and log2 (fold change) > 1 or < -1. Figure 19B The volcano plot represents the dynamic changes in significant DEGs in infected samples compared to controls. Compared to the corresponding controls, 988, 3,352, 5,193, and 5,618 DEGs (both upregulated and downregulated) were identified in samples infected for 3, 6, 9, and 12 days, respectively. The longer the infection period, the more significant the DEGs became. A peak in upregulated DEGs was observed on day 9 (n = 2,868), and a peak in downregulated DEGs was observed on day 12 (n = 3,062). Figure 21B The MA diagram in the figure shows similar results.

[0477] To further explore the core DEG shared by the four infection time points, red and blue dots from the volcano diagram were plotted using a Venn diagram. For example... Figure 19CAs shown in the figure, 465 upregulated DEGs (bottom left) and 71 downregulated DEGs (bottom right) were found in the core of the figure, accounting for 47.1% and 31.7% of the DEGs in the samples on day 3 post-infection, respectively. ShinyGO gene ontology enrichment analysis of the core shared genes (both upregulated and downregulated DEGs, n = 539) confirmed significant precursors to several immune response pathways, such as chemokine signaling, cytokine-cytokine receptor interactions, cell adhesion molecules, necrosis and apoptosis, TNF signaling, and IL-17 signaling. The raw expression values ​​of the shared upregulated and downregulated DEGs are shown in Table S3.

[0478] Among the top-ranked upregulated and downregulated DEGs, CCL8, ADAM7-AS1, CXCL10 / 11, and VCAM1 showed a rapid response to early infection, with significant (>100) increases in gene expression on day 3 post-infection. By day 12 post-infection, CCL8 showed a 10,000-fold increase in gene expression, indicating its crucial role in granuloma formation and the immune response to mycobacterial infection. As infection progressed, several genes (such as TNFAIP6, IGFBP3, IFI27 / 44L, IFIT1, CCL4, IL6, CXCL2 / 8 / 14, MMP8, SUGCT-AS1, AIM2, MET, and IER3) showed a trend of increasing expression, reaching peak expression levels on day 12 post-infection. IDO1, MMP1 / 12, ITGB8, IL1A, CCR7, CCL20, CXCL1 / 3, and IL1B showed increased or peak expression around day 9 post-infection, but their expression decreased by day 12 post-infection. Our study demonstrates the dynamic changes in the abundance of immune response-related transcripts during granulomatous lesion development.

[0479] To identify the DEGs most biologically relevant to the immune response against pathogenic infection, all DEGs (red and blue dots) from the volcano plot were subjected to gene set enrichment analysis (GSEA). Some studies have shown that the same pathway from different databases may contain other annotated genes. Variations in enrichment levels in each database significantly affect enrichment results and predictive modeling

[25] . Since our study is of most interest in identifying disease-related immune responses, we performed enrichment analyses using two methods: Integrity Pathway Analysis (IPA) and GSEA via gseKEGG from the Clusterprofiler package (R). IPA is a robust web-based application that provides information on pathways, diseases, and other characteristics that may be significantly altered in different samples

[26] . We performed classical pathway analysis using z-scores representing activation (positive) or inhibition (negative). gseKEGG uses computational methods to determine whether a set of genes shows statistically significant regulation in a specific pathway (KEGG pathway in this study), where the enrichment score represents activation or inhibition of the pathway.

[0480] Figure 19D The bar chart shows the dynamic changes in activation or inhibition of the top 45 most enriched classical pathways identified by IPA. Overall, 25 pathways were persistently activated throughout the 12-day infection period and were primarily associated with the innate immune response of monocytes and macrophages to infection (i.e., phagosome formation, IL17 signaling, idiopathic pulmonary fibrosis signaling, pyroptosis signaling, Toll-like receptor and chemokine signaling). Several novel pathways were identified. For example, two neurally related signaling pathways (neuroinflammatory signaling pathways in neurons and CREB signaling) were significantly activated throughout the infection period (red dots). Five cholesterol-related pathways (cholesterol biosynthesis I, II, and III, glycolysis I, and the cholesterol biosynthesis super-pathway (purple triangles)) were not activated early but were significantly upregulated during granuloma formation. These results suggest that changes in lipid and cholesterol metabolism may play a key role in granuloma formation in a 3D model. Two pathways (CREB signaling in neurons and G protein-coupled receptor signaling (blue asterisks)) were activated early at the onset of infection. However, these pathways diminish as infection progresses, suggesting their role in the early innate immune response against the pathogen. Eight suppressed pathways during infection are shown at the bottom of the figure.

[0481] The KEGG database is used in parallel, allowing DEGs from the same group to undergo gseKEGG analysis in R. For example... Figure 19EAs shown, the dynamic changes of the top 40 enriched KEGG pathways are presented using normalized enrichment scores (NES). Both the IPA and gseKEGG analyses share ten pathways (red dots). Some fundamental signaling pathways of the innate immune response to infection were only found in the gseKEGG analysis (blue asterisks), such as TNF signaling, cytokine-cytokine receptor interactions, cell adhesion molecules, apoptosis, and focal adhesion pathways. Interestingly, the repressed pathways in the gseKEGG analysis differed significantly from those in the IPA analysis, indicating that database selection plays a crucial role in the GSEA results.

[0482] The heatmap shows the abundance of transcripts from selected pathways of both IPA and gseKEGG. Figures 22A-22H ). DEGs in these selected pathways are primarily involved in inducing innate immune responses against mycobacterial infection; some key genes are cell adhesion molecules (ICAM1 / 2 / 3, VCAM1, SIGLEC1, CADM1, SELL), angiogenesis (ANGPTL4, ANGPT1, ANGPT2, ANGPT4), necrosis and apoptosis (TNF, IFNB1, TLR3, TNFSF10), inflammasomes and pyroptosis (IFI27, AIM2, GBP1 / 2 / 3 / 5, CASP1 / 3 / 4 / 6 / 8), MMPs (MMP1 / 2 / 3 / 8 / 9 / 10 / 12 / 13 / 14 / 19), neuroactive ligand-receptor interactions (ADM, SSTR2 / 3), chemokines and cytokines (CD40, CCL1 / 2 / 3 / 7 / 14 / 17 / 20 / 22 / 24, IL2 / 5 / 7 / 10 / 23 / 36, CXCL1 / 3 / 8 / 12, TGFB1). Use the R Path View tool to visualize gene expression changes in a selected pathway (data not shown).

[0483] Our transcriptomic results in the THP-1-Mm 3D model showed good agreement with other in vitro and in vivo TB studies. Using the same filtering parameters (adjusted P < 0.05, |log2 fold change > 1|), our model shared 529 DEGs (data not shown) with the ECM-incorporated polymer-encapsulated PBMC 3D model proposed by Elkington et al., and 728 DEGs (data not shown) with transcriptomic studies of TB clinical samples (lymph nodes with granulomas) from the same cohort of studies. The THP1 3D model also shared 288 DEGs (data not shown) with TB-infected mouse and non-human primate (NHP) samples. Among the 16 characteristic genes of TB blood RNA identified by Zak et al., our RNAseq data showed upregulation of 11 genes (68.8%) (data not shown). In the UK London and Leicester cohort dataset studied by Berry and colleagues, among 70 characteristic genes with persistently upregulated active TB and LTBI, the THP1 3D model showed upregulation in approximately 90% of the genes (data not shown). These results suggest that RNA sequencing in the model provides insights into transcriptional changes associated with granuloma formation.

[0484] 3D in vitro tuberculoma models can be used to study training immunity against TB and other mycobacterial infections.

[0485] Training immunity is a type of heterologous immunity induced by epigenetic and metabolic imprinting and reprogramming in monocytes, macrophages, NK cells, and other innate immune cells following exposure to live attenuated vaccines, vector vaccines, one or more microorganisms, specific cytokines, or therapeutic agents, resulting in long-term innate immune memory and an effective immune response. This concept was first well-documented in 2012, and BCG vaccination against TB has been shown to provide cross-protection via innate immunity and reduce mortality in severely immunocompromised mice (lacking T and B lymphocytes and therefore unable to mount an adaptive immune response) caused by fatal Candida albicans infection or lung cancer (melanoma). In addition to pathogenic stimuli, endogenous molecules such as DAMPs (e.g., vimentin) and cytokines (e.g., granulocyte-macrophage colony-stimulating factor) can also induce training immunity. We found that in 3D cell cultures, macrophages derived from THP-1 monocytes trained with a live, attenuated, and non-toxic BCG vaccine significantly prevented the development of secondary infections induced by toxic mycobacteria (i.e., GFP-expressing Mm 1218) compared to THP-1 cells trained with RPMI alone, and also prevented the development of granulomatous lesions in a 3D tuberculoma model. Figure 23These results indicate that 3D in vitro tuberculoma models can be used to study training immunity induced by live attenuated and subunit TB vaccines, immunotherapeutic agents, biologics, and host-targeted therapies developed for TB and other mycobacterial diseases. A 3D in vitro tuberculoma model can be used as an HTS-compatible platform to identify potential HDT compounds.

[0486] The screening of biosimilars provides a feasibility indicator that 3D models can be used as an HTS bioplatform for identifying potential anti-TB therapeutics, potentially offering advantages over existing in vitro granuloma models in discovering the effects of these therapeutics on both mycobacterial load and granulomatous lesions. Given the macrophage-rich framework of natural granulomas and the difficulty of targeting bacteria in tuberculomas, we investigated whether this platform could identify and characterize HDTs that modulate macrophage responses in the physiological tuberculous environment. Considering the delayed onset and entry of adaptive immune cells in granulomas, such HDTs that modulate the disrupted granuloma-macrophage defense pathway may have the greatest impact in controlling early mycobacterial infection. Here, we used a custom library of 65 known potential HDT compounds developed using a 3D co-culture of THP-1 monocytes and MmM (tdTomato) to screen solid tuberculomas. Figure 24A The library comprises 44 FDA-approved drugs, 10 compounds in clinical trials or for various indications in humans, and 11 investigational compounds not yet studied in humans. These compounds were selected based on their known ability to reduce mycobacterial load in 2D cell cultures or their potential to induce host-directed anti-mycobacterial immune responses in human or animal models, after reviewing literature published between 2000 and 2020. However, their anti-mycobacterial activity in relevant 3D tuberculoma settings has not been investigated in head-to-head comparisons. Based on known mechanisms of action, many of these compounds exhibit diverse and numerous host-directed effects and belong to a broad class of drugs inducing anticancer, anti-inflammatory, kinase-modulating, ion channel blocking, neuroleptic, antioxidant, epigenetic regulation, antidiabetic, and other effects. Furthermore, at least 13 compounds have previously been reported to possess some direct mycobacterial activity in pure cultures, although not necessarily bactericidal. We evaluated the efficacy of each of the 65 compounds in reducing bacterial growth and granulomatous lesions after a single treatment at a concentration of 20 µM on day 6 by performing FI readings and imaging of the 3D co-cultures 6–8 days post-treatment.

[0487] From four independent screenings, we found that only 31 of these compounds reduced the mean Mm load in the 3D environment by >25% compared to the untreated control, and only 18 of these significantly (p < 0.05) reduced the Mm load compared to the negative control treated with the drug carrier DMSO. Figure 24A Interestingly, all seven compounds increased the Mm load compared to the untreated control. Importantly, we detected significant antibacterial activity of the known pathogen- and host-targeting drug nitrozonide (20 µM / well) and the antibiotic rifampin (1 µg / ml) in this cell model. Figure 25A These serve as positive controls and inform us of the reliability of these results. When we evaluated a widely used statistical measure describing the degree of separation between positive and negative controls, assays performed using an Mm infection model with an MOI range of 0.007 to 0.012 showed a Z' factor > 0.5, which is considered excellent in terms of robustness for HTS assays. Figure 25A When we investigated the optimal MOI for four Mtb (tdTomato) strains exhibiting different growth rates in THP-1 cells, we found that for assays demonstrating superior Z' factor performance, the slow-growing Mtb strains H37Rv, Erdman, and Beijing F2 required relatively higher MOIs (0.012–0.05) than Mm. For the CDC1551 strain (which proliferates in small clusters outside the 3D structure and exhibits evasion of encapsulation within spheroids), even with an MOI of 0.05 and despite well-tissued lesion formation within spheroids, the assay quality did not reach a superior level. Figure 25B Next, we confirmed the assay performance using Mtb H37Rv with an optimal MOI of 0.025 in a large number (n = 10) of 3D spherical plates. Reproducible inhibition of Mtb growth with nitrozanide and rifampin was detected in a large number of wells, and the results in each of these plates demonstrated excellent assay quality. Figure 25C These results collectively demonstrate the applicability of tuberculoma models as HTS-compatible biological platforms. They further allow us to expand our research on compounds in the BSL-3 laboratory using four Mtb strains representing a heterogeneous set of genotypes.

[0488] Using Mtb strains H37Rv, Erdman, and Beijing F2 (which demonstrated excellent assay quality and were selected from 2–4 independent screenings for each strain), we identified 29–31 compounds (data not shown) that reduced the mean Mtb load by >25% compared to the untreated control, although there were strain-specific differences in the inhibitory effects of HDT compounds on bacterial growth. Using the MtbCDC1551 infection model and the same parameters, we identified 39 compounds. Since this platform did not demonstrate excellent assay quality, we did not consider using these results and strains for further research. Comparison of screening results in the BSL-2 (Mm M) and BSL-3 (three Mtb strains) platforms particularly revealed considerable overlap among the “preferred scalpers” that reduced bacterial load by >50%, although there were strain-specific variations dependent on host cytokines for intracellular survival. These preferred scare drugs include the following 12 drugs with different known host-directing effects that are effective in all four infection models: AT9283, aurinofen, clomastine fumarate, chlorpromazine hydrochloride, 3'4' dichlorobenzamidine hydrochloride, dovirtinib, doxycycline, H89, lansoprazole, methaxaton hydrochloride, nizoxinide, and zuclothiasol dihydrochloride. Figure 24B In all infection models, we will use a list of spurs identified by >50% bacterial inhibition, with statistical significance (p < 0.05) and robust cutoff values. < The composite z-score of -4 was used to compare the potential leads, and encouragingly, the lists of "preferred leads" identified using both methods were found to be virtually identical. Figure 24C The high concordance (>90%) among the preferred scare compounds identified in the Mm and Mtb infection models reassured us of the performance and validity of the results in our BSL-2 and -3 platforms. We then considered the ability of 65 compounds to alleviate immunopathology by reducing granulomatous lesions (Figure 26 and...). Figure 27 Twelve "preferred symptomatic" compounds also effectively reduced the lesion burden, except for chlormastine fumarate and methaxone hydrochloride (which did not effectively reduce the lesion burden in the Mtb-Erdman infection model). Although they consistently reduced the mean lesion burden by >25% in all infection models, the effects of lansoprazole, dovirtinib, 3',4' dichlorobenzamidil hydrochloride, and AT9283 were not statistically significant in the Erdman model.

[0489] Lymphocytes can influence the antimycobacterial response in tuberculous granulomas, and the performance of HDTs in the context of innate and adaptive primary human immune cells is crucial. Therefore, the use of primary human CD14 cells infected with Mtb Erdman (tdTomato) is essential. +Blood mononuclear cells were then replenished, including lymphocytes and CD14. - In a 3D tuberculoma model generated from autologous PBMC subsets of myeloid cells, we further investigated 65 compounds ( Figure 24D We found considerable consistency between the compound screening results in THP-1 monocytes and the PBMC platform. Of the 65 compounds screened, 29 inhibited Mtb Erdman load > 25% in the THP-1 model and 23 were also effective in the PBMC model. However, considering individual donor variability, only 14 of these compounds caused a significant reduction in Mtb load in the PBMC model compared to the DMSO-treated control (p < 0.05), compared to 23 compounds in the THP-1 model. Nevertheless, no significant difference in efficacy was observed for 64 compounds when comparing the normalized percentage of Mtb load for individual compounds in both cell models. One drug, methaxazone hydrochloride, reduced Mtb load significantly more in THP-1 compared to the PBMC model (p < 0.05), suggesting that this drug may be ineffective in the context of primary human immunity.

[0490] Next, we considered the consistency of the compounds' efficacy in the five infection models described above using THP-1 monocytes or PBMCs (Figure 24). Interestingly, in these models, none of the FDA-approved antidiabetic drugs (metformin, glibenclamide, sitagliptin) and nonsteroidal or steroidal anti-inflammatory drugs (dexamethasone, prednisone, aspirin, ibuprofen, indomethacin, diclofenac sodium) tested reduced the bacterial load by >25%. Furthermore, previously identified potential HDT agents, including 1-methyl-D-tryptophan (IDO inhibitor), zileutone (5-lipoxygenase inhibitor), BzATP (P2X7 receptor agonist), verapamil (calcium channel blocker), zoledronic acid (γδ T cell response inducer), N-acetyl-L-cysteine ​​(antioxidant), sodium valproate (anticonvulsant and sodium channel blocker), L-citrulline (nitric oxide enhancer), sodium chlorate (histone deacetylase inhibitor), maraviro (CCR5 receptor antagonist), and irbesartan (antihypertensive and PPARγ activator) (Figure 24), were also ineffective as stand-alone treatments, except for indomethacin and L-citrulline (which moderately suppressed bacterial load in single THP-1 models using Mtb Beijing-F2 or Erdman).

[0491] Interestingly, not all the lead compounds identified in previous studies using 2D cell culture screening with mycobacterial infection were effective in our 3D model. While those previous studies primarily treated cell cultures with compounds during or shortly after mycobacterial uptake by host cells, our screening investigated a late-infection time window involving the establishment of a granulomatous environment. For example, in 2D cell cultures of the Mtb H37Rv-infected melanoma cell line MelJuSo or primary human monocyte-derived macrophages exposed to compounds immediately after bacterial uptake, nine compounds from our custom library of 1260 pharmacologically active compounds (LOPACs) were previously identified as preferred lead compounds. However, only four compounds (devitinib, H89, 3'4' dichlorobenzamidine hydrochloride, and GW5074) consistently inhibited bacterial loads of 25%–90% in all of our infection models (Figure 24). In one to three, but not all, infection models, three compounds (quinacrine dihydrochloride, the tyrphostin AG459 inhibitor, and haloperidol) reduced bacterial load by >25%, while SU 6656 and SB 216763 were ineffective in our models. These results require further in vivo validation as the efficacy of these compounds in animal models of TB remains to be determined. Of particular importance are the efficacy of lead compounds previously reported as stand-alone therapeutics against mycobacterial load in animal models in our infection models. For example, the gastric proton pump inhibitor (PPI) lansoprazole (previously identified as a potential lead in Mtb-infected 2D lung fibroblast (MRC-5) cultures after screening 1280 FDA-approved drugs in the Prestwick Chemicals library and exhibiting partial inhibitory activity against Mtb in mice) was also highly effective in all our infection models. Similarly, in a zebrafish juvenile model infected with *M. m.*, chlorpromazine hydrochloride, methaxanthin hydrochloride, chlormastine fumarate, and zucloxacillin dihydrochloride were identified as lead HDTs in a screening using the FDA-approved Prestwick library. These drugs were also identified as first-line leads in all our infection models, except for methaxanthin hydrochloride (which was ineffective in the PBMC model), as described above. Furthermore, the antimicrobial drugs nizoxin and doxycycline (each previously reported to have host-directed effects and inhibit mycobacterial load in animal models) demonstrated potential antimycobacterial activity in our infection models. These results support the ability of our platform to identify the in vivo effects of known host-directed and pathogen-targeting drugs in a 3D in vitro tuberculoma setting.To further explore compounds that better inhibit bacterial growth in 3D microenvironments compared to 2D environments, we screened compounds in 2D THP-1 cultures infected with Mtb H37Rv during a late time window involving infection establishment (day 6 post-infection), similar to that in 3D co-cultures (data not shown). While AT9283 and simvastatin reduced bacterial load better in the 3D tuberculoma model, imatinib and quinacrine dihydrochloride were more effective in 2D cell cultures. Interestingly, several compounds identified in published studies involving treatment during the early time window of infection in 2D cell cultures were ineffective after a sustainable balance was established between the pathogen and the host cellular environment.

[0492] To differentiate between drugs with host-directed effects and those that directly target bacteria in a 3D environment, we treated Mm "M" or Mtb Erdman (tdTomato) broth cultures in 3D ULA microplates with the compounds. Bacterial growth was monitored as pure 3D cultures, and FI was measured on days 6 and 8 after compound treatment (data not shown). Thirteen compounds from a custom library, including antibiotics and the MMP inhibitor doxycycline, have previously been described in broth cultures for their antimycobacterial activities in addition to host-directed effects (data not shown). As expected, the antibiotics doxycycline and rifampin exhibited significant inhibitory activity against Mm "M" and Mtb Erdman in pure 3D cultures compared to the untreated control, validating the ability of this assay to reproduce the effects of known anti-TB drugs in 3D broth cultures. Interestingly, at the tested concentration of 20 µM, aurinolone (an antirheumatic drug), nitrozonide (an antiparasitic drug), and lansoprazole (a PPI with known antimicrobial activity) exhibited significant inhibitory activity against Mtb Erdman, but were ineffective against Mm (data not shown). However, these drugs significantly inhibited both species in the 3D tuberculoma bioplatform. Chlorpromazine hydrochloride (an antipsychotic and acid sphingomyelinase inhibitor) and fingolimod hydrochloride (an acid sphingosine-1-phosphate receptor modulator) have been reported to have direct antimycobacterial activity and have also shown strong intracellular mycobacterial growth inhibitory activity in the 3D tuberculoma bioplatform; however, these drugs were ineffective in pure 3D cultures. Among other compounds with known direct antimycobacterial activity, N-acetyl-L-cysteine, verapamil, sodium valproate, and irbesartan were ineffective in both 3D culture medium and the tuberculoma bioplatform. Interestingly, in 3D pure cultures, five compounds previously identified as potential HDT drugs (chlormastine fumarate, porphyrin IX, tyrosine phosphorylation inhibitor AG 494, methaxaton hydrochloride, and fluoxetine hydrochloride) exhibited significant growth inhibition against one or two species compared to the control, suggesting that these HDT compounds have some pathogen-targeting activity and require further investigation. By excluding any direct mycobacterial activity in pure cultures, these results indicate that among the 12 "preferred seed" compounds (Figure 24), AT9283, zuclothiasol dihydrochloride, dovirtinib, 3'4' dichlorobenzamid hydrochloride, and H89 primarily exert their intracellular mycobacterial inhibitory effects through host macrophages.

[0493] Given the high attrition rate of lead compounds due to host toxicity, and to verify whether any observed effects stemmed from impacts on host cell viability, we treated uninfected 3D cultures of THP-1 cells or PBMCs with the individual compounds and measured cytotoxicity using the CytoTox Glo assay. Cytotoxicity screening results showed high consistency between the two cell cultures, regardless of whether monocyte cell lines or primary host cells were used. Of the 15 compounds that caused >50% cytotoxicity in these cell cultures, 13 were cytotoxic to both cell types at a concentration of 20 µM (data not shown). Interestingly, 11 of these compounds are FDA-approved or evaluated in clinical trials and found safe for human use, highlighting the inconsistency between in vitro toxicity and in vivo testing results. While toxicity results are an integral part of the therapeutic evaluation process, these results also suggest that in vitro cytotoxicity methods and dosages cannot capture all the complexities encountered in the human body during in vivo testing. Furthermore, although there was a positive correlation between compound-induced cytotoxicity and normalized bacterial load in the Mm-infected THP-1 model (data not shown), only nine of the 17 lead compounds that reduced the average Mm load by >50% resulted in a >50% reduction in THP-1 viability. In contrast, the four compounds that increased Mm load also caused >50% cytotoxicity. To further determine the cytotoxic concentration (CC) that resulted in a 50% reduction in THP-1 cell viability, further research was conducted. 50 We tested the concentration range of each cytotoxic compound in 3D cultures of THP-1-RFP cells (data not shown). Of the 12 “preferred seed” compounds, six were cytotoxic at 20 µM concentrations, and effective concentrations (ECs) of nitrozonidine, doxycycline, and dovirtinib were obtained. 50 Values. At non-toxic doses, auronoxine and 3',4'-dichlorobenzamidine hydrochloride reduced Mm load by >35%, but AT9283 did not inhibit Mm growth. Considering that Mm and Mtb can grow intracellularly after macrophage death, and that Mm can rapidly exceed globular structures through extracellular proliferation, we inferred, after considering all results, that compound-induced cytotoxicity in in vitro assays may not always be associated with a reduction in intracellular mycobacterial load. We further investigated that host-targeted therapeutic agent-induced cell death associated with innate immune mechanisms (e.g., apoptosis, autophagy, and pyroptosis) can reduce cell viability while inhibiting intracellular pathogen survival. Since an ideal biological platform should be able to characterize such immune mechanisms and identify therapeutic efficacy within 3D granulomas, we next investigated innate macrophage defense activated by HDT compounds. The results of the immune mechanism of HDT can be incorporated in situ into the 3D tuberculoma biological platform.

[0494] In infected human macrophages and animal models, physiological or pharmacological activation of hypoxia, phagocytosomal fusion, lysosomal acidification, autophagy, and inflammasome pathways can severely reduce intracellular Mtb survival. Therefore, we investigated whether the results of such innate immune mechanisms could be incorporated into our models. We generated 3D co-cultures of THP-1 monocytes and WTMtb Erdman cells and incorporated fluorescent probes that fluoresce in positive microenvironments. 3D tuberculomas exposed to hypoxia probes fluoresced red when oxygen levels in the multicellular core were <4%, while exposure to lysosomal acidification probes fluoresced red when lysosomal numbers or acidification increased in the cell scaffold. We also generated 3D co-cultures of Mtb Erdman (WT) and THP-1-Difluo hLC3 reporter cells to monitor autophagy flux. In these reporter cells, RFP-GFP tandemly labeled LC3 probes emitted a red plus green (yellow) signal in the cytosol and autophagosomes, but only red fluorescence in autolysosomes, because acid-sensitive GFP is more easily quenched or degraded in acidic environments than acid-stable RFP. Yellow spots accumulated after autophagosome and lysosome fusion failure, while both yellow and red spots decreased under autophagy inhibition. Furthermore, we used a 3D co-culture of Mtb Erdman (tdTomato) and THP-1-ASC-GFP reporter cells to monitor ASC-dependent inflammasome formation. In these reporter cells, Mtb infection or antigen exposure led to ASC-GFP expression and ASC-spot formation following inflammasome activation. We used high-content imaging in a 96-well format to spatiotemporally resolve components of the innate immune pathway in 3D tuberculomas and their regulation by HDT compounds. Although treatment reduced cell viability and may inhibit reporter probe activity, we did not exclude cytotoxic drugs in this screening.

[0495] Following treatment with six individual "preferred spur" noncytotoxic compounds at a dose of 20 µM, 3D tuberculomas exhibited increased hypoxia in the core or cell aggregates (blue arrows) on day 6 post-treatment, compared to DMSO or untreated lesions, unlike bacterial permissive necrosis lesions (white arrows). Figure 28A and Figure 28BTuberculomas treated with these compounds also showed increased autolysosome formation and acidification, as revealed by increased red fluorescence relative to green or yellow fluorescence. Rifampicin-treated tuberculomas exhibited similar hypoxic and autophagy-activated features, but their scaffolds displayed a less acidic microenvironment, likely due to reduced Mtb-induced cell death. In contrast, tuberculomas treated with six representative non-cytotoxic “non-leader” compounds showed more cell aggregates with autophagosome accumulation (yellow arrows) or no acidification (although the tuberculoma core was acidified), indicating that downstream steps of autophagy were blocked. They also showed less hypoxic staining on day 6 post-treatment, as in DMSO-treated or untreated controls (…). Figure 28B Because autophagy induction is a dynamic, multi-step process, we performed a series of imaging studies in tuberculomas over six days to monitor autophagy flux. In tuberculomas treated with the preferred lead compound, autophagy flux progressed relatively rapidly within three days compared to negative controls or tuberculomas treated with the "non-leader" compound (data not shown), particularly with lansoprazole and H89; and by day 6 post-treatment, more cells expressing ASC-GFP persisted with reduced Mtb growth, indicating increased inhibition of Mtb-induced cell death. In tuberculomas treated with the "non-leader" compound, Mtb growth was unrestrained, and autophagy flux was gradually generated between days 3 and 6, with several cell clusters lacking autophagosome maturation. In rifampicin-treated tuberculomas, autophagy developed slowly by day 6, possibly due to nutrient starvation, with some cell aggregates lacking autophagosome maturation or allowing Mtb persistence, exhibiting strong inflammasome activation, indicating delayed autophagy induction and incomplete bactericidal activity. In summary, the screening results of non-cytotoxic "preferred lead" HDT compounds expanded early discovery in 2D cell cultures infected with Mtb by establishing pharmacologically induced susceptibility of Mtb to autophagy and lysosomal acidification in a 3D tuberculoma model.

[0496] Tuberculomas treated with six "preferred precursor" cytotoxic compounds showed relatively less hypoxia. On day six post-treatment, they still exhibited autophagy induction and acidification at the center. Figure 28C and Figure 28DThis may be due to reduced cell viability. In these tuberculomas, autophagy flux appeared to progress between 1 and 3 days relative to the DMSO-treated control, with a decrease in ASC-GFP-expressing cells on day 6, while Mtb growth was controlled (data not shown). We also investigated six remaining cytotoxic compounds that exhibited species-specific heterogeneity in mycobacterial growth inhibition but resulted in a 25%–50% reduction in Mtb Erdman load. Tuberculomas treated with simvastatin or fingolimod hydrochloride showed autophagy induction between 3 and 6 days. In contrast, histone deacetylase inhibitors resveratrol and vorinostat, as well as previously described autophagy inhibitors quinacrine dihydrochloride and sunitinib malate (which emits autofluorescence in the GFP channel), induced incomplete autophagy flux in 3D tuberculomas.

[0497] Since excessive hypoxia can lead to pathogenic necrosis of macrophages infected with mycobacteria, and autophagy and inflammasome activity can lead to non-apoptotic programmed cell death and pyroptosis, we carefully examined the results of all 65 compounds to further characterize the associations between these Mtb-controlled processes in 3D tuberculomas. When we considered these mechanisms individually, no direct association with inhibition of Mtb Erdman loading was found by day 6 post-treatment. For example, KN62, loperamide, and glibenclamide increased hypoxia compared to the DMSO control (data not shown), and imatinib, KN62, and loperamide induced autophagy with autolysosome formation or core acidification, but did not significantly inhibit Mtb loading. Similarly, of the nine compounds previously reported to activate autophagy in Mtb-infected 2D cultures (data not shown), only gefitinib and everolimus induced autophagy with autolysosome formation in the tuberculoma core by day 6. Incomplete autophagy flux was observed in numerous cell clusters with the remaining seven drugs, indicating inconsistent abilities to induce autophagy in the 3D tuberculoma environment. Except for SRT1720, none of the nine compounds significantly inhibited Mtb Erdman loading. Notably, autophagy flux was delayed in 3D tuberculomas treated with these HDT compounds (data not shown). These results collectively suggest that rapid pharmacological induction of complete autophagy flux (rather than just autophagosome initiation) is associated with inhibition of Mtb growth in 3D tuberculomas. They also demonstrate the versatility of the biological platform to incorporate therapeutically induced in situ innate immune mechanisms. In 3D biological platforms, HDT compounds can be classified into functional clusters based on immune mechanisms and therapeutic efficacy.

[0498] In more than one of the five mycobacterial infection models studied, several compounds reduced bacterial load and lesion load (data not shown). In all infection models, six “first-choice spur” compounds (nitrozonide, auronoxine, doxycycline, dovirtinib, 3',4' dichlorobenzamidine hydrochloride, and AT9283) that caused significant cytotoxicity in THP-1 and PBMC cultures inhibited bacterial load by >50% and significantly, except for AT9283 (which reduced the average bacterial load by 42.2% but significantly in the PBMC model). In three of the four infection models using THP-1 cells, these six cluster 1 compounds also reduced lesion load by >50%. They induced rapid autophagy flux and lysosomal acidification at a cytotoxic 20 µM dose, but did not induce hypoxia (…). Figure 29 Using a range of concentrations, we confirmed that these compounds, at CC levels, were effective in THP-1 monocytes infected with WT cells transfected with the autophagy sensor LC3B-RFP (BacMam 2.0). 50 or EC 50 Concentration-induced autophagy (data not shown). In 3D tuberculomas of THP-1-Difluo autophagy reporter cells, autophagy flux decreased and autophagosome accumulation increased with decreasing concentrations of auronoxine and AT9283 (data not shown). In 3D tuberculomas of ASC-GFP inflammasome reporter cells, the decrease in autophagy flux roughly corresponded to a reduction in the inhibition of Mtb Erdman loading. Although cell viability increased with decreasing compound concentrations, no corresponding increase in hypoxia-induced autophagy was observed, confirming that the efficacy of these preferred lead compounds in the 3D tuberculoma model is associated with autophagy induction but not with hypoxia.

[0499] In all mycobacterial infection models, six other “first-choice spur” compounds (methoxazole hydrochloride, H89, chlorpromazine hydrochloride, lansoprazole, zuciclothiasol dihydrochloride, and chlormastine fumarate, which caused <50% toxicity in THP-1 cultures) also reduced bacterial load by >50% compared to untreated controls, with the exception of methoxazole hydrochloride, zuciclothiasol dihydrochloride, and chlormastine fumarate (which did not significantly inhibit Mtb Erdman load in PBMC models). Interestingly, the FDA-approved drug lansoprazole induced >50% toxicity in 3D PBMC cultures. In the three infection models using THP-1, these six cluster 2 compounds inhibited >50% of the lesion load and induced hypoxia, rapid autophagy flux, and lysosomal acidification. Figure 29Notably, as lansoprazole and H89 concentrations decreased, hypoxia, autophagy, and ASC spot formation decreased on day 6, and Mtb Erdman loading increased, suggesting that these pathways contribute to the efficacy of these compounds in the 3D tuberculoma model (data not shown).

[0500] Five potential “sprout” cytotoxic compounds (sunitinib malate, fingolimod hydrochloride, dasatinib, GW 5074, and SRT 1720) formed a cluster. In all mycobacterial infection models, they reduced bacterial load by >25% compared to untreated controls, except for dasatinib (which failed to inhibit Mtb Erdman load in the THP-1 and PBMC models). In at least three infection models using THP-1, they also inhibited >25% of lesion load and induced autophagy flux (although slow between 3–6 days post-treatment) and lysosomal acidification, but did not induce hypoxia. Figure 29 Five potential “sprout” non-cytotoxic compounds (fluoxetine hydrochloride, gefitinib, loperamide, haloperidol, tin protoporphyrin IX, and ezetimibe) formed a cluster. In at least three mycobacterial infection models using THP-1 cells, they suppressed >25% of bacterial load compared to untreated controls. However, except for fluoxetine hydrochloride and tin protoporphyrin IX, the other three compounds did not suppress >25% Mtb Erdman load in the PBMC model. Furthermore, they showed heterogeneous efficacy in reducing lesion load and inducing autophagy flux, and slowly induced autophagy flux and hypoxia and lysosomal acidification between 3 and 6 days.

[0501] The comparison also revealed the species-specific heterogeneity of HDT compounds in inhibiting bacterial growth. Figure 29 Four cytotoxic compounds (simvastatin, vorinostatin, resveratrol, and quinacrine dihydrochloride) exhibited species-specific heterogeneity in mycobacterial growth inhibition in 3D tuberculomas and formed clusters. In all four or at least one of the four Mtb infection models in THP1 and PBMC, they did not reduce Mm load but inhibited Mtb load by >25%. Conversely, using this criterion, pazopanib, all-trans retinoic acid (ATRA), imatinib, omeprazole, and sildenafil were effective only in Mm infection models and formed clusters. In the THP-1 infection model, compounds in both clusters exhibited heterogeneity in inhibiting lesion load and inducing innate defense. These observations are consistent with reports of subtle differences in the response of human macrophages to infected Mm and Mtb strains.

[0502] In summary, our screening showed that kinase inhibitors (dovirtinib, H89, AT9283, sunitinib malate, and GW5074), as well as antipsychotics and antidepressants (chlorpromazine hydrochloride, methaxaton hydrochloride, zucloxacillin dihydrochloride, haloperidol, and fluoxetine hydrochloride), and antimicrobial agents with HDT activity (nizoxinide, doxycycline, and auronoxine) consistently suppressed bacterial load between 25% and 90% in all or more of the three infection models. AT9283 effectively inhibited Mtb burden and granulomatous lesions in mice in vivo.

[0503] One of the “preferred lead” compounds and a multi-targeted kinase inhibitor currently in clinical trials for the treatment of hematologic malignancies, AT9283 significantly inhibited Mm and Mtb burden and lesions in THP-1 3D tuberculomas. Compared to conventional 2D monolayer cultures in a host-directed manner, the compound preferentially exhibited anti-Mtb activity in the 3D tuberculoma environment, while showing no obvious direct bacterial-targeting activity in pure cultures. To determine whether AT9283 has Mtb-specific efficacy in vivo, we investigated its anti-mycobacterial activity during early infection in a C3Heb / FeJ mouse model. We chose to examine early infection before the onset of peak adaptive immunization, as this is the time period best simulated by our simple THP-1 3D co-culture model. ...

Claims

1. A three-dimensional (3D) cell co-culture comprising a variety of human immune cells and a variety of mycobacteria.

2. The 3D cell coculture according to claim 1, wherein the 3D cell coculture comprises a mycobacterial structure in a spheroid formed by the human immune cells and the plurality of mycobacteria.

3. The 3D cell co-culture according to claim 1, wherein the human immune cells comprise cells selected from monocytes, macrophages, and peripheral blood mononuclear cells (PBMCs).

4. The 3D cell co-culture according to claim 3, wherein the 3D cell co-culture comprises monocytes, wherein the monocytes include THP-1 cells, U937 tissue cells, and purified CD14. + Monocytes, or monocyte subsets in PBMCs.

5. The 3D cell co-culture according to claim 1, wherein the mycobacteria include Mycobacterium marineum (Mm), Mycobacterium tuberculosis (Mtb), or a Mycobacterium tuberculosis complex.

6. The 3D cell co-culture according to claim 5, wherein the 3D cell co-culture comprises Mm, wherein Mm comprises strain 1218 or strain M.

7. The 3D co-culture according to claim 5, wherein the 3D co-culture comprises Mtb, wherein the Mtb includes strain H37Rv, strain Erdman, strain CDC1551, strain Beijing F2, or clinical isolates of drug-sensitive or drug-resistant Mtb.

8. The 3D co-culture according to claim 1, wherein the 3D co-culture comprises 500-5000 colony-forming units (cfu) of mycobacteria / 10 5 Individual immune cells result in a multiplicity of infection (MOI) of 0.005-0.

05.

9. The 3D co-culture according to claim 8, wherein the 3D co-culture comprises Mm strain 1218 or strain M, wherein the MOI is about 0.006-0.

012.

10. The 3D coculture of claim 8, wherein the 3D coculture comprises Mtb strain H37Rv, wherein the MOI is about 0.015-0.

05.

11. The 3D co-culture of claim 10, wherein the MOI is about 0.

025.

12. The 3D co-culture according to claim 8, wherein the 3D co-culture comprises Mtb Erdman or Beijing F2, wherein the MOI is about 0.12-0.

05.

13. The 3D co-culture of claim 12, wherein the 3D co-culture comprises Mtb Erdman, wherein the MOI is about 0.

05.

14. The 3D cell co-culture according to claim 5, wherein the mycobacterium expresses green fluorescent protein (GFP), red fluorescent protein (RFP), a fluorescent marker, or a luminescent marker.

15. The 3D cell co-culture of claim 14, wherein the RFP comprises tdTomato or far-red fluorescent protein.

16. The 3D cell coculture of claim 1, wherein the 3D cell coculture is contained in a U-shaped bottom well, the U-shaped bottom well containing a hydrogel coating, wherein the hydrogel is hydrophilic, neutrally charged and bioinert.

17. The 3D cell co-culture of claim 16, wherein the hydrogel comprises a perfluorinated polymer, an olefin, or a combination thereof.

18. The 3D co-culture of claim 17, wherein the hydrogel comprises CORNING® PURAMATRIX™ peptide hydrogel.

19. The 3D co-culture of claim 16, wherein the hydrogel comprises one or more of collagen, fibroin, and alginate.

20. The 3D cell co-culture according to claim 16, wherein the wells are CORNING® ultra-low adsorption spherical microplates or S-BIO PRIMESURFACE® 3D culture spherical plates.

21. The 3D cell co-culture according to claim 1, wherein the 3D cell co-culture further comprises one or more additional cell types selected from the following: A549 human lung epithelial cells, HUVEC-1 human fetal endothelial cells, HULEC human lung endothelial cells and MRC-5 human lung fibroblasts or primary human epithelial cells, endothelial cells and fibroblasts.

22. The 3D cell co-culture according to claim 1, wherein the 3D cell co-culture is prepared by a method comprising: (a) The human immune cells are suspended in Roswell Park Memorial Institute cell culture medium, which further comprises one or more of L-glutamine, FBS, sodium pyruvate and HEPES buffer; (b) The mycobacteria are mixed with the human immune cells at an MOI of 0.005-0.5; wherein the mixture is contained in polystyrene U-bottom ultra-low adsorption pores of a microplate, wherein the pores contain a hydrogel coating, and wherein the hydrogel is hydrophilic, neutrally charged, and bioinert; and (c) Incubating the mixture at 37ºC for a time sufficient to allow the human immune cells and mycobacteria to form mycobacterial structures and granulomatous lesions within the spheroids.

23. A method for preparing a 3D cell co-culture, wherein the 3D cell co-culture comprises multiple human cells and multiple mycobacteria, the method comprising: (a) The human immune cells are suspended in a cell culture medium, the cell culture medium further comprising one or more of L-glutamine, FBS, sodium pyruvate and HEPES buffer; (b) The mycobacteria are mixed with the human immune cells at an MOI of 0.005-0.5; wherein the mixture is contained in the wells of a microplate; as well as (c) Incubating the mixture at 37ºC for a time sufficient to allow the human immune cells and mycobacteria to form mycobacterial structures and granulomatous lesions within the spheroids.

24. The method of claim 23, wherein the human immune cells comprise cells selected from monocytes, macrophages, and peripheral blood mononuclear cells (PBMCs).

25. The method of claim 24, wherein the human immune cells comprise monocytes, and wherein the monocytes comprise THP-1 cells, U937 tissue cells, and CD14 cells. + Monocytes, or monocyte subsets in PBMCs.

26. The method of claim 23, wherein the mycobacterium comprises Mycobacterium marineum (Mm) or Mycobacterium tuberculosis (Mtb).

27. The method of claim 26, wherein the mycobacterium comprises Mm, wherein Mm comprises strain 1218 or strain M.

28. The method of claim 27, wherein the Mm is mixed with the human immune cells at an MOI of about 0.006-0.

012.

29. The method of claim 23, wherein the mycobacterium comprises Mtb, and wherein the Mtb comprises strain H37Rv, strain Erdman, strain CDC1551, strain Beijing F2, or a clinical isolate of drug-sensitive or drug-resistant Mtb.

30. The method of claim 29, wherein the Mtb is mixed with the human immune cells at an MOI of about 0.012-0.

05.

31. The method of claim 30, wherein the MOI is about 0.

025.

32. The method of claim 29, wherein the Mtb comprises strain H37Rv, and the Mtb is mixed with the human immune cells at an MOI of about 0.015-0.

05.

33. The method of claim 23, wherein the mycobacterium expresses GFP, RFP, tdTomato, far-red fluorescent protein, or another fluorescent protein.

34. The method of claim 33, wherein the RFP comprises Tdtomato.

35. The method of claim 23, wherein the cell culture medium comprises Roswell Park Memorial Institute medium.

36. The method of claim 23, wherein the pores are made of polystyrene.

37. The method of claim 23, wherein the hole has a U-shaped bottom.

38. The method of claim 23, wherein the pore comprises a hydrogel coating.

39. The method of claim 38, wherein the hydrogel is hydrophilic, neutrally charged, and bioinert.

40. The method of claim 39, wherein the hydrogel comprises CORNING® PURAMATRIX™ peptide hydrogel.

41. The method of claim 39, wherein the pores are CORNING® ultra-low adsorption spherical microplates or S-BIO PRIMESURFACE® 3D culture spherical plates.

42. The method of claim 23, further comprising contacting the mycobacterial structure in the spheroid with the extracellular matrix (ECM).

43. The method of claim 42, wherein the ECM is added to the mycobacterial structure in the spheroid three days after the human immune cells and the mycobacteria are mixed.

44. The method of claim 23, further comprising placing and storing the mixture of human immune cells and mycobacteria at a temperature of -80ºC and optionally at a temperature of ≤-160ºC within 30 minutes of mixing the human immune cells and mycobacteria.

45. The method of claim 23, further comprising, after mixing the human cells and the mycobacteria, incubating the mixture at 37ºC for 16-72 hours; removing the cell culture medium; adding a cryopreservative to the co-culture of human cells and mycobacteria; and freezing the co-culture of spheroids.

46. ​​The method of claim 45, wherein the cryoprotectant comprises 5% (v / v) dimethyl sulfoxide (DMSO) in RPMI-1640 or 3D cell culture medium.

47. The method of claim 46, further wherein the cryopreservative further comprises heat-inactivated fetal bovine serum.

48. A method for screening molecules capable of preventing, treating, or reducing mycobacterial infections, the method comprising: (a) Contacting the 3D bio-platform according to claim 2 with the molecules; (b) Measuring one or more characteristics of the mycobacterial structure within the spheroid; and (c) Comparing one of the one or more properties with a control, wherein a change in at least one of the one or more properties indicates that the molecule is able to treat or reduce the mycobacterial infection or one or more of its pathological features.

49. The method of claim 48, wherein the one or more properties include one or more of the following: (a) The amount of fluorescence produced by the mycobacterium, wherein the mycobacterium expresses fluorescent molecules; (b) The integrity of the granuloma; (c) Growth of mycobacteria; (d) Granuloma formation; (e) Granulomatous growth; (f) Number and size of granulomas; (g) Formation of autolysosomes; (h) Inflammasome and pyroptosis induction; (i) Hypoxia-induced or inhibited; (j) Lysosomal acidification; (k) Host cell toxicity; and (l) The expression level of one or more gene or protein biomarkers expressed by cells of the 3D biological platform.

50. The method of claim 49, wherein the one or more properties include the amount of fluorescence produced by the mycobacterium, wherein the amount of fluorescence indicates mycobacterial load, and wherein a reduction in the mycobacterial load of the molecule to a threshold, compared to the control, indicates that the molecule is capable of treating or reducing mycobacterial infection or one or more pathological features thereof.

51. The method of claim 50, wherein the threshold is reduced by at least 25%.

52. The method of claim 50, wherein the threshold is a z-score less than -2.

53. The method of claim 52, wherein the threshold is a z-score less than -4.

54. The method of claim 50, wherein when the mycobacterium comprises Mycobacterium marineum, the human immune cells comprise THP-1 cells, and wherein the MOI of the mycobacterium is 0.007-0.

012.

55. The method of claim 50, wherein when the mycobacterium comprises Mycobacterium tuberculosis strain H37Rv, Erdman, or Beijing F2, the human immune cells comprise THP-1 cells, and wherein the MOI of the mycobacterium is 0.012-0.

05.

56. The method of claim 55, wherein the mycobacterium comprises Mycobacterium tuberculosis strain H37Rv, and the MOI is 0.

025.

57. The method of claim 55, wherein the mycobacterium comprises Mycobacterium tuberculosis strain Erdman or Beijing F2, and the MOI is 0.

05.

58. The method of claim 49, wherein the one or more characteristics include the expression levels of one or more genes expressed by cells of the 3D biological platform, wherein changes in the expression levels of the one or more genes, proteins, or immune processes compared to the control indicate that the molecules are capable of treating or reducing mycobacterial infection or one or more pathological features thereof.

59. The method of claim 58, wherein the control comprises uninfected human immune cells.

60. The method of claim 49, wherein the molecule is added approximately 0-6 days after co-incubation of the mycobacteria and the human immune cells.

61. The method of claim 49, wherein after contacting the 3D bio-platform with the molecule, the one or more of the properties are observed once or more within 1-14 days, optionally once on days 7, 9, and 12.

62. A method of treating or reducing tuberculomatous lesions in a subject in need, the method comprising administering to the subject an antimycobacterial agent selected from: antiCD11a antibody, anti-α4β7 integrin antibody, antiCD30 antibody, antiIGF1R antibody, antiIL-6R antibody, AT9283, tinizonide (the active metabolite of nitrozonide), dasatinib, quinacrine dihydrochloride, all-trans retinoic acid (ATRA), vorinostat, sitagliptin, and lansoprazole.

63. The method of claim 62, wherein the subject has a mycobacterial infection.

64. A method of treating or reducing tuberculomatous lesions in a subject of need, the method comprising administering to the subject an antimycobacterial agent selected from: antiCD11a antibody, anti-α4β7 integrin antibody, antiCD30 antibody, antiIGF1R antibody, antiIL-6R antibody, AT9283, tinizonide (the active metabolite of nitrozonide), dasatinib, quinacrine dihydrochloride, all-trans retinoic acid (ATRA), vorinostat, sitagliptin, H89, and lansoprazole.

65. The method of claim 64, wherein the subject has a mycobacterial infection.

66. A method for screening candidate agents that induce training immunity in human immune cells, the method comprising measuring the growth of pathogenic mycobacteria in a 3D platform of claim 1 after co-culturing the human immune cells and pathogenic mycobacteria, wherein the human immune cells have been contacted with the candidate agent, wherein reduced growth of the pathogenic mycobacteria or reduced granulomatous lesions produced by the co-culturing indicates that the candidate agent induces training immunity in the human immune cells.

67. The method of claim 66, wherein the candidate agent is a vaccine, an immunotherapeutic agent, a biological agent, or a host-targeted therapeutic agent.

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