Cell labeling and annotation in minute organoid spheres

Abstract

Provided herein are methods and materials for labeling and annotating living cells in minute organoid spheres (MOSs), as well as methods for assessing MOS containing labeled living cells.

Description

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 449,666, filed March 3, 2023. The disclosure of the prior application is considered part of the disclosure of this application and is hereby incorporated by reference into the disclosure of this application. TECHNICAL FIELD

[0002] This document relates to methods and materials for labeling cells in three-dimensional (3D) microorganoid spheroids (MOSs), and to methods and materials for using MOSs containing labeled live cells. For example, this document provides methods and materials for labeling specific types of cells (e.g., tumor cells, immune cells, fibroblasts, endothelial cells, or epithelial cells) in MOSs, methods and materials for determining the effects of various therapeutic agents on labeled cells in MOSs, and methods and materials for treating a mammal based at least in part on the determined effects. BACKGROUND

[0003] Model cell and tissue systems can be used in biological and medical research. Immortalized cell lines can be extracted from tissues and cultured under two-dimensional (2D) conditions (e.g., culture dishes or well plates), but 2D cell lines have poor correlation with individual patient responses to therapy. In contrast, three-dimensional (3D) cell culture models (e.g., spheroids and organoids) are particularly useful in the fields of developmental biology, disease pathology, regenerative medicine, drug toxicity and efficacy testing, and personalized medicine. Multicellular tumor spheroids can be obtained by culturing cancer cell lines under non-attachment conditions. Spheroids typically form free-floating cell aggregates in ultra-low attachment plates and have been shown to retain more stem cell-related properties than 2D cell culture. Organoids are multicellular, three-dimensional aggregates of cells of a specific tissue origin that include a population of stem cells that can differentiate into cells of the major lineages. Organoids typically have a diameter exceeding 1 mm and can be cultured through multiple passages. However, research and testing of drug sensitivity of organoids often takes months of time, which can reduce their clinical applicability because, ideally, testing should be completed within about 7 to 10 days after a single core biopsy. In addition, the number of organoids obtained from a clinically relevant 18-gauge core biopsy is often insufficient for high-throughput drug screening. In vitro Sources of Cell Aggregates SUMMARY

[0004] This document is based at least in part on the development of methods and materials for staining cells (e.g., live cells) in microorganoid spheres (MOS), and methods of using stained MOS. For example, this document provides a rapid live cell staining method that can be used for annotation of cell types in MOS and / or for phenotypic screening of cells in MOS. The methods provided herein allow for live cells cultured in MOS to be observed in real time without the need to fix the cells. In addition, the methods provided herein can also be used, for example, to observe behavior of cells within MOS, to track interactions between cells within MOS, and to assess persistence of cell responses to drugs or other therapeutic agents. In some cases, the methods and materials provided herein include the use of patient-derived microorganoid spheres (PMOS) containing cells derived from a mammal. For example, PMOS (also referred to herein simply as “MOS”) can comprise cells extracted from small patient biopsy tissue, resected patient tissue (e.g., resected primary tumor or tissue from a dysfunctional organ), and / or established patient-derived tumor models (PDMC), including patient-derived xenografts (PDX) and organoids. In some cases, MOS can comprise cells from a cell line (e.g., a genetically engineered cell line). The characterization of MOS and the labeling of cells (e.g., live cells) therein can allow for the exploration of biological processes, including the assessment of various responses of the immune system to new and existing therapies.

[0005] An example of the assays described herein that include the use of live cell staining in MOS is the detection and measurement of phagocytosis in 3D MOS. For example, as shown herein, phagocytosis of labeled cells (e.g., tumor cells) by phagocytic cells initially contained in or added to MOS generated from a multiple myeloma patient (MM) sample was observed and assessed. In addition, PMOS generated from MM tissue were labeled to determine the effect of a candidate agent (e.g., a therapeutic agent such as a drug) on phagocytosis.

[0006] Generally, one aspect of this document features a method comprising labeling a population of cells within a micro-organoid sphere (MOS) with a detectable label, wherein at least a portion of the cells within the population comprise the detectable label. The labeling can comprise adding the detectable label to a MOS that has already been formed. The labeling can comprise adding the detectable label to a population of cells prior to MOS formation. The portion of cells can comprise cancer cells, immune cells, fibroblasts, endothelial cells, stromal cells, or epithelial cells. The MOS can comprise multiple myeloma cells. The detectable label can be a fluorescent dye. The detectable label can be a pH-sensitive label. The detectable label can be attached to the cells via an antibody or antibody fragment, wherein the antibody or antibody fragment has an antigen-binding region that interacts with an epitope on the cells. The detectable label can be attached to the cells via a Fab antibody fragment. The epitope can be CD38, CD138, CD33, CS1, BCMA, CD45, a cell adhesion molecule, EpCAM, TROP2, CD3, CD8, PD-1, CD-31, CD19, FAPa, ASGR1, or HER2. The label can be a pH-sensitive label, and the method can further comprise measuring a signal level over time to determine a rate or extent of phagocytosis. The label can be a pH-sensitive label, and the method can further comprise detecting a presence or absence of a signal output from the pH-sensitive detectable label, wherein a presence of the signal indicates phagocytosis of cells within the population, and wherein an absence of the signal indicates a lack of phagocytosis of cells within the population.

[0007] In another aspect, the present document features a method comprising or consisting essentially of providing a MOS comprising a population of cells, wherein at least a portion of the cells within the population comprise a pH-sensitive detectable label; measuring a first signal level from the pH-sensitive detectable label, wherein the first signal level is directly related to a level of phagocytosis of cells within the portion of cells; contacting the MOS with a candidate agent; measuring a second signal level from the pH-sensitive detectable label, wherein the second signal level is directly related to a level of phagocytosis of cells within the portion of cells after the contacting; and comparing the first measured signal level to the second measured signal level, and identifying the candidate agent as a phagocytosis enhancer for the population of cells when the second measured signal level is increased as compared to the first measured signal level. The portion of cells can comprise cancer cells. The MOS can comprise multiple myeloma cells. The pH-sensitive label can be a fluorescent dye. The pH-sensitive detectable label can be attached to the cells via an antibody or antibody fragment, wherein the antibody or antibody fragment has an antigen-binding region that interacts with an epitope on the cells. The pH-sensitive detectable label can be attached to the cells via a Fab antibody fragment. The epitope can be CD38, CD138, CD33, CS1, BCMA, CD45, a cell adhesion molecule, EpCAM, TROP2, CD3, CD8, PD-1, CD-31, CD19, FAPa, ASGR1, or HER2. The method can further comprise measuring the signal level over time to determine a rate of phagocytosis.

[0008] In another aspect, the document features a method for treating a mammal. The method can include or consist essentially of providing a MOS comprising a population of cells from the mammal, wherein at least a portion of the cells within the population comprise a pH-sensitive detectable marker; measuring a first signal level from the pH-sensitive detectable marker, wherein the first signal level is directly related to a level of phagocytosis of cells within the portion of cells; contacting the MOS with a candidate agent; measuring a second signal level from the pH-sensitive detectable marker, wherein the second signal level is directly related to a level of phagocytosis of cells within the portion of cells after the contacting; determining that the second measured signal level is increased compared to the first measured signal level; and administering the candidate agent to the mammal. The plurality of cells can include cancer cells. The MOS can include multiple myeloma cells. The pH-sensitive marker can be a fluorescent dye. The pH-sensitive detectable marker can be attached to the cells via an antibody or antibody fragment, wherein the antibody or antibody fragment has an antigen-binding region that interacts with an epitope on the cells. The pH-sensitive detectable marker can be attached to the cells via a Fab antibody fragment. The epitope can be CD38, CD138, CD33, CS1, BCMA, CD45, a cell adhesion molecule, EpCAM, TROP2, CD3, CD8, PD-1, CD-31, CD19, FAPa, ASGR1, or HER2. The method can further include measuring the signal level over time to determine a rate of phagocytosis.

[0009] In yet another aspect, the present document features a method for treating a mammal, the method comprising or consisting essentially of administering a therapeutic agent to a mammal identified as having cells that experience an increased level of phagocytosis upon contact with the therapeutic agent, wherein the mammal is identified by providing a MOS comprising a population of cells from the mammal, wherein at least a portion of the cells within the population comprise a pH-sensitive detectable marker; measuring a first signal level from the pH-sensitive detectable marker, wherein the first signal level is directly related to the level of phagocytosis of the cells within the portion of cells; contacting the MOS with a candidate agent; measuring a second signal level from the pH-sensitive detectable marker, wherein the second signal level is directly related to the level of phagocytosis of the cells within the portion of cells after contact; and determining that the second measured signal level is increased compared to the first measured signal level. The plurality of cells can comprise cancer cells. The MOS can comprise multiple myeloma cells. The pH-sensitive marker can be a fluorescent dye. The pH-sensitive detectable marker can be attached to the cells via an antibody or antibody fragment, wherein the antibody or antibody fragment has an antigen-binding region that interacts with an epitope on the cells. The pH-sensitive detectable marker can be attached to the cells via a Fab antibody fragment. The epitope can be CD38, CD138, CD33, CS1, BCMA, CD45, a cell adhesion molecule, EpCAM, TROP2, CD3, CD8, PD-1, CD-31, CD19, FAPa, ASGR1, or HER2. The method can further comprise measuring the signal level over time to determine the rate of phagocytosis.

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the application, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0011] The details of one or more embodiments of the application are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the application will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figures 1A to 1D Establishment of live MOS staining technique is demonstrated. Figure 1A Workflow of live MOS staining method is demonstrated. Figure 1BRepresentative images of EpCAM / CD45 staining of MOS including different ratios of PBMC and colorectal cancer (CRC) cells. Magenta indicates EpCAM staining, while green indicates CD45 staining. Figure 1C Representative images showing live cell staining of MOS coated with different types of cells or primary tissue-derived cells. Top left: MOS coated with CRC and tumor infiltrating lymphocytes stained with EpCAM and CD45; Top middle: MOS derived from primary lung tissue stained with EpCAM and CD45; Top right: Mixed culture MOS coated with tumor-associated fibroblasts or CRC cells stained with FAPa and EpCAM; Bottom left: MOS coated with CRC and hepatocytes stained with EpCAM and ASGR1; Bottom middle: MOS coated with CRC cells, HUVECs, and tumor-associated fibroblasts stained with CD31 and EpCAM; Bottom right: MOS coated with primary lung-derived cells stained with EpCAM and CD45. Figure 1D Representative images showing the duration of EpCAM staining signal in CRC MOS cultures over 7 days.

[0013] Figures 2A to 2G EpCAM staining of live MOS does not affect cell growth or drug response of MOS. Figures 2A to 2C Graph showing the growth rate of three CRC MOS cultures (501299, 500040, and 501304, respectively) with or without EpCAM staining. Figures 2D to 2G Graph showing the survival rate of CRC MOS after treatment with the chemotherapy drug 5-fluorouracil (5-FU) with or without EpCAM staining.

[0014] Figures 3A to 3C Live MOS staining shows the response of tumor cells to anticancer therapy in MOS coated with CRC in combination with non-cancer cells (fibroblasts, endothelial cells, and PBMCs). Figure 3A Representative images showing live MOS staining of CRC cells co-coated with fibroblasts, endothelial cells, and PBMCs, stained with EpCAM, and treated with vehicle or oxaliplatin for three days, followed by staining with calcein AM (CAM) and ethidium homodimer (EtH). Figure 3B Graph showing the difference in drug response observed for CRC groups only and for CRC groups co-coated with non-cancer cells as measured by CTG detection. Figure 3C Graph showing the drug response of CRC groups only and co-coated groups, indicating that they have similarities based on EpCAM staining and live-dead cell imaging analysis.

[0015] Figure 4ATo illustrate, steps in a representative assay to label MM myeloid cells using PHRODO TM labeled CD38 antibody are shown: when myeloid cells are phagocytosed by macrophages, a signal detectable by flow cytometry (in this case, green fluorescence) is produced. Steps in the illustrated method include: (1) labeling CD38 antibody with PHRODO TM green; (2) adding PHRODO TM labeled CD38 antibody to MM cell culture to label CD38 + myeloid cells; and (3) mixing PHRODO TM labeled MM cells with or without PBMC-derived macrophages to generate MOS. Co-culture with monocyte-derived macrophages (MDM) can be used to ensure phagocytosis occurs. Figure 4B To illustrate, steps in a representative assay to assess phagocytosis using zymosan are shown. Zymosan is a glucan with repeating glucose units linked by beta-1,3-glycosidic bonds and can be taken up by macrophages. Labeling zymosan allows measurement of total phagocytosis, which can serve as a control. Steps in the illustrated method include: (1) labeling zymosan with PHRODO TM green; (2) co-encapsulating PHRODO TM labeled zymosan and MM sample into MOS; and (3) using flow cytometry to determine whether phagocytosis occurred, if it did, PHRODO TM labeled zymosan will turn green and can be detected by flow cytometry. Figure 4C To illustrate, a graph showing assay readouts that can be made for the methods depicted in Figure 4A and Figure 4B Specifically, flow cytometry can be performed to measure cells labeled with fluorescein isothiocyanate (FITC), CD11b (a marker for myeloid cells), and CD16 (a marker for natural killer cells, neutrophils, monocytes, macrophages, and certain T cells) on day 1 of MOS culture (about 24 hours after MOS generation) and day 5 of MOS culture. In addition, imaging of MOS can be performed on day 5.

[0016] Figure 5A Representative images of MM MOS cultured with zymosan (top) or PHRODO TM labeled anti-CD38 antibody (bottom) are included as depicted in Figure 4B and Figure 4A respectively. Figure 5B Flow cytometry readouts for unlabelled MM MOS (top), PHRODO TMLabeled anti-CD38 antibody (middle) or PHRODO TM Labeled zymosan (bottom) generated histograms.

[0017] Figure 6A and Figure 6B Phagocytic cells (CD11b Figure 6A ) or CD11b + cells in the indicated samples were plotted. Figure 6B PHRODO™-based phagocytosis assays were performed as described for Figure 4A and Figure 4B Data was collected at 24 hours for: (1) un-labeled MM sample (no PHRODO TM labeled anti-CD38 antibody); (2) PHRODO TM labeled MM sample; (3) zymosan-labeled MM sample co-encapsulated with PHRODO TM labeled zymosan; and (4) MM sample with MDM (PBMC-derived macrophages co-encapsulated with MM sample). Figure 6A Plots of green cells in live cells after 24 hours of incubation under each condition were plotted. Figure 6B Plots of CD11b Figure 6A gated green cells from + were plotted.

[0018] Figure 7 Plots of the percentage of PHRODO™+ CD11b+ cells after 4 hours of treatment with IgG4 or anti-CD47 or no treatment were plotted.

[0019] Figure 8A and Figure 8B Plots of the percentage of PHRODO TM+ cells (CD11b Figure 8A ) and phagocytosis (CD11b Figure 8B ) in the indicated samples were plotted. All data was generated by flow cytometry from a second frozen MM sample. The freeze-thawed MM sample was labeled with PHRODO TM labeled antibody and a MOS was generated. Phagocytosis assays were performed on day 0 and day 5 of the MOS culture. Data was collected at 24 hours. Conditions used were: (1) no PHRODO TM : no PHRODO TM labeled antibody added; (2) zymosan: MM sample co-encapsulated with PHRODO TM labeled zymosan (data collected only on day 1); and (3) untreated: vehicle control. Figure 8A Plots of green cells in live cells after 24 hours of incubation under each condition were plotted.Figure 8B FIG. 16A is a graph plotting the percentage of CD11b Figure 8A + cells gated for CD11b

[0020] Figure 9A FIG. 16B is a graph plotting the percentage of CD11b Figure 9B + cells gated for CD11b + DETAILED DESCRIPTION

[0021] Method for generating MOS The present document provides methods and materials for staining live cells in MOS, as well as methods for using live cell-stained MOS. For example, the live cell staining methods described herein can be used to annotate cell types in MOS, to phenotype screen cells in MOS, to observe live cells in MOS in real time, to track the behavior of cells within MOS, to observe interactions between cells within MOS, and / or to assess the response of cells to drugs or other therapeutic agents. In some cases, the methods and materials provided herein include the use of PMOS containing cells derived from mammals, such as cells extracted from patient biopsy tissue, cells obtained from resected patient tissue (e.g., tissue from a resected primary tumor or a dysfunctional organ), and / or cells from established PDMCs, PDXs, and organoids. Labeling of cells in MOS can allow for the exploration of biological processes, including the assessment of various responses of the immune system to new and existing therapies.

[0022] ​​​Generally, the methods and materials provided herein relate to an antibody-based staining method for labeling viable cells in MOS droplets, which enables MOS fluorescent antibody cell tracking (MOS-FACT). As described herein, antibodies conjugated to fluorescent labels can efficiently diffuse into MOS droplets and can stain surface markers of target cells. Thus, using fluorescently conjugated primary antibodies against specific cell surface markers, different types of cells (e.g., tumor cells, immune cells, fibroblasts, endothelial cells, cholangiocytes, hepatocytes, etc.) in MOS droplets can be specifically labeled and annotated, typically within 1 to 4 hours after staining, without the need to fix or kill the cells. The live MOS staining signal can also reflect and guide histological diagnosis of the primary tissue. As described herein, the antibody fluorescent signal can persist for up to 7 days, and the antibody staining of cells in MOS does not affect the drug response of MOS to chemotherapeutic drugs or tyrosine kinase inhibitors (TKIs). In combination with imaging (e.g., high-content confocal imaging), the methods described herein enable longitudinal tracking and phenotyping of cells in MOS cultures. MOS do not adhere to the culture surface but are suspended in the culture medium, thus MOS can be transferred by pipetting and can be easily transferred to other vessels, containers, or devices. Without being bound to a particular mechanism of action, this property, in combination with the stability of fluorescent labels over time and the fact that labeled cells remain viable in MOS, means that analysis of labeled MOS populations is not limited to imaging but can also include downstream analyses such as flow cytometry, single-cell RNA sequencing, high-throughput analysis, etc.

[0023] Methods for making MOS can employ, for example, cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology techniques. Examples of such techniques are described in other literature. See, e.g., Molecular Cloning A Laboratory Manual (1989), Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Patent No. 4,683,195; DNA Cloning Glover, ed., 1985; Oligonucleotide Synthesis Gait, ed., 1984; Nucleic Acid Hybridization Hames and Higgins, eds., 1984; Transcription and Translation, Hames and Higgins, eds., 1984; Culture of Animal Cells Freshney, Alan R. Liss, Inc., 1987;Immobilized Cells And Enzymes , IRL Press, 1986; Perbal (1984), A Practical Guide To Molecular Cloning; See Ausubel et al. (1999) Short Protocols in Molecular Biology, 4th Ed., John Wiley & Sons, Inc. Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells , Miller and Calos eds., Cold Spring Harbor Laboratory, 1987; Methods in Enzymology , Vols. 154 and 155, Wu et al. eds., Academic Press Inc., N.Y. Immunochemical Methods in Cell and Molecular Biology , Mayer and Walker eds., Academic Press, London, 1987; and Handbook of Experimental Immunology , Vols. I-IV, Weir and Blackwell eds., 1986.

[0024] MOS can be formed from normal primary cells (e.g., normal organ tissue) or tumor tissue. For example, in some cases, PMOS can be formed from a cancerous tumor biopsy, enabling the use of the particular tumor tissue examined to select a personalized treatment regimen. The methods for generating MOS as described herein allow for the formation of hundreds, thousands, or even tens of thousands (e.g., 500, 750, 1000, 2000, 5000, 10,000, or more) of MOS from a single tissue biopsy within a few hours after the biopsy is removed from the patient. Generally, dissociated primary cells from a patient biopsy can be combined with a fluid matrix material, such as a basement membrane matrix, e.g., MATRIGEL ®A soluble basement membrane matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, commercially available from Sigma, is combined to form MOS. The resulting plurality of MOS can have a predetermined size range (e.g., a diameter of about 10 pm to about 700 pm, and any sub-range therein) and initial primary cell number (e.g., about 1 to about 1000, particularly lower cell numbers such as about 1 to about 200). The cell number and / or diameter can be controlled to within, for example, ± 5%, ± 10%, ± 15%, ± 20%, ± 25%, ± 30%, etc. The PMOS can have a very high survival rate (e.g., >75%, >80%, >85%, >90%, >95%) and can be used and tested stably in a very short time, typically within the first 1 to 14 days after formation (e.g., within 1 day, within 2 days, within 3 days, within 4 days, within 5 days, within 6 days, within 7 days, within 8 days, within 9 days, within 10 days, within 11 days, within 12 days, within 13 days, or within 14 days). This can allow for rapid testing of potentially large numbers of patient-specific and biologically relevant PMOS, which can save critical time in developing and deploying patient treatments, such as cancer treatment plans. The PMOS can rapidly form 3D cell structures that mimic and correspond to the tissue environment from which they are biopsied, such as 3D tumor microenvironments. In some cases, the PMOS can be referred to as “droplets.” Each PMOS can include (e.g., as part of the fluid matrix material) growth factors and structural proteins (e.g., collagen, laminin, entactin, etc.) that can mimic the original tissue (e.g., tumor) environment.

[0025] In some cases, the MOS used in the methods provided herein can comprise cancer cells and one or more other types of cells (e.g., fibroblasts, endothelial cells, and / or immune cells, including cells capable of phagocytosis such as macrophages, neutrophils, monocytes, dendritic cells, and / or osteoclasts). The MOS can be derived from, for example, a bone marrow tissue sample of a mammal having multiple myeloma (MM) (referred to herein as “MM MOS”), from an acute myeloid leukemia (AML) bone marrow sample, from a solid tumor sample (e.g., a biopsy of a colorectal tumor, a breast tumor, a kidney tumor, a liver tumor, a lung tumor, an ovarian tumor, or a prostate tumor), or from any other appropriate type of sample. In some cases, the MOS can comprise cells from a cell line (e.g., a genetically engineered cell line). Methods and materials for generating genetically engineered cell lines (e.g., using CRISPR components) are described, for example, in PCT Publication No. WO2024 / 040145, which is incorporated by reference herein in its entirety. In some cases, the MOS can not comprise cells from a cell line.

[0026] MOS that can be labeled and used in the methods provided herein are generally spheroids formed from dissociated cells (e.g., dissociated primary cells distributed within a biopsy material). MOS can have a diameter of about 50 pm to about 500 pm (e.g., about 50 pm to about 400 pm, about 50 pm to about 300 pm, about 50 pm to about 250 pm, about 100 pm to about 500 pm, about 100 pm to about 250 pm, or about 50 pm to about 200 pm). MOS initially can comprise about 1 to about 1000 dissociated primary cells distributed within a base material (e.g., about 1 to about 750, about 1 to about 500, about 1 to about 400, about 1 to about 300, about 1 to about 200, about 1 to about 150, about 1 to about 100, about 25 to about 200, about 50 to about 150, or about 25 to 100 primary cells).

[0027] Despite the small size (typically about 50 pm to about 250 pm) and low cell density (e.g., about 50 to about 200 cells per MOS typically) of MOS, they can be used directly or cultured for a relatively short period of time (e.g., 14 days or less, 10 days or less, 7 days or less, or 5 days or less). Moreover, the cells within MOS can survive while retaining most or even all of the characteristics of the tissue from which they were extracted. Cell survival within MOS is typically high, and MOS can be cultured through multiple passages for days or even weeks, with cells dividing, clustering, and forming structures that resemble the parent tissue. Moreover, in some cases, cells from the dissociated MOS source tissue can even form morphological structures within the minimal MOS. While in some applications the presence of such structures is not necessary for these MOS to be useful (such as when used before substantial structural reorganization occurs), in some cases such structures can be particularly useful.

[0028] MOS can be generated using any suitable method, including those described herein. The following definitions are set forth to facilitate explanation of the subject matter of the present disclosure.

[0029] The term "unpolymerized mixture" is used herein to refer to a composition comprising a biologically relevant material, including a dissociated tissue sample and a first fluid matrix material. The fluid matrix material is generally a material that can be polymerized to form a support or support network for the dissociated tissue and / or cells dispersed within it. Once polymerized, the polymerized material can form a hydrogel and can be formed and / or can include proteins other than cells that form a biocompatible medium. Biocompatible media suitable for use in the methods disclosed herein can be formed from any biocompatible material that is a gel, semi-solid, or liquid (such as a low viscosity liquid) at room temperature (e.g., 25 °C) and can serve as a three-dimensional matrix for cells, tissues, proteins, and other biological materials of interest. Exemplary materials that can be used to form a biocompatible medium in accordance with the subject matter of the present disclosure include, but are not limited to, polymers and hydrogels including collagen, fibrin, chitosan, MATRIGEL® (BD Biosciences, San Jose, CA), polyethylene glycol (PEG), dextran (e.g., dextran that can be chemically cross-linked or photo-cross-linkable), and the like, as well as electrospun biocomposites or biocomposite mixtures. In some cases, the biocompatible medium can be a hydrogel. ® (BDBiosciences, San Jose, CA), polyethylene glycol (PEG), dextran (e.g., dextran that can be chemically cross-linked or photo-cross-linkable), and the like, as well as electrospun biocomposites or biocomposite mixtures. In some cases, the biocompatible medium can be a hydrogel.

[0030] The term "hydrogel" as used herein refers to a two- or multi-component gel having a three-dimensional network of polymer chains, in which water acts as a dispersion medium and fills the spaces between the polymer chains. Hydrogels used in accordance with the subject matter of the present disclosure can generally be selected for a particular application based on the intended use of the structure, while taking into account the parameters to be used to form the MOS and the effect that the selected hydrogel will have on the behavior and activity of the biological materials (e.g., cells) to be incorporated into the biological suspension to be placed in the structure. Exemplary hydrogels for use with the subject matter of the present disclosure can include polymeric materials such as, but not limited to, alginate, collagen (including type I and type VI collagen), elastin, keratin, fibronectin, proteoglycans, glycoproteins, polylactide, polyethylene glycol, polycaprolactone, polycocohistate, polydioxanone, polyacrylate, polyurethane, polysulfone, peptide sequences, proteins and derivatives, oligopeptides, gelatin, elastin, fibrin, laminin, polymethacrylate, polyacetate, polyesters, polyamides, polycarbonates, polyanhydrides, polyamino acid carbohydrates, polysaccharides and modified polysaccharides, and derivatives and copolymers thereof, and inorganic materials such as glasses (such as bioactive glasses), ceramics, silica, alumina, calcite, hydroxyapatite, calcium phosphate, bone, and any combination thereof.

[0031] Further, with respect to hydrogels used to generate MOS, in some cases, the hydrogel can comprise a material selected from the group consisting of agarose, alginates, type I collagen, polyoxyethylene-polyoxypropylene block copolymers (e.g., PLURONIC F127; BASF Corporation, Mount Olive, NJ), silicones, polysaccharides, polyethylene glycol, and polyurethanes. In some cases, the hydrogel can consist of alginates. ® F127; BASF Corporation, Mount Olive, NJ), silicones, polysaccharides, polyethylene glycol, and polyurethanes. In some cases, the hydrogel can consist of alginates.

[0032] In some cases, the MOS can comprise a biologically relevant material. The phrase "biologically relevant material" refers to a material that can be contained in a biocompatible medium as defined herein and subsequently interact with and / or affect a biological system. For example, the biologically relevant material can be a magnetic bead (such as a bead that is itself magnetic or contains a material that is magnetic in response to a magnetic field (such as iron particles)) that can be combined as part of the unpolymerized material to aid in the generation of the MOS (e.g., for isolation and purification of the MOS). As another example, in some cases, the biologically relevant material can include cells in addition to the dissociated tissue sample (e.g., biopsy material). In the unpolymerized mixture, the dissociated tissue sample and additional biologically relevant material can exist as a homogenous mixture or as a distributed mixture (e.g., on one half or the other of the MOS, such as only in the core or only in the outer region of the MOS formed). In some cases, the additional biologically relevant material within the unpolymerized material can be suspended with the dissociated tissue sample in suspension (e.g., prior to polymerization of the droplets forming the MOS). In some cases, the biologically relevant material that can optionally be contained in the dissociated tissue sample (e.g., biopsy tissue) can contain a variety of cell types, including pre-adipocytes, mesenchymal stem cells (MSCs), endothelial progenitor cells, T cells, B cells, mast cells, and / or adipose tissue macrophages, MDMs, and small blood vessels or microvessel fragments found within the stromal vascular fraction.

[0033] As used herein, the term "passage" refers to the average number of cell doublings within a MM MOS. While traditional passage numbers refer to the transfer or subculturing of cells from one culture vessel to another, cells within a MOS can remain stably within the same MOS and can continue to grow and divide. Thus, passage number as used herein generally refers to the average number of doublings experienced by dissociated cells from a tissue biopsy within a MOS. Population doublings are an approximation of the number of doublings a population of cells has undergone since dissociation (e.g., since the MOS was formed from a freshly dissociated biopsy). Generally, MOS described herein can be cultured for a shorter period of time (e.g., less than 10 passages, less than 9 passages, less than 8 passages, less than 7 passages, less than 6 passages, less than 5 passages, less than 4 passages, or less than 3 passages) relative to the growth (e.g., doublings) of some or all of the cells within the MOS.

[0034] In some cases, the tissue sample (and cells contained therein) used to generate a MOS can be collected from a mammal. The tissue sample can be from any suitable mammal (e.g., a human or other mammal, such as a dog, cat, mouse, rat, or rabbit, suffering from MM), typically collected by biopsy. The tissue can be derived from, without limitation, a biopsy, a surgical specimen, an aspirate, a drain, or a cell-containing fluid. Typically, a MOS will contain a plurality of cell types present in the original tissue, although a MOS can also be formed from one particular cell type. The cells can be obtained directly from the subject, without an intermediate step of subculture, or the cells can first be subjected to an intermediate culture step to produce a primary culture. Any suitable method for obtaining cells from biological tissue and / or cell-containing fluid can be used. For example, suitable techniques for obtaining cells from biological tissue include the techniques described in Mahesparan, P. et al. (1999) 97:231-239. Acta Neuropathol (1999) 97:231-239.

[0035] The cells in the tissue sample are typically dissociated or separated from one another prior to formation of the MOS. Dissociation of the cells can be accomplished using any suitable method. For example, the cells can be subjected to mechanical and / or chemical treatment, such as by treatment with an enzyme. "Mechanical" dissociation typically involves the use of, for example, a surgical knife, scissors, or a machine such as a homogenizer to disrupt the connections between associated cells. "Enzymatic" dissociation can involve treatment of the cells with one or more enzymes to disrupt the connections between associated cells (e.g., using collagenase, dispase, DNase, and / or hyaluronidase). The one or more enzymes can be used under any suitable reaction conditions, such as incubation in a 37 °C water bath or at room temperature.

[0036] The dissociated tissue can be processed to remove dead / necrotic cells and / or cellular debris. Any suitable method such as magnetic bead methods and / or antibody methods can be used to remove such dead and / or necrotic cells. For example, apoptotic cells can be separated from live cells using annexin V biotin binding, followed by binding of the biotin to streptavidin magnetic beads, since phosphatidylserine can be redistributed from the inner to the outer leaflet of the plasma membrane in apoptotic or dead cells. Removal of cellular debris can be achieved by techniques such as, for example, filtration.

[0037] The dissociated cells can be suspended in a carrier material prior to being combined with the fluid matrix material. Alternatively, the fluid matrix material can be referred to as the carrier material. In some cases, the carrier material can be a material having a level of viscosity that delays sedimentation of the cells in the cell suspension prior to polymerization and formation of the MOS. In some cases, the carrier material can have sufficient viscosity to allow the dissociated biopsy tissue cells to remain suspended in the suspension until polymerization. The viscosity required to achieve this can be optimized by monitoring the sedimentation rate at different viscosities and selecting a viscosity that gives an appropriate sedimentation rate for the expected time delay between loading the cell suspension into the device that forms the MOS by polymerizing the un-polymerized material including the cells. In some cases, the un-polymerized material can be flowed or shaken by the device (e.g., when using lower viscosity materials) to keep the cells suspended and / or distributed as needed.

[0038] As noted above, in some cases, the un-polymerized mixture comprising the dissociated tissue sample and the fluid matrix material can comprise one or more other components, such as one or more biologically relevant materials. Biologically relevant materials that can be included in the un-polymerized mixture include, but are not limited to, one or more extracellular matrix proteins (e.g., fibronectin), drugs (e.g., small molecules), peptides, antibodies (e.g., to modulate any one of cell survival, proliferation, or differentiation), and / or inhibitors of specific cellular functions. Such biologically relevant materials can be used, for example, to increase cell viability or otherwise mimic the in vivo environment by reducing cell death and / or activation of cell growth / reproduction. In vivoenvironment. The biologically relevant material can include or can mimic one or more of the following components: serum, interleukins, chemokines, growth factors, glucose, physiological salts, amino acids, and hormones. In some cases, the biologically relevant material can supplement one or more agents in the fluid matrix material. In some cases, the fluid matrix material can be a synthetic gel (hydrogel) and can be supplemented with one or more biologically relevant materials. In some cases, the fluid matrix can be a natural gel. Thus, the gel can include one or more extracellular matrix components such as collagen, fibrinogen, laminin, fibronectin, vitronectin, hyaluronic acid, fibrin, alginate, agarose, and / or chitosan. For example, MATRIGEL ® include a biologically relevant material. The biologically relevant material can include or can mimic one or more of the following components: serum, interleukins, chemokines, growth factors, glucose, physiological salts, amino acids, and hormones. In some cases, the biologically relevant material can supplement one or more agents in the fluid matrix material. In some cases, the fluid matrix material can be a synthetic gel (hydrogel) and can be supplemented with one or more biologically relevant materials. In some cases, the fluid matrix can be a natural gel. Thus, the gel can include one or more extracellular matrix components such as collagen, fibrinogen, laminin, fibronectin, vitronectin, hyaluronic acid, fibrin, alginate, agarose, and / or chitosan. For example, MATRIGEL

[0039] The MOS typically includes dissociated biopsy tissue (e.g., cells), the number or concentration of cells within the MOS (e.g., cells / ml or cells / mm 3) is fixed or known. In some cases, the number of cells initially contained in a MOS can range from 1 cell to hundreds of cells. Specifically, for use in some assays (e.g., drug toxicity assays), it can be beneficial to generate MOS containing about 1 to about 75 cells or about 1 to about 50 cells (i.e., a relatively low number of cells) per MOS. The number of cells per MOS can be set or selected by the technician generating the MOS. In some cases, the device used to generate the MOS can contain one or more controls to set the number of cells from the primary tissue to be contained in each MOS. The number of cells can be selected or set based on the intended use of the MOS. For example, MOS with very low numbers of cells (e.g., 1 cell per MOS or 1 to 5 cells per MOS) can be particularly suitable for studying clonal diversity (e.g., for tumor heterogeneity). In some cases, each MOS is grown from a single cell, in which case it can be observed which clones are resistant, and then these particular MOS can be examined (e.g., by genomic sequencing) to determine the genomic (mutational) diversity associated with the particular clone. MOS with low to moderate numbers of cells per MOS (e.g., about 3 to about 30 cells, about 5 to about 30 cells, about 5 to about 25 cells, about 5 to about 20 cells, or about 10 to about 25 cells) can be particularly useful for rapid drug testing (including toxicity testing) because these MOS generally grow rapidly. MOS with higher numbers of cells per MOS (e.g., about 20 to about 100 cells, about 30 to about 100 cells, about 40 to about 100 cells, or more than 50 cells) can be particularly suitable for mimicking the tissue composition in each MOS, as the MOS can contain different lineages - which can include epithelial cells (e.g., cancer cells) and mesenchymal cells (e.g., stromal cells, immune cells, or vascular cells). It should be noted that for MM MOS, it can be beneficial for each MOS to contain a relatively high number of cells (e.g., about 50 to about 200 cells per MOS) to provide different cell types for each MOS, which can help to maintain the MM cells.

[0040] In some cases, a MOS can be generated by forming a droplet of a disaggregated tissue sample and an unpolymerized mixture (in some cases, a frozen mixture) of a fluid matrix material in an immiscible material, such as a fluid hydrophobic material (e.g., oil). For example, a MOS can be formed by combining a stream of unpolymerized material with one or more streams of immiscible material to form a droplet. The density of cells present in the droplet can be determined by the dilution of the disaggregated material (e.g., cells) in the unpolymerized material. The size of the MOS can be related to the size of the droplet formed. Generally, a MOS is a spherical structure having a stable geometry.

[0041] The MOS can have any suitable size, which can be matched to the number of cells to be included. For example, the MOS can be relatively small in size, having a diameter of about 20 pm to about 500 pm (e.g., on average about 50 pm or about 100 pm, or about 100 pm to about 200 pm). In some cases, the MOS can be about 300 pm in size, in which case about 10 to about 50 cells (e.g., about 10 to about 30 cells) can be included in each MOS. The number of cells and the size can be varied and / or can be controlled. In some cases, the number of cells and / or the size of the MOS can be set by one or more controls on the device used to form the MOS. For example, the size of the MOS and / or the density of cells within the MOS can be adjusted by adjusting the flow rate and / or concentration of the dissociated tissue sample (e.g., cells from a biopsy).

[0042] During culture, cells from the dissociated biopsy tissue within the MOS can aggregate, cluster, or assemble within the MOS. The aggregation of cells can be highly organized and can form a determined morphology or can be a cluster of cells that have clustered or adhered together. This organization can reflect the original tissue. In some cases, the MOS can comprise a single cell type (homotypic), while in other cases, the MOS can comprise more than one cell type (heterotypic).

[0043] Additional description of methods and devices for making MOS can be found in U.S. Patent Publication No. 2021-0285054. For example, See Paragraphs

[0165] to

[0183] and Figures 6 to 10 of U.S. Patent Publication No. 2021-0285054.

[0044] In some cases, the MOS can be generated from a MM tissue sample. It is generally difficult to replicate tumor cells using MM tissue because MM cells are often difficult to work with and it is challenging to culture MM cells in 3D cultures. Furthermore, MM cells cannot survive without bone marrow stromal cells. However, as described in Examples 1 to 3 of U.S. Provisional Application No. 63 / 404,472 (incorporated by reference in its entirety herein), MOS can be generated from MM biopsy tissue, which is surprising given the aforementioned challenges. This application and the techniques described herein can provide a tumor microenvironment for MM cells, allowing MM cells to grow. Cells within the MM immune microenvironment (e.g., macrophages) are also captured in the MOS, which expands the range of drugs to screen the MOS. This range includes, but is not limited to, agents that are able to modulate phagocytosis by macrophages and other cells within the MM MOS (e.g., drugs that are able to induce this phagocytosis).

[0045] Accordingly, in some cases, tissue (e.g., biopsy) samples used to generate MOS (e.g., dissociated tissue) can be derived from bone marrow obtained from a mammal having MM. In such cases, the tissue used in the MOS can include cells of the immune system, such as T lymphocytes, B lymphocytes, polymorphonuclear leukocytes, macrophages, and dendritic cells. These cells can include stem cells, progenitor cells, or somatic cells. These tissues and resulting dissociated cells can be primary cells extracted from a patient biopsy tissue (e.g., obtained by a needle biopsy). The dissociated cells can be incorporated into the MM MOS and can include cells commonly found in bone marrow. In this regard, exemplary cells that can be incorporated into the MM MOS include plasma cells (both normal and cancerous), stromal cells, hematopoietic stem cells, monocytes, macrophages, neutrophils, basophils, eosinophils, red blood cells, dendritic cells, T cells, B cells, and natural killer (NK) cells. The tissue can be dissociated using any suitable technique.

[0046] As described in U.S. Provisional Application No. 63 / 404,472, in some cases, it can be convenient to prepare and use MOS generated from MM bone marrow biopsy tissue by (1) using about 50 to about 150 cells per MOS (e.g., about 50 to 100 cells, about 75 to about 125 cells, about 100 to about 150 cells, or about 100 cells), (2) increasing the concentration of added MATRIGEL ® to about 80% to 100% (e.g., about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 95% to about 100%, about 80% to about 90%, or about 90% to about 100%), and / or (3) using ultra-low attachment plates. Without being bound by a particular mechanism, using a relatively larger number of cells can increase the likelihood that all cells in the bone marrow microenvironment (e.g., stromal cells and immune cells) are incorporated into the MM MOS, which is important from the perspective of drug screening. Further, without being bound by a particular mechanism, using a higher concentration of MATRIGEL ® helps retain MM cells within the MOS, while using low or ultra-low attachment plates can reduce the likelihood that the MOS adheres to the bottom of the culture well. Further, in some cases, the methods disclosed herein for preparing MM MOS can include adding a small amount of MATRIGEL ® (e.g., 1%) to the culture medium rather than the MOS mixture, and / or adding one or more cytokines (e.g., IL-6, GM-CSF, IL-2, B cell activating factor (BAFF), or IL-4) to the MATRIGEL ® ® ​whether added to the MOS mixture or to the culture medium. These strategies can also help retain MM cells within the MOS and / or prevent MOS attachment to the bottom of the culture well. In view of this, MM MOS can have a cell number of about 50 to about 150 per MOS, with the MOS cell population comprising MM plasma cells, stromal cells, and immune cells. Generally, MOS can be generated directly from a biopsy tissue and typically maintain similar cell proportions as the patient biopsy tissue. In some cases, the ratio of stromal cells to MM cells in the MOS can be about 6: 1 to about 3: 1 (e.g., about 5: 1 or about 4: 1). In some cases, the ratio of immune cells (e.g., macrophages) to MM cells in the MOS can be about 1:50 to about 1:200 (e.g., about 1: 100).

[0047] Once MOS are formed, they can be cryopreserved and / or cultured. Generally, cultured MOS can be maintained in suspension, either static suspension (e.g., in a well, vial, or other suitable container) or in motion suspension (e.g., rolling or shaking). Any appropriate technique can be used to culture MOS. Exemplary techniques can be found in (but are not limited to) Freshney, Culture of Animal Cells, A Manual of Basic Techniques , 4th Ed., Wiley Liss, John Wiley & Sons, 2000; Basic Cell Culture: A Practical Approach , Davis, Ed., Oxford University Press, 2002; Animal Cell Culture: A Practical Approach , Masters, Ed., 2000; and U.S. Patent Nos. 5,516,681 and 5,559,022, all of which are incorporated by reference herein in their entireties.

[0048] Labelled MOS Cells within the MOS (e.g., tumor cells or non-tumor cells), or cells in a biological sample comprising dissociated cells that will be used to generate MOS, can be stained with a detectable label directed against a selected cell surface marker. Any appropriate detectable label (e.g., fluorescent label) can be coupled to the dissociated cells or cells within a cultured MOS. In some cases, a pH-sensitive detectable label can be used. An exemplary pH-sensitive label is PHRODO TM (Thermo Fisher), a fluorescent dye that acts as an intracellular pH indicator and can be used to measure pH within live cells. Specifically, PHRODO TMThe fluorescence is weak at neutral pH, but increases as the pH decreases (e.g., during phagocytosis). It should be noted that as an alternative to fluorescent dye conjugated antibodies, any dye capable of staining unfixed cells can be used in the methods described herein, including live cell or dead cell (e.g., fixed cell) fluorescent dyes, dyes for staining cytoskeleton or intracellular organelles, and / or dyes for staining lipid droplets.

[0049] Any appropriate method can be used to conjugate a detectable label to a particular cell (e.g., a cancer cell, an immune cell, a fibroblast cell, an endothelial cell, etc.) in a dissociated biological sample (e.g., a dissociated tumor sample) or a cell within a cultured MOS. For example, a detectable label can be conjugated to an antibody or antibody fragment (e.g., a V H H fragment, also known as a NANOBODY ® ) and the conjugate can then be attached to a cell in a dissociated tissue sample or MOS that has been formed via the antigen binding domain of the antibody or antibody fragment. The term "antibody" as used herein includes full-length antibody molecules (e.g., IgA, IgD, IgE, IgG, or IgM type antibodies, including IgG or IgM types such as, but not limited to, IgGl, IgG2, IgG3, IgG4, IgMl, and IgM2 types) and also includes antibody-like molecules containing engineered antibody subdomains or naturally occurring antibody variants. The antibody-like molecules can be single domain antibodies such as V H domain only or V L domain only that are derived from natural sources such as camelids (Muyldermans et al. (2001) Rev. Mol. Biotechnol. 74:277-302) or obtained by phage display from libraries from humans, camelids, or other species (Holt et al. (2003) In vitro Trends Biotechnol. 21:484-90). In particular embodiments, the polypeptide structure of the antigen binding protein can be based on an antibody, including but not limited to minibodies, synthetic antibodies (sometimes referred to as "synthetic antibodies"), human antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and fragments of each of these.

[0050] ​An "Fv fragment" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a significantly lower affinity than the entire binding site. A "Fab fragment" also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH1). H 1) The "Fab fragment" differs from the "Fab' fragment" by the addition of a few residues at the carboxy terminus of the heavy chain C H 1 domain, including one or more cysteines from the antibody hinge region. The "F(ab')2 fragment" originally was produced as a pair of Fab' fragments which have hinge cysteines between them. Methods for producing such antibody fragments can include digestion with, for example, papain or pepsin.

[0051] Nanobodies are recombinantly expressed antigen binding V H H domains from heavy chain IgG. Nanobodies typically have a molecular weight of about 12 kDa to 15 kDa and are one of the smallest antigen binding fragments that occur naturally. These isolated V H H domains retain the ability to bind to antigen.

[0052] Further discussion of antibody fragments, See for example, Bates and Power, Antibodies , 8:28, 2019. In addition, kits useful for coupling a label (e.g., PHRODO TM ) to an antibody (e.g., PHRODO TM Red Antibody Labeling Kit from ThermoFisher Scientific) are commercially available.

[0053] An antibody or antibody fragment used to couple a detectable label to a cell (e.g., a cancer cell) can target any appropriate cell surface marker (e.g., a cell surface polypeptide or portion thereof) on the cell. For example, the antigen binding domain of the antibody or antibody fragment can target an antigen (e.g., a cancer cell specific antigen), such as, but not limited to, CD38, CD138, CS1, BCMA, CD45, a cell adhesion molecule, EpCAM, TROP2, CD3, CD8, PD-1, CD-31, CD33, CD19, FAPa, ASGR1, or HER2.

[0054] In some cases, the antigen binding domain of the antibody or antibody fragment can target a cell-specific antigen, such as CD33, which is a marker of AML. In some cases, the antigen binding domain of the antibody or antibody fragment can target CD31 (a marker of endothelial cells), CD19 (a marker of B-cell lymphoma and leukemia), or HER2 (a marker of breast cancer cells).

[0055] In some cases, live cells can be labeled by adding an appropriate amount of one or more antibodies or antibody fragments (each coupled to a detectable label) to a medium containing cells dissociated from a tissue sample. For example, about 0.005 pg to about 5 pg (e.g., about 0.005 pg to about 0.05 pg, about 0.05 pg to about 0.5 pg, or about 0.5 pg to about 5 pg) of one or more antibody-coupled detectable labels can be added to a well of a plate (e.g., a 96-well plate) containing about 100 to about 100,000 cells (e.g., about 100 to about 500, about 500 to about 1000, about 1000 to about 5000, about 5000 to about 10,000, about 10,000 to about 50,000, or about 50,000 to about 100,000 cells). In some cases, live cells within a MOS can be labeled by adding an appropriate amount of one or more antibodies or antibody fragments (each coupled to a detectable label) to a MOS that has already been formed and is in culture. For example, 0.005 pg to about 5 pg (e.g., about 0.005 pg to about 0.05 pg, about 0.05 pg to about 0.5 pg, or about 0.5 pg to about 5 pg) of antibody-coupled detectable labels can be added to a well of a plate (e.g., a 96-well plate) containing about 50 to about 400 MOS (e.g., about 50 to about 100, about 100 to about 200, about 200 to about 300, or about 300 to about 400 MOS). It should be noted that when two or more labeled antibodies or antibody fragments targeting different cellular antigens are used, each antibody or antibody fragment can target a different antigen. In some cases, the different antigens can be present on different types of cells. Furthermore, each of the two or more antibodies or antibody fragments can be coupled to a different detectable label, such that differentially labeled cells can be distinguished from one another. Cells or MOS can be incubated with the detectable labels for any suitable period of time (e.g., about 30 minutes to about 4 hours). In some cases, the cells or MOS can then be washed to remove excess label. When one or more antibody-coupled labels are applied to dissociated cells, these cells can then be utilized to generate MOS. MOS can be imaged using any suitable method (e.g., flow cytometry or fluorescence microscopy) to detect the label, regardless of whether the cells labeled prior to or after MOS formation are contained within the MOS. In some cases, MOS can be used for other detection, depending on the cells to which the detectable label binds.

[0056] Method for using labelled MOS Cells (e.g., living cells) containing MOS, after being detectably labeled as described herein, can be used in a variety of assays. For example, particular cells (e.g., tumor cells, immune cells, fibroblasts, or endothelial cells) can be labeled before or after integration into the MOS, and the MOS can be imaged to observe where the cells are located, how they interact with other cells within the MOS, etc. In some cases, MOS containing labeled cells can be used to screen various therapeutic agents to predict which therapeutic agents can be effective and safe for use in a patient from which the tissue sample was provided. For example, the MOS can be used for toxicity screening of drugs (e.g., immuno-oncology or “IO” drugs) or other chemical compositions, and based at least in part on the reaction of the cells within the MOS to one or more drugs, it can be determined whether the one or more drug compositions can be effective to treat the patient before the patient receives the drug treatment. This enables a quick screening of a patient before they receive months of treatment that can be ineffective. Methods of using MOS for screening can be performed automatically or manually. A general description of screening methods using MOS can be found in, e.g., paragraphs

[0190] to

[0194] and FIG. 17 of U.S. Patent Publication No. 2021 / 0285054.

[0057] In some cases, the methods and materials provided herein can include using MOS containing labeled living cells to assess phagocytosis of cells in the MOS, and / or to assess the effect of a candidate agent (e.g., a therapeutic agent such as a drug) on phagocytosis of the labeled cells. Phagocytosis is a cellular process of uptake and clearance of particles at least 0.5 pm in diameter, such as apoptotic cells, microorganisms, and foreign bodies. Phagocytosis is primarily performed efficiently by specialized cells, including macrophages, neutrophils, monocytes, dendritic cells, and osteoclasts (referred to as phagocytes), which express receptors that activate signaling pathways that initiate phagocytosis. Other cell types, including fibroblasts, epithelial cells, and endothelial cells, can also accomplish phagocytosis, but less efficiently. See For example, Uribe-Querol and Rosales, Front. Immunol , 11:1066, 2020; doi.org / 10.3389 / fimmu.2020.01066.

[0058] In some cases, PMOS containing labeled cells (e.g., MM MOS) can be used for screening assays to determine the effect of one or more potential therapeutic agents on phagocytosis of labeled cancer cells by phagocytic cells within the PMOS. For example, the composition to be tested can comprise a drug, a dilution of a drug, a formulation of a drug, a combination of drugs (e.g., multiple active ingredients), a dosage form of a drug, a concentration of a drug, etc. In some cases, the drug preparation can be a preparation comprising a mixture of a drug and one or more non-active ingredients. MOS containing labeled cells can also be used for longitudinal high-throughput screening, e.g., to identify additional (e.g., improved) labeling reagents for use in technology and assay development. For example, MOS containing labeled cells can be used for longitudinal studies to identify new functional dyes, to track specific cell populations, and / or to longitudinally capture dynamic responses, such as by tracking the effect of a drug on specific cell populations over time in a complex mixture of cells from the original sample contained within the MOS.

[0059] In some cases, immune cells encapsulated in a MOS or exogenously added to a pre-formed MOS can be labeled using the methods described herein, and the label can be detected / measured to assess the behavior of the immune cells. For example, a labeled anti-CD45 antibody can be used to stain white blood cells, a labeled anti-CD3 antibody can be used to stain T cells, a labeled anti-CD8 antibody can be used to stain cytotoxic T cells, a labeled anti-CD4 antibody can be used to stain helper T cells, a labeled anti-CD56 antibody can be used to stain NK cells, and a labeled anti-CD19 antibody can be used to stain B cells. In some cases, such labeled antibodies can be added to a MOS coated with immune cells and cancer cells. In some cases, such labeled antibodies can be used to stain immune cells to be exogenously added to a MOS culture coated with cancer cells. After staining, the label can be detected and / or measured to track the interaction of the immune cells with the cancer cells in the MOS. In some cases, the label can be detected / measured to assess the response of the cells to a drug treatment (e.g., an IO drug treatment).

[0060] The steps of the assays provided herein can include, for example, placing MOS containing labeled live cells into the wells of a multi-well plate (e.g., a 96-well plate) or a multi-well grid (e.g., a 10,000 micro-well array formed from a 100 x 100 well grid). The MOS (e.g., gel droplets) can be added to the micro-well array, or in some cases onto the micro-well array, and incubated with media. The MOS can be incubated for a period of time ranging from about 1 day to about 14 days. On a selected day (e.g., day 3), one or more candidate phagocytosis stimulating agents can be added to the wells to detect the effect of these agents on phagocytosis of cancer cells within the MOS. The wells can be imaged (e.g., via fluorescence microscopy) to assess the signal output of the pH sensitive label and determine whether phagocytosis has occurred and / or whether the level of phagocytosis has been affected by the one or more candidate agents. In some cases, the signal output can be measured over time in order to assess the rate of phagocytosis (e.g., to determine whether a drug is able to increase the level or rate of phagocytosis of tumor cells by phagocytic cells within a MOS from a cancer patient). An increase in the level or rate of phagocytosis after addition of an agent can indicate that the agent can be useful for treating cancer cells in a mammal from which the biological sample used to generate the MOS was obtained.

[0061] The application will be further described in the following examples, which do not limit the scope of the application described in the claims.

[0062] Example 1 - labelling of MOS containing CRC According to Figure 1A Live MOS were stained according to the method shown. Different antibodies were coupled to different colors of fluorescent dyes at a ratio of about 1 : 100 to about 1 :2000. The antibody mixture was then added to the wells containing MOS generated from a CRC cell line. After incubation on a shaker in an incubator for 1 to 2 hours, the labeled MOS were imaged using an IMAGEXPRESS ® Micro Confocal High Content Imaging System (IXM; MOLECULAR DEVICES ® , San Jose, CA) or other suitable imaging platform. Figure 1BRepresentative images of MOS stained with CD45 (green) and EpCAM (magenta) are shown. The top panel of the figures, from left to right, shows MOS coated with PBMCs, MOS coated with CRC cells, and MOS coated with both PBMCs and CRC cells at a 1:10 ratio. The bottom panel, from left to right, shows MOS coated with both PBMCs and CRC cells at a 1:5 ratio, MOS coated with both PBMCs and CRC cells at a 1:1 ratio, and MOS coated with both PBMCs and CRC cells at a 2:1 ratio. Antibody mixtures (EpCAM and CD45) were added to wells containing different types of MOS, and the wells were imaged using IXM approximately one hour after staining. These studies demonstrate that CD45 can be used to label cells within a PBMC population, and EpCAM can be used to label CRC cells. Figure 1C Additional representative images of antibody-stained MOS are shown. The top panel, from left to right, shows MOS stained with EpCAM and CD45 and coated with CRC and tumor-infiltrating lymphocytes; MOS stained with EpCAM and CD45 and derived from primary lung tissue; and MOS stained with FAPα and EpCAM and coated with mixed cultures of tumor-associated fibroblasts or CRC cells. The bottom panel, from left to right, shows MOS stained with EpCAM and ASGR1 and coated with both CRC and hepatocytes; MOS stained with CD31 and EpCAM and coated with CRC cells, HUVECs, and tumor-associated fibroblasts; and MOS stained with EpCAM and CD45 and coated with primary lung-derived cells. Furthermore, Figure 1D Representative images of MOS generated from two CRC cell lines (501304 and 500040) stained with EpCAM are shown, with CELIGO used in the images. ® An imaging cytometer (RedwoodCity, CA) was used to capture the fluorescence signal from EpCAM staining. These studies further demonstrate that antibodies targeting different cell markers can be used to appropriately stain cells within MOS.

[0063] Further research was conducted to determine how MOS staining might affect MOS growth or drug response. For example... Figures 2A to 2C As shown, CTG measurements revealed no significant growth differences in MOS cells generated from three different CRC cell lines during the 7-day culture period, regardless of whether EpCAM staining was used. Furthermore, when MOS cells generated from two different CRC cell lines were stained with EpCAM at a 1:500 dilution or unstained, and then treated with different doses of 5-FU for 3 days, no differences in drug response were observed. Drug response was measured by CTG. Figure 2D Targeting 501304 cells and Figure 2FFor 500040 cells) or by staining with live / dead cell dyes ( Figure 2E Targeting 501304 cells and Figure 2G Measurements were performed on 500040 cells.

[0064] Additional images of stained live MOS are shown in Figure 3A The study included representative images of live MOS cells containing CRC cells co-coated with fibroblasts, endothelial cells, and PBMCs. The MOS cells were stained with EpCAM and treated for three days with either a solvent or oxaliplatin (1.23 µM or 33.3 µM), followed by staining with calcein AM (CAM) and ethinyl bromide dimer (EtH). Higher oxaliplatin doses resulted in stronger EtH staining and weaker calcein AM staining, indicating greater cytotoxicity. When MOS cells containing only CRC cells or those co-coated with CRC cells, fibroblasts, endothelial cells, and PBMCs were treated with oxaliplatin, CTG measurements revealed differences in the response to treatment between CRC-coated MOS cells and co-coated MOS cells. Figure 3B However, based on the ratio of live cell dye (CAM) to dead cell dye (EtH) in EpCAM-positive cells, the drug response of MOS cells coated only with CRC cells was more similar to that of MOS cells co-coated with CRC cells and non-cancer cells. Figure 3C ).

[0065] Example 2 - use of labelled MM MOS in phagocytosis assays like Figure 4A As shown, PHRODO was generated. TM MM MOS of labeled myeloid cells. Phagocytosis by labeled myeloid cells produces a green fluorescent signal detectable by flow cytometry. Figure 4A As shown, using PHRODO TM To label CD38 antibodies, MOS is generated from MM samples and used with PHRODO. TM The labeled CD38 antibody was cultured together to label CD38 within MOS. + Myeloid cells, and before imaging, PHRODO TM The labeled MOS was cultured with or without PBMC-derived macrophages. Figure 4C Co-culturing with monocyte-derived macrophages (MDM macrophages) was included as a positive control to demonstrate the occurrence of phagocytosis.

[0066] Still using PHRODO TM Yeast polysaccharides were used to label MM cells to provide overall measurements of phagocytosis, such as... Figure 4B As shown. Using PHRODO TM Green is used to label yeast polysaccharides and PHRODOTM Labeled yeast glycans were co-encapsulated with MM samples, and the level of phagocytosis was then assessed by flow cytometry.

[0067] Figure 4C Listed Figure 4A and Figure 4B Readings were obtained using the method shown. Specifically, flow cytometry was performed on day 1 of MOS culture (approximately 24 hours after MOS generation) and day 5 of MOS culture to measure cells labeled with fluorescein isothiocyanate (FITC), CD11b, and CD16. MOS was also imaged on day 5. Figure 5A Representative images are shown. MM MOS with PHRODO TM Labeled yeast polysaccharides (top) or PHRODO TM The labeled anti-CD38 antibody (bottom) was cultured together. Figure 5B This demonstrates the application of flow cytometry to label-free MM MOS (top) and PHRODO. TM Labeled anti-CD38 antibody (intermediate) or PHRODO TM Histograms of labeled yeast glycans (bottom). These studies indicate the detection of CD38 in MM MOS. + Phagocytosis of cells.

[0068] Phagocytes and CD11b were measured. + The percentage of phagocytes, plotted on Figure 6A and Figure 6B As shown in the figure, MOS is generated directly from the freeze-thawed MM sample, and PHRODO is applied before MOS generation. TM Labeled antibodies or PHRODO TM Labeled yeast glycans were added to the culture medium. Data were collected from the following samples 24 hours later: (1) unlabeled MM samples, (2) yeast glycan-labeled MM samples, and (3) PHRODO. TM The labeled MM samples, and (4) MM samples containing macrophages derived from PBMCs. Figure 6A The percentage of green cells among the live cells under each condition was plotted after 24 hours of incubation. (In "Tumor" and "Tumor") + An increased percentage of phagocytes was observed in the MDM sample, indicating that phagocytosis occurred in the MM sample. Figure 6B Drawn Figure 6A CD11b in green cells + The percentage of cells shows that almost all green cells are CD11b. +These studies show that phagocytosis was detected in the MM MOS on day 1, most of the phagocytes were CD11b positive, and the addition of MDM increased the percentage of phagocytosis positive events (less than 5% of total cells were macrophage-derived cells).

[0069] After 5 days of culture, additional studies were performed to determine the percentage of phagocytes (and more specifically, CD11b positive phagocytes), and the results are plotted in Figure 7 MOS were generated directly from frozen MM samples and cultured for 5 days. Anti-CD47 or IgG4 control was added to the media starting on day 0 (the day the MOS was generated). On day 5, PHRODO TM labeled antibody was added to the media and data was collected after 24 hours. The conditions used were: untreated, IgG4 added on day 5 after MOS generation, and 10 pg / ml anti-CD47 added on day 5 after MOS generation. Figure 7 The percentage of green CD11b + cells in live cells after 24 hours of incubation was plotted for each condition. All conditions were in MM samples co-encapsulated with PBMC-derived macrophages. An increase in the percentage of phagocytosis was observed in the “untreated” sample, indicating that phagocytosis occurred, while an increase in the percentage of phagocytosis in the anti-CD47 sample indicates that anti-CD47 induced more phagocytosis. These studies show that phagocytosis was detected in the MM MOS on day 5, the addition of MDM increased the percentage of phagocytosis positive events, and anti-CD47 induced phagocytosis.

[0070] A frozen MM sample from a second patient was used to validate PHRODO TM anti-CD38 detection. All data was generated by flow cytometry. MOS were generated directly from frozen MM samples and PHRODO TM labeled antibody was added to the media either immediately after MOS generation or on day 0 or day 5 of MOS culture. Data was collected after 24 hours. The conditions used were: (1) no PHRODO TM labeled antibody added; (2) MM co-encapsulated with PHRODO TM labeled zymosan; and (3) untreated (no drug added). Figure 8A The percentage of green cells in live cells after 24 hours of incubation was plotted for each condition. An increase in the percentage of green cells was observed in the “untreated” sample, indicating that phagocytosis occurred in this MM sample. Figure 8B A plot of the CD11b + cell gating of the green cells from Figure 8A was plotted, indicating that almost all green cells were CD11b+ Consistent with the above work, these studies showed that phagocytosis was detected at day 1 and day 5 in MM MOS, and most of the phagocytes were CD1 lb positive.

[0071] In further studies, a time course assay was performed in which MM myeloid cells in MOS were labeled with PHRODO™ labeled CD38 antibody and the MOS were treated with anti-CD47 antibody, and then flow cytometry was performed at 2, 4, 6, 8, 16 and 24 hours post-dosing (FIG. 6). Figure 9A This assay showed that the PHRODO™ + percentage of CD1 lb + positive cells was higher in MOS treated with PHRODO™ anti-CD38 than in MOS treated with isotype PHRODO™ staining. Figure 9B EMBODIMENTS

[0072] It is to be understood that while the application has been described in conjunction with the specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the application. Other aspects, advantages and modifications are within the scope of the following claims.​

Claims

1. A method comprising: A population of cells within a microorganoid sphere (MOS) is labeled with a detectable marker, wherein at least a portion of the cells within the population contain the detectable marker.

2. The method of claim 1, wherein the marking comprises adding the detectable marker to an already formed MOS.

3. The method of claim 1, wherein the marking comprises adding the detectable marker to the cell population prior to the formation of the MOS.

4. The method according to any one of claims 1 to 3, wherein the partial cells include cancer cells, immune cells, fibroblasts, endothelial cells, stromal cells, or epithelial cells.

5. The method according to any one of claims 1 to 4, wherein the MOS comprises multiple myeloma cells.

6. The method according to any one of claims 1 to 5, wherein the detectable marker is a fluorescent dye.

7. The method according to any one of claims 1 to 6, wherein the detectable marker is a pH-sensitive marker.

8. The method according to any one of claims 1 to 7, wherein the detectable marker is attached to a cell via an antibody or antibody fragment, wherein the antibody or antibody fragment comprises an antigen-binding region that interacts with an epitope on the cell.

9. The method of claim 8, wherein the detectable marker is attached to the cell via a Fab antibody fragment.

10. The method according to claim 8 or claim 9, wherein the epitope is CD38, CD138, CD33, CS1, BCMA, CD45, cell adhesion molecule, EpCAM, TROP2, CD3, CD8, PD-1, CD-31, CD19, FAPα, ASGR1, or HER2.

11. The method according to any one of claims 1 to 10, wherein the marker is a pH-sensitive marker, and wherein the method further comprises measuring the signal level over time to determine the rate or extent of phagocytosis.

12. The method of any one of claims 1 to 10, wherein the marker is a pH-sensitive marker, and wherein the method further comprises detecting the presence or absence of a signal output from the pH-sensitive detectable marker, wherein the presence of the signal indicates phagocytosis of cells within the population, and wherein the absence of the signal indicates a lack of phagocytosis of cells within the population.

13. A method comprising: A MOS comprising a cell population, wherein at least a portion of the cells within the population contains a pH-sensitive detectable marker. A first signal level from the pH-sensitive detectable marker is measured, wherein the first signal level is directly correlated with the level of phagocytosis within the subset of cells. Contact the MOS with the candidate drug. The level of a second signal from the pH-sensitive detectable marker is measured, wherein the second signal level is directly correlated with the level of phagocytosis within the cells after contact. The signal level of the first measurement is compared with the signal level of the second measurement, and when the signal level of the second measurement increases compared with the signal level of the first measurement, the candidate drug is identified as a phagocytic enhancer for the cell population.

14. The method of claim 13, wherein the subset of cells comprises cancer cells.

15. The method of claim 13 or claim 14, wherein the MOS comprises multiple myeloma cells.

16. The method according to any one of claims 13 to 15, wherein the pH-sensitive marker is a fluorescent dye.

17. The method of any one of claims 13 to 16, wherein the pH-sensitive detectable marker is attached to a cell via an antibody or antibody fragment, wherein the antibody or antibody fragment comprises an antigen-binding region that interacts with an epitope on the cell.

18. The method of claim 17, wherein the pH-sensitive detectable marker is attached to the cell via a Fab antibody fragment.

19. The method of claim 17 or claim 18, wherein the epitope is CD38, CD138, CD33, CS1, BCMA, CD45, cell adhesion molecule, EpCAM, CD3, CD8, PD-1, CD-31, CD19, FAPα, ASGR1, or HER2.

20. The method according to any one of claims 13 to 19, further comprising measuring the signal level over time to determine the rate of phagocytosis.

21. A method for treating mammals, the method comprising: Provide a MOS comprising a cell population from said mammal, wherein at least a portion of the cells within said population contains a pH-sensitive detectable marker. A first signal level from the pH-sensitive detectable marker is measured, wherein the first signal level is directly correlated with the level of phagocytosis within the subset of cells. Contact the MOS with the candidate drug. A second signal level from the pH-sensitive detectable marker is measured, wherein the second signal level is directly correlated with the level of phagocytosis within the cells after contact. Compared to the signal level measured in the first measurement, the signal level measured in the second measurement increased, and The candidate drug was administered to the mammal.

22. The method of claim 21, wherein the plurality of cells include cancer cells.

23. The method of claim 21 or claim 22, wherein the MOS comprises multiple myeloma cells.

24. The method according to any one of claims 21 to 23, wherein the pH-sensitive marker is a fluorescent dye.

25. The method of any one of claims 21 to 24, wherein the pH-sensitive detectable marker is attached to a cell via an antibody or antibody fragment, wherein the antibody or antibody fragment comprises an antigen-binding region that interacts with an epitope on the cell.

26. The method of claim 25, wherein the pH-sensitive detectable marker is attached to the cell via a Fab antibody fragment.

27. The method of claim 25 or claim 26, wherein the epitope is CD38, CD138, CD33, CS1, BCMA, CD45, cell adhesion molecule, EpCAM, TROP2, CD3, CD8, PD-1, CD-31, CD19, FAPα, ASGR1, or HER2.

28. The method according to any one of claims 21 to 27, further comprising measuring the signal level over time to determine the rate of phagocytosis.

29. A method for treating a mammal, the method comprising administering the therapeutic agent to a mammal identified as having cells that experience an increased level of phagocytosis upon contact with a therapeutic agent, wherein the mammal is identified by: Provide a MOS comprising a cell population from said mammal, wherein at least a portion of the cells within said population contains a pH-sensitive detectable marker. A first signal level from the pH-sensitive detectable marker is measured, wherein the first signal level is directly correlated with the level of phagocytosis within the subset of cells. Contact the MOS with the candidate drug. The level of a second signal from the pH-sensitive detectable marker is measured, wherein the second signal level is directly correlated with the level of phagocytosis within the cells after contact. The signal level of the second measurement is determined to be higher than that of the first measurement.

30. The method of claim 29, wherein the plurality of cells include cancer cells.

31. The method of claim 29 or claim 30, wherein the MOS comprises multiple myeloma cells.

32. The method according to any one of claims 29 to 31, wherein the pH-sensitive marker is a fluorescent dye.

33. The method of any one of claims 29 to 32, wherein the pH-sensitive detectable marker is attached to a cell via an antibody or antibody fragment, wherein the antibody or antibody fragment comprises an antigen-binding region that interacts with an epitope on the cell.

34. The method of claim 33, wherein the pH-sensitive detectable marker is attached to the cell via a Fab antibody fragment.

35. The method of claim 33 or claim 34, wherein the epitope is CD38, CD138, CD33, CS1, BCMA, CD45, cell adhesion molecule, EpCAM, TROP2, CD3, CD8, PD-1, CD-31, CD19, FAPα, ASGR1, or HER2.

36. The method according to any one of claims 29 to 35, further comprising measuring the signal level over time to determine the rate of phagocytosis.

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