Labeling and annotation of cells in microorganospheres

Abstract

Provided herein are methods and materials for labeling and annotating live cells in microorganospheres (MOS), as well as methods for evaluating MOS containing labeled live cells, adapted for use in detecting disease, particularly cancer treatment, in mammals.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from U.S. Provisional Patent Application No. 63 / 449,666, filed March 3, 2023. The disclosure of that prior application is considered part of (and is incorporated by reference into) the disclosure of this application.

[0002] This specification relates to methods and materials for labeling cells within three-dimensional (3D) microorganospheres (MOS), as well as methods and materials for using MOS containing labeled live cells. For example, this specification provides methods and materials for labeling specific cell types (e.g., tumor cells, immune cells, fibroblasts, endothelial cells, or epithelial cells) in MOS, methods and materials for determining the effects of various therapeutic agents on labeled cells in MOS, and methods and materials for treating mammals based at least in part on the determined effects. [Background technology]

[0003] Model cell and tissue systems are useful in biological and medical research. Immortalized cell lines can be derived from tissues and cultured in two-dimensional (2D) conditions (e.g., Petri dishes and well plates), but 2D cell lines do not correlate well with individual patient responses to treatment. In contrast, three-dimensional (3D) cell culture models (e.g., spheroids and organoids) can be particularly useful in 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-adherent conditions. Spheroids typically form as free-floating cell aggregates in ultra-low attachment plates and have been shown to maintain properties associated with stem cells better than 2D cell cultures. Organoids are in vitro-derived cell aggregates containing populations of stem cells capable of differentiating into cells of major cell lineages. Organoids typically have a diameter greater than 1 mm and can be cultured for multiple passages. However, because assays ideally should be performed from a single core biopsy within approximately 7-10 days, it typically takes several months to develop and test drug sensitivity in organoids, which may reduce their clinical applicability. Additionally, the number of organoids obtained from a clinically relevant 18-gauge core biopsy is generally insufficient to perform high-throughput drug screening. Summary of the Invention

[0004] The present disclosure is based, at least in part, on the development of methods and materials for staining cells (e.g., live cells) in microorganospheres (MOS), as well as methods for using stained MOS. For example, the present disclosure provides a rapid live cell staining approach that can be used for cell type annotation in MOS and / or phenotypic screening of cells in MOS. The methods provided herein allow for real-time observation of live cells in culture in MOS without the need for cell fixation. In addition, the methods provided herein can be used, for example, to observe the behavior of cells in MOS, track interactions between cells in MOS, and evaluate the durability of cellular responses to drugs or other therapeutic agents. In some cases, the methods and materials provided herein involve the use of patient-derived microorganospheres (PMOS) containing cells derived from a mammal. For example, PMOS (also referred to herein simply as "MOS") can contain cells extracted from small patient biopsies, from excised patient tissue (e.g., tissue from a resected primary tumor or a dysfunctional organ), and / or from established patient-derived models of cancer (PDMCs), including patient-derived xenografts (PDXs) and organoids. In some cases, MOS can contain cells derived from cell lines (e.g., genetically engineered cell lines). Characterization of MOS and labeling of cells (e.g., living cells) therein allows for the investigation of biological processes, including evaluation of various immune system responses to new and existing therapies.

[0005] One example of an assay described herein involving the use of live cell staining in MOS is the detection and measurement of phagocytosis in 3D MOS. As demonstrated herein, for example, phagocytosis of labeled cells (e.g., tumor cells) by phagocytes initially contained within or added to MOS generated from multiple myeloma (MM) patient samples was observed and evaluated. In addition, PMOS generated from MM tissue was labeled to determine the effect of a candidate agent (e.g., a therapeutic agent such as a drug) on ​​phagocytosis.

[0006] In general, one aspect of the present disclosure features a method including labeling a population of cells in a microorganosphere (MOS) with a detectable label, wherein at least a portion of the cells in the population include the detectable label. Labeling can include adding the detectable label to an already formed MOS. Labeling can also include adding the detectable label to the cell population before the MOS is formed. The portion of cells can include cancer cells, immune cells, fibroblasts, endothelial cells, stromal cells, and epithelial cells. The MOS can include 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, where 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, FAPα, ASGR1, or HER2. The label can be a pH-sensitive label, and the method can further include measuring signal levels over time to determine the rate or extent of phagocytosis. The label can be a pH-sensitive label, and the method can further include detecting the presence or absence of a signal output from the detectable pH-sensitive label, wherein the presence of the signal indicates phagocytosis of cells in the population and the absence of the signal indicates a lack of phagocytosis of cells in the population.

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

[0008] In another aspect, this description 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, at least a portion of the cells comprising a detectable pH-sensitive label; measuring a first signal level from the detectable pH-sensitive label, where the first signal level directly correlates to the level of phagocytosis of cells within the portion of the cells; contacting the MOS with a candidate agent; measuring a second signal level from the detectable pH-sensitive label, where the second signal level directly correlates to the level of phagocytosis of cells within the portion of the cells after contact; 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 label can be a fluorescent dye. The detectable pH-sensitive label can be bound to the cells via an antibody or antibody fragment, where the antibody or antibody fragment has an antigen-binding region that interacts with an epitope on the cells. The detectable pH-sensitive label can be bound to cells via a Fab antibody fragment. The epitope can be CD38, CD138, CD33, CS1, BCMA, CD45, cell adhesion molecule, EpCAM, TROP2, CD3, CD8, PD-1, CD-31, CD19, FAPα, ASGR1, or HER2. The method can further include measuring the signal level over time to determine the rate of phagocytosis.

[0009] In yet another aspect, this description features a method for treating a mammal, the method including, or consisting essentially of, administering a therapeutic agent to a mammal identified as having cells that undergo an increased level of phagocytosis when contacted with the therapeutic agent, wherein the mammal is identified by: providing a MOS comprising a population of cells from the mammal, at least a portion of the cells comprising a detectable pH-sensitive label; measuring a first signal level from the detectable pH-sensitive label, the first signal level directly correlating with the level of phagocytosis of cells within the portion of the cells; contacting the MOS with a candidate agent; and measuring a second signal level from the detectable pH-sensitive label, the second signal level directly correlating with the level of phagocytosis of cells within the portion of the 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 include cancer cells. The MOS can include multiple myeloma cells. The pH-sensitive label can be a fluorescent dye. The detectable pH-sensitive label can be bound to cells via an antibody or antibody fragment, where the antibody or antibody fragment has an antigen-binding region that interacts with an epitope on the cells. The detectable pH-sensitive label can be bound to cells via a Fab antibody fragment. The epitope can be CD38, CD138, CD33, CS1, BCMA, CD45, cell adhesion molecule, EpCAM, TROP2, CD3, CD8, PD-1, CD-31, CD19, FAPα, ASGR1, or HER2. The method can further include 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 those skilled in the art to which this invention pertains. Although the present invention can be practiced using methods and materials similar or equivalent to those described herein, 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. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0011] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the detailed description below. Other features, objects, and advantages of the invention will be apparent from the detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0012] Figure 1A-Figure 1D demonstrate the establishment of the live MOS staining technique. [Figure 1A] The workflow of the live MOS staining method is illustrated. [Figure 1B] Representative epithelial cell adhesion molecule (EpCAM) / CD45 staining images of MOS containing different ratios of PBMCs and colorectal cancer (CRC) cells are included. Magenta indicates EpCAM staining, and green indicates CD45 staining. [Figure 1C]Representative images showing live cell staining of MOS containing different cell types or primary tissue-derived cells are included. Top left: MOS containing CRC and tumor-infiltrating lymphocytes stained with EpCAM and CD45; top center: MOS derived from primary lung tissue stained with EpCAM and CD45; top right: mixed-culture MOS containing cancer-associated fibroblasts or CRC cells stained with FAPα and EpCAM; bottom left: MOS containing CRC and hepatocytes stained with EpCAM and ASGR1; bottom center: MOS containing CRC cells, HUVECs, and cancer-associated fibroblasts stained with CD31 and EpCAM; bottom right: MOS containing primary lung cells stained with EpCAM and CD45. [Figure 1D] Representative images showing the persistence of EpCAM staining signal in CRC MOS cultures over 7 days are included. Figures 2A-G demonstrate that EpCAM staining of live MOS does not affect MOS cell proliferation or drug response. [Figure 2A] 1 is a graph plotting the proliferation rate of CRC MOS cultures (501299) with and without EpCAM staining. [Figure 2B] 1 is a graph plotting the proliferation rate of CRC MOS cultures (500040) with and without EpCAM staining. [Figure 2C] 1 is a graph plotting the proliferation rate of CRC MOS cultures (501304) with and without EpCAM staining. [Figure 2D] 1 is a graph plotting survival rates of CRC MOS after treatment with the chemotherapy drug 5-fluorouracil (5-FU) in the presence or absence of EpCAM staining. [Figure 2E] 1 is a graph plotting survival rates of CRC MOS after treatment with the chemotherapy drug 5-fluorouracil (5-FU) in the presence or absence of EpCAM staining. [Figure 2F] 1 is a graph plotting survival rates of CRC MOS after treatment with the chemotherapy drug 5-fluorouracil (5-FU) in the presence or absence of EpCAM staining. [Figure 2G]Figures 3A-C show that viability of CRC MOS after treatment with the chemotherapy drug 5-fluorouracil (5-FU) is plotted against the presence or absence of EpCAM staining. Figures 3A-C show that viable MOS staining reveals tumor cell response to anticancer treatment in CRC-harboring MOS combined with non-cancerous cells (fibroblasts, endothelial cells, and PBMCs). [Figure 3A] Representative images showing live MOS staining of CRC cells encapsulated with fibroblasts, endothelial cells, and PBMCs stained with EpCAM and treated with vehicle or oxaliplatin for 3 days followed by staining with calcein AM (CAM) and ethidium homodimer (EtH) are included. [Figure 3B] 1 is a graph plotting the differential drug response observed for CRC-only conditions and CRC co-encapsulated with non-cancerous cells, as measured by CTG assay. [Figure 3C] Graph plotting drug response of the included group alongside the CRC-only condition, showing that they were similar based on EpCAM staining and viable cell imaging analysis. [Figure 4A]

[0023] Figure 1 is a schematic diagram of the steps of a representative assay in which PHRODO™-labeled CD38 antibody is used to tag MM myeloid cells, resulting in the generation of a signal (in this case, green fluorescence) detectable by flow cytometry when the myeloid cells are phagocytosed by macrophages. The steps of the illustrated method include (1) labeling the CD38 antibody with PHRODO™, (2) adding the PHRODO™-labeled CD38 antibody to an MM cell culture to tag CD38+ myeloid cells, and (3) mixing the PHRODO™-tagged MM cells with or without PBMC-derived macrophages to generate MOS. Co-culture with monocyte-derived macrophages (MDMs) can be used to confirm that phagocytosis is occurring. [Figure 4B]Schematic diagram of steps in a representative assay to assess phagocytosis using zymosan. Zymosan is a glucan consisting of repeating glucose units linked by β-1,3-glycosidic bonds and can be taken up by macrophages. Labeling zymosan allows for measurement of total phagocytosis, which serves as a control. The steps of the illustrated method include (1) labeling zymosan with PHRODO™ green, (2) co-encapsulating PHRODO™-labeled zymosan and MM samples in MOS, and (3) determining whether phagocytosis is occurring using flow cytometry; if phagocytosis is occurring, the PHRODO™-labeled zymosan will turn green and can be detected by flow cytometry. [Figure 4C] 4A and 4B are charts showing assay results that can be performed using the methods illustrated in FIG. 4A and FIG. 4B. In particular, flow cytometry can be 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 (a marker for myeloid cells), and CD16 (a marker for natural killer cells, neutrophils, monocytes, macrophages, and certain T cells). In addition, on day 5, MOS can be imaged. [Figure 5A] Representative images of MM MOS cultured with zymosan (top) or PHRODO™-labeled anti-CD38 antibody (bottom) as illustrated in Figures 4A and 4B, respectively. [Figure 5B]Figures 6A and 6B show histograms generated by flow cytometry of MM MOS for no labeling (top), PHRODO™-labeled anti-CD38 antibody (middle), or PHRODO™-labeled zymosan (bottom). Figures 6A and 6B are plots of the percentage of phagocytes (Figure 6A) or CD11b+ phagocytes (Figure 6B) in the indicated samples. PHRODO™-based phagocytosis assays were performed as described for Figures 4A and 4B. Data were collected 24 hours later for (1) unlabeled MM samples (no PHRODO™-labeled anti-CD38 antibody), (2) PHRODO™-labeled MM samples, (3) zymosan-labeled MM samples encapsulated with PHRODO™-labeled zymosan, and (4) MM samples with MDM (PBMC-derived macrophages encapsulated with MM samples). [Figure 6A] 1 is a graph plotting the percentage of green cells among live cells for each condition after 24 hours of incubation. [Figure 6B] FIG. 6B is a graph plotting the gating of CD11b+ cells from the green cells in FIG. 6A. [Figure 7] Figures 8A and 8B plot the percentage of PHRODO™+CD11b+ cells after 4 hours of treatment with IgG4 or anti-CD47, or left untreated for 4 hours. Figures 8A and 8B plot the percentage of PHRODO™+ cells (Figure 8A) and the percentage of phagocytosis (Figure 8B) for the indicated samples. All data were generated by flow cytometry from a second frozen MM sample. Frozen-thawed MM samples were tagged with PHRODO™-labeled antibodies to generate MOS. Phagocytosis assays were performed on days 0 and 5 of MOS culture. Data were collected after 24 hours. The following conditions were used: (1) No PHRODO™: no PHRODO™-labeled antibody added; (2) Zymosan: MM samples encapsulated with PHRODO™-labeled zymosan (data collected on day 1 only); (3) Untreated: vehicle control. [Figure 8A]1 is a graph plotting the percentage of green cells among live cells for each condition after 24 hours of incubation. [Figure 8B] FIG. 8B is a graph plotting the gating of CD11b+ cells from the green cells in FIG. 8A. [Figure 9A] FIG. 1 is a schematic diagram of the steps in a representative time course assay in which MM myeloid cells in MOS were tagged with a PHRODO™-labeled CD38 antibody, and MOS were treated with an anti-CD47 antibody followed by flow cytometry at the indicated time points. [Figure 9B] Graph plotting the results of a time course assay, plotting the percentage of CD11b+ cells that were PHRODO™+ in MOS stained with PHRODO™ anti-CD38, isotype PHRODO™ stained, or unstained. DETAILED DESCRIPTION OF THE INVENTION

[0013] Method for generating MOS The present specification provides methods and materials for staining live cells in MOS, as well as methods for using live-stained MOS. For example, the live-cell staining methods described herein can be used to annotate cell types within MOS, perform phenotypic screening of cells within MOS, observe live cells within MOS in real time, track cell behavior within MOS, observe interactions between cells within MOS, and / or evaluate cellular responses to drugs or other therapeutic agents. In some cases, the methods and materials provided herein include the use of PMOS containing mammalian-derived cells, such as cells extracted from patient biopsies, cells obtained from excised patient tissue (e.g., tissue from a resected primary tumor or a dysfunctional organ), and / or cells from established PDMCs, PDXs, and organoids. Labeling cells in MOS enables the investigation of biological processes, including the evaluation of various immune system responses to new and existing therapies.

[0014] Generally, the methods and materials provided herein relate to an antibody-based staining approach for labeling live cells in MOS droplets, enabling MOS fluorescent antibody cell tracking (MOS-FACT). As described herein, antibodies conjugated with fluorescent labels can efficiently diffuse into MOS droplets and stain surface markers on target cells. Thus, by using fluorescently conjugated primary antibodies targeted to specific cell surface markers, different cell types (e.g., tumor cells, immune cells, fibroblasts, endothelial cells, cholangiocytes, hepatocytes, etc.) in MOS droplets can be specifically labeled and annotated, generally within 1–4 h after staining, without the need for cell fixation or killing. Live MOS staining signals can also be reflected and informative for histological diagnosis of primary tissues. As described herein, antibody fluorescent signals can persist for up to 7 days, and antibody staining of cells in MOS does not affect the drug response of MOS treated with chemotherapeutic drugs or tyrosine kinase inhibitors (TKIs). When combined with imaging (e.g., high-content confocal imaging), the approach described herein enables longitudinal tracking and phenotypic screening of cells in MOS cultures. Because MOS are not attached to a culture surface but rather suspended in the culture medium, they can be pipetted and easily transferred to other vessels, containers, or devices. Without being bound to a specific mechanism of action, this property, combined with the stability of the fluorescent label over time and the fact that labeled cells remain viable within the MOS, means that analysis of labeled MOS populations is not limited to imaging but can rather include downstream analyses such as flow cytometry, single-cell RNA sequencing, and high-throughput analysis.

[0015] Methods for producing MOS can use techniques from, for example, cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, examples of which are described elsewhere.See, for example, Molecular Cloning A Laboratory Manual (1989), 2nd Ed., Sambrook, Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Patent Application No. 4,683,195; DNA Cloning, Volumes I and II, Glover, ed., 1985; Oligonucleotide Synthesis, Gait, ed., 1984; Nucleic Acid Hybridization, Hames and Higgins, eds., 1984; Transcription and Translation, Hames and Higgins, 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, Methods In Enzymology (Academic Press, Inc., NY); 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., NY, Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987, and Handbook of Experimental See Immunology, Volumes I-IV, Weir and Blackwell, eds., 1986.

[0016] MOSs can be formed from primary cells that are normal (e.g., normal organ tissue) or from tumor tissue. For example, in some cases, PMOSs can be formed from cancerous tumor biopsy tissue, allowing for customized treatments that can be selected using the specific tumor tissue examined. Methods for generating MOSs allow for the formation of hundreds, thousands, or even tens of thousands (e.g., 500, 750, 1000, 2000, 5000, 10,000, or more) of MOSs from a single tissue biopsy within hours of the biopsy being removed from the patient. Generally, dissociated primary cells obtained from a patient biopsy can be combined with a fluid matrix material, such as a basement membrane matrix (e.g., MATRIGEL®, a solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, available from Sigma), to form the MOS. The resulting MOSs can have a predefined range of size (e.g., diameters of about 10 μm to about 700 μm, and any subrange therebetween) and initial number of primary cells (e.g., about 1 to about 1000, particularly smaller numbers of cells, such as about 1 to about 200). Cell number and / or diameter can be controlled within a range of, for example, ±5%, ±10%, ±15%, ±20%, ±25%, ±30%, etc. The MOSs can have very high viability (e.g., greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%) and can be stable for use and testing within a very short period of time, generally within the first 1 to 14 days after formation (e.g., within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days). This can enable the rapid testing of potentially vast numbers of patient-specific, biologically relevant PMOS, potentially saving critical time in the development and deployment of patient therapies, such as cancer treatment plans. PMOS can rapidly form 3D cellular structures that replicate and represent the biopsied tissue environment, such as the 3D tumor microenvironment. In some instances, 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, nidogen, etc.) that can mimic the original tissue (e.g., tumor) environment.

[0017] In some cases, MOS used in the methods provided herein can contain not only cancer cells but also one or more other types of cells (e.g., immune cells, including fibroblasts, endothelial cells, and / or cells capable of phagocytosis, such as macrophages, neutrophils, monocytes, dendritic cells, and / or osteoclasts). MOS can be derived, for example, from a bone marrow tissue sample from a mammal with multiple myeloma (MM) (referred to herein as "MM MOS"), an acute myeloid leukemia (AML) bone marrow sample, a solid tumor sample (e.g., a biopsy from a colorectal tumor, breast tumor, kidney tumor, liver tumor, lung tumor, ovarian tumor, prostate tumor), or any other suitable type of sample. In some cases, MOS can contain 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 WO 2024 / 040145, the entire contents of which are incorporated herein by reference. In some cases, the MOS may not contain cells from a cell line.

[0018] MOS that can be labeled and used in the methods provided herein are typically spheroids formed from dissociated cells (e.g., dissociated primary cells distributed within a biopsy). MOS can have a diameter of about 50 μm to about 500 μm (e.g., about 50 μm to about 400 μm, about 50 μm to about 300 μm, about 50 μm to about 250 μm, about 100 μm to about 500 μm, about 100 μm to about 250 μm, or about 50 μm to about 200 μm). The MOS can initially contain about 1 to about 1000 dissociated primary cells (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, or about 50 to about 150, or about 25 to about 100 primary cells) distributed within the substrate.

[0019] Despite their small size (often about 50 to about 250 μm) and low cell density (e.g., often about 50 to about 200 cells per MOS), MOS can be used immediately or cultured for relatively short periods (e.g., 14 days or less, 10 days or less, 7 days or less, or 5 days or less). Furthermore, cells within MOS can survive while maintaining most or even all of the characteristics of the tissue from which they were extracted. The viability of cells within MOS is typically high, and MOS can be cultured for days or even weeks, through multiple passages during which cells can divide, cluster, and form structures similar to the parent tissue. Furthermore, in some cases, cells from the dissociated tissue that generated the MOS can form morphological structures even within the smallest MOS. While the presence of such structures is not necessary for the usefulness of these MOS in some applications (e.g., when used before substantial structural reorganization has occurred), in some cases, such structures can be particularly useful.

[0020] Any suitable method can be used to generate a MOS, including those described herein. To facilitate description of the presently disclosed subject matter, the following definitions are provided herein.

[0021] The term "unpolymerized mixture" is used herein to refer to a composition containing biologically relevant materials, including a dissociated tissue sample and a first fluid matrix material. The fluid matrix material is typically a material that can be polymerized to form a support or support network for the dissociated tissue and / or cells dispersed therein. Upon polymerization, the polymerized material can form a hydrogel and can form and / or contain proteins in addition to cells, forming a biocompatible medium. Biocompatible media suitable for use in the methods disclosed herein can be formed from any biocompatible material that is a liquid, such as a gel, semi-solid, or low-viscosity liquid, at room temperature (e.g., 25°C) and can be used as a three-dimensional substrate for cells, tissues, proteins, and other biomaterials of interest. Exemplary materials that can be used to form a biocompatible medium in accordance with the presently disclosed subject matter include, but are not limited to, polymers and hydrogels containing collagen, fibrin, chitosan, MATRIGEL® (BD Biosciences, San Jose, Calif.), polyethylene glycol (PEG), dextran (e.g., chemically or photocrosslinkable dextran), and the like, as well as electrospun biomaterials, synthetic materials, or biomaterial-synthetic blends. In some cases, the biocompatible medium can be a hydrogel.

[0022] The term "hydrogel" is used herein to refer to a two- or multi-component gel having a three-dimensional network of polymer chains in which water acts as a dispersion medium, filling the spaces between the polymer chains. Hydrogels used in accordance with the presently disclosed subject matter can generally be selected for a particular application based on the intended use of the structure, taking into account the parameters used to form the MOS and the effect of the selected hydrogel on the behavior and activity of biomaterials (e.g., cells) incorporated into the biological suspension that will be disposed within the structure. Exemplary hydrogels for use with the presently disclosed subject matter include, but are not limited to, polymeric materials such as alginate, collagen (including type I collagen and type VI collagen), elastin, keratin, fibronectin, proteoglycans, glycoproteins, polylactides, polyethylene glycols, polycaprolactones, polycolides, polydioxanones, polyacrylates, polyurethanes, polysulfones, peptide sequences, proteins and derivatives, oligopeptides, gelatin, elastin, fibrin, laminin, polymethacrylates, polyacetates, polyesters, polyamides, polycarbonates, polyanhydrides, polyamino acids, carbohydrates, polysaccharides and modified polysaccharides, and derivatives and copolymers thereof, and inorganic materials such as glasses, including bioactive glasses, ceramics, silica, alumina, calcite, hydroxyapatite, calcium phosphate, bone, and any combination thereof.

[0023] Further with regard to the hydrogel used to generate the MOS, in some cases the hydrogel can contain a material selected from the group consisting of agarose, alginate, type I collagen, polyoxyethylene-polyoxypropylene block copolymer (e.g., PLURONIC® F127; BASF Corporation, Mount Olive, NJ), silicone, polysaccharide, polyethylene glycol, polyurethane. In some cases, the hydrogel can be made from alginate.

[0024] In some cases, the MOS can contain biologically relevant materials. The phrase "biologically relevant materials" refers to materials that can be included in a biocompatible medium, as defined herein, and subsequently interact with and / or affect a biological system. For example, the biologically relevant materials can be magnetic beads (e.g., beads that are themselves magnetic or beads that contain a material that responds 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 the isolation and purification of the MOS). As another example, in some cases, the biologically relevant materials can include cells in addition to a dissociated tissue sample (e.g., a biopsy). In the unpolymerized mixture, the dissociated tissue sample and additional biologically relevant materials can be present as a homogeneous mixture or as a dispersed mixture (e.g., in one half or another portion of the MOS, e.g., only within the core or outer region of the formed MOS). In some cases, the additional biologically relevant materials in the unpolymerized material can be suspended with the dissociated tissue sample in the suspension (e.g., before the droplets that form the MOS are polymerized). In some cases, biologically relevant materials that may optionally be included with a dissociated tissue sample (e.g., a biopsy) may include preadipocytes, mesenchymal stem cells (MSCs), endothelial progenitor cells, T cells, B cells, mast cells, and / or adipose tissue macrophages, MDMs, and small blood vessels or microvascular fragments found within the stromal vascular fraction.

[0025] As used herein, the term "passage" refers to the average number of doublings of cells within a MM MOS. While traditional passage number refers to the transfer or subculture of cells from one culture vessel to another, cells within a MOS can be stably maintained within the same MOS and continue to grow and divide. Thus, as used herein, passage number typically refers to the average number of doublings by dissociated cells from biopsy tissue within the MOS. Population doublings are the approximate number of doublings a cell population has undergone since isolation (e.g., since the formation of the MOS from freshly dissociated biopsy tissue). Generally, the MOS described herein can be cultured for a short 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, less than 3 passages) for the growth (e.g., doubling) of some or all of the cells within the MOS.

[0026] In some cases, the tissue sample (and cells contained therein) used to generate MOS can be obtained from a mammal. The tissue sample can be obtained from any suitable mammal (e.g., a human or other mammal with MM, such as a dog, cat, mouse, rat, or rabbit), and is typically obtained by biopsy. The tissue can be derived from, but is not limited to, a biopsy, a surgical specimen, an aspiration, a drainage, or a cell-containing fluid. Typically, MOS contains multiple cell types resident in the original tissue, although it is also possible to form MOS from a specific cell type. Cells can be obtained directly from a subject without the intermediate step of subculture, or the cells can first undergo an intermediate culture step to generate a primary culture. Any suitable method for harvesting cells from biological tissue and / or cell-containing fluid can be used. For example, suitable techniques used to obtain cells from biological tissue include those described in Mahesparan, Acta Neuropathol (1999) 97:231-239.

[0027] Cells in a tissue sample are typically first dissociated or separated from one another before forming MOS. Cell dissociation can be accomplished by any suitable method. For example, cells can be treated mechanically and / or chemically, such as by treatment with an enzyme. "Mechanical" dissociation typically involves using a machine, such as a scalpel, scissors, or homogenizer, to sever the connections between attached cells. "Enzymatic" dissociation involves treating cells with one or more enzymes (e.g., using collagenase, dispase, DNAse, and / or hyaluronidase) to sever the connections between attached cells. 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.

[0028] Dissociated tissue can be treated to remove dead / dying cells and / or cell debris.Removal of such dead and / or dying cells can be accomplished by any suitable means, such as bead and / or antibody methods.For example, phosphatidylserine can be redistributed from the inner leaflet to the outer leaflet of the plasma membrane in apoptotic or dead cells, so the use of Annexin V-biotin binding, followed by the binding of biotin to streptavidin magnetic beads, can allow the separation of apoptotic cells from live cells.Removal of cell debris can be achieved by techniques such as filtration.

[0029] The dissociated cells can be suspended in a carrier material before being combined with the fluid matrix material. Alternatively, the fluid matrix material can be referred to as a carrier material. In some cases, the carrier material can be a material with a viscosity level that retards the settling of cells in the cell suspension prior to polymerization and formation of the MOS. In such cases, the carrier material can have a viscosity sufficient to allow the dissociated biopsy tissue cells to remain suspended in suspension until polymerization. The viscosity required to achieve this can be optimized by monitoring the settling rate at various viscosities and selecting a viscosity that provides an appropriate settling rate for the expected time delay between filling the device with the cell suspension and forming the MOS by polymerizing the droplets of unpolymerized material containing the cells. In some cases, the unpolymerized material can be flowed or agitated with the device (e.g., when a lower viscosity material is used) to maintain the cells in suspension and / or distribute them as desired.

[0030] As mentioned above, in some cases, the unpolymerized mixture containing the dissociated tissue sample and the fluid matrix material can contain one or more other components, such as one or more biologically relevant materials. Biologically relevant materials that can be contained in the unpolymerized mixture include, but are not limited to, one or more of extracellular matrix proteins (e.g., fibronectin), drugs (e.g., small molecules), peptides, antibodies (e.g., for regulating cell survival, proliferation, or differentiation), and / or inhibitors of specific cellular functions. Such biologically relevant materials may be used, for example, to increase cell viability by reducing cell death and / or activation of cell growth / replication, or to otherwise mimic an in vivo environment. The biologically relevant materials can include or mimic one or more of the following components: serum, interleukins, chemokines, growth factors, glucose, physiological salts, amino acids, and hormones. The biologically relevant materials can, in some cases, be supplemented with one or more drugs 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 may 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® includes bioactive polymers important for cell viability, proliferation, development, and migration. In some cases, the matrix material can be a gel including type I collagen, such as type I collagen obtained from rat tail. The gel can be a pure type I collagen gel or a gel containing type I collagen in addition to other components, such as other extracellular matrix proteins. In some cases, the fluid matrix can be a synthetic gel that does not occur in nature.Examples of synthetic gels include gels derived from polyethylene glycol (PEG), polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), or polyethylene oxide (PEO). In some cases, the matrix material can include one or more natural polymers, such as alginate, agarose, hyaluronic acid, collagen, gelatin, fibrin, and elastin, or can include one or more synthetic polymers, such as PEG and polyacrylamide. Both organic and inorganic synthetic polymers can be used.

[0031] The MOS is generally expressed as a fixed or known number of cells or cell concentration (e.g., cells / mL or cells / mm) within the MOS. 3) containing dissociated biopsy tissue (e.g., cells). In some cases, the number of cells initially included in a MOS can range from one cell up to several hundred cells. Particularly for use in some assays (e.g., drug toxicity assays), it may be beneficial to generate MOSs containing about 1 to about 75 cells or about 1 to about 50 cells (i.e., a relatively small number of cells). The number of cells per MOS can be set or selected by the technician generating the MOS. In some cases, the device for generating MOSs can include one or more controls for setting the number of cells from the primary tissue to be included in each MOS. The number of cells can be selected or set based on how the MOS is intended to be used. For example, MOSs with very low numbers of cells (e.g., one cell per MOS or 1 to 5 cells per MOS) may be particularly suitable for studying clonal diversity (e.g., tumor heterogeneity). In some cases, each MOS is grown from a single cell, in which case it can be observed which clones are drug resistant, and these specific MOSs can then be examined (e.g., by genome sequencing) to determine the genomic (mutational) diversity associated with the particular clone. Because these MOSs typically grow rapidly, 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) may be particularly useful for rapid drug testing, including toxicity testing. Because MOSs may contain different lineages, potentially including epithelial (e.g., cancer) cells and mesenchymal (e.g., stromal, immune, or vascular) cells, larger 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) may be particularly suitable for mimicking the tissue composition of each MOS. Note that in the case of MM MOSs, including a relatively large number of cells per MOS (e.g., about 50 to about 200 cells per MOS) can provide each MOS with different cell types that can act to maintain MM cells.

[0032] In some cases, MOSs can be generated by forming droplets of an unpolymerized mixture (in some cases, a cooled mixture) of a dissociated tissue sample and a fluid matrix material in an immiscible material, such as a fluid hydrophobic material (e.g., oil). For example, MOSs may be formed by combining a stream of unpolymerized material with one or more streams of immiscible materials to form droplets. The density of cells present in the droplets can be determined by dilution of the dissociated material (e.g., cells) in the unpolymerized material. The size of the MOS can be correlated to the size of the droplets formed. Generally, MOSs are spherical structures with stable geometric shapes.

[0033] The MOS can have any suitable size that can accommodate the number of cells to be contained. For example, the size of the MOA can be relatively small, having a diameter of about 20 μm to about 500 μm (e.g., an average of about 50 μm or about 100 μm, or about 100 μm to about 200 μm). In some cases, the size of the MOS can be about 300 μm, in which case about 10 to about 50 cells (e.g., about 10 to about 30 cells) can be contained in each MOS. The number and size of cells can be varied and / or controlled. In some cases, the number and / or size of cells in the MOS can be set by one or more controllers 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).

[0034] During culture, cells from dissociated biopsy tissue in MOS can aggregate, cluster, or aggregate within the MOS. The cell aggregates can be highly organized and may form a defined morphology, or may be clumps of cells clustered or attached together. The tissue may reflect the original tissue. In some cases, MOS can contain a single cell type (homotypic), while in other cases, MOS can contain two or more cell types (heterotypic).

[0035] Additional description of methods and apparatus for fabricating MOS can be found in U.S. Patent Application Publication No. 2021-0285054. See, for example, paragraphs

[0165] to

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

[0036] In some cases, MOS can be generated from MM tissue samples. Using MM tissue to replicate tumor cells is generally challenging because MM cells are typically difficult to handle and culturing them in 3D culture can be challenging. Furthermore, without bone marrow stromal cells, MM cells cannot survive. However, as described in Examples 1-3 of U.S. Provisional Patent Application No. 63 / 404,472 (incorporated herein by reference in its entirety), MOS can be generated from MM biopsies, which was surprising given the challenges described above. The technology described in that patent and herein can provide MM cells with a tumor microenvironment, thereby enabling them to grow. Cells within the MM immune microenvironment (e.g., macrophages) can also be captured in MOS, expanding the range of drugs that can be screened for MOS. This range includes, but is not limited to, agents that can modulate phagocytosis by macrophages and other cells within MM MOS (e.g., drugs that can induce phagocytosis).

[0037] Thus, in some cases, the tissue (e.g., biopsy) sample (e.g., dissociated tissue) used to generate MOS may be derived from bone marrow obtained from a mammal with MM. In some cases, the tissue used in MOS may include cells of the immune system, such as T lymphocytes, B lymphocytes, polymorphonuclear leukocytes, macrophages, and dendritic cells. The cells may include stem cells, progenitor cells, or somatic cells. The tissue and resulting dissociated cells may be primary cells obtained from a patient biopsy (e.g., by needle biopsy). The dissociated cells may be incorporated into MM MOS and may include cells commonly found in bone marrow. In this regard, exemplary cells that may be incorporated into MM MOS include plasma cells (normal and cancerous), stromal cells, hematopoietic stem cells, monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, T cells, B cells, and natural killer (NK) cells. The tissue may be dissociated using any suitable technique.

[0038] As described in U.S. Provisional Patent Application No. 63 / 404,472, in some cases, preparation and use of MOS generated from MM bone marrow biopsies can be facilitated by (1) using about 50 to about 150 cells per MOS (e.g., about 50 to about 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 binding plates. Without being bound by any particular mechanism, using a relatively large number of cells can increase the likelihood that the full complement of cells from the bone marrow microenvironment (e.g., stromal and immune cells) will be included in the MM MOS, which may be important from a drug screening perspective. Additionally, without being bound by any particular mechanism, using higher concentrations of MATRIGEL® can help retain MM cells within the MOS, and using low- or ultra-low-binding plates can reduce the likelihood that the MOS will adhere to the bottom of the culture well. Additionally, in some cases, the methods disclosed herein for generating 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®, regardless of whether the MATRIGEL® is added to the MOS mixture or the culture medium. These strategies may also help retain MM cells within the MOS and / or prevent the MOS from adhering to the bottom of the culture well. With this in mind, MM MOS can have a cell population of about 50 to about 150 cells per MOS, with MM plasma cells, stromal cells, and immune cells included in the MOS cell population.Generally, MOS can be generated quickly from a biopsy and generally retain similar cell ratios to the patient biopsy. 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).

[0039] Once formed, MOS can be cryopreserved and / or cultured. Generally, cultured MOS can be maintained in suspension either statically (e.g., in a well, vial, or other suitable container) or dynamically (e.g., by rolling or agitation). MOS can be cultured using any suitable technique. Exemplary techniques can be found in, but are not limited to, Freshnev, 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. Pat. Nos. 5,516,681 and 5,559,022, all of which are incorporated herein by reference in their entirety.

[0040] MOS labeling Cells in MOS (e.g., tumor cells or non-tumor cells) or cells in a biological sample containing dissociated cells to be used to generate MOS can be stained with a detectable label targeting a selected cell surface marker. Any suitable detectable label (e.g., a fluorescent label) can be attached to dissociated cells or cells in MOS in culture. In some cases, a detectable pH-sensitive label can be used. An exemplary pH-sensitive label is PHRODO™ (Thermo Fisher), a fluorescent dye that functions as an intracellular pH indicator and can be used to measure the pH of live cells. In particular, PHRODO™ is weakly fluorescent at neutral pH but becomes more fluorescent as the pH decreases (e.g., during phagocytosis). As an alternative to fluorescent dye-conjugated antibodies, it should be noted that any dye capable of staining unfixed cells can be used in the methods described herein, including fluorescent dyes for live or dead cells (e.g., fixed cells), dyes for staining the cytoskeleton or intracellular organelles, and / or dyes for staining lipid droplets.

[0041] Any suitable means can be used to attach the detectable label to specific cells (e.g., cancer cells, immune cells, fibroblasts, endothelial cells, etc.) in a dissociated biological sample (e.g., a dissociated tumor sample) or to cells in MOS in culture. For example, the detectable label can be attached to an antibody or antibody fragment (e.g., V HThe antibody or antibody fragment may be conjugated to an H fragment, also referred to as a NANOBODY®, and the conjugate may bind to cells in the dissociated tissue sample or cells in the already formed MOS 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 IgG antibodies, including but not limited to, IgG or IgM types such as IgG1, IgG2, IgG3, IgG4, IgM1, and IgM2), as well as antibody-like molecules containing engineered subdomains of antibodies or naturally occurring antibody variants. Antibody-like molecules have been developed either from natural sources such as camelids (Muydermans et al. (2001) Rev. Mol. Biotechnol. 74:277-302) or through in vitro display of libraries from humans, camelids, or other species (Hot et al. (2003) Trends Biotechnol. 21:484-90). H Only domain or V L In certain embodiments, the polypeptide structure of the antigen-binding protein can be based on an antibody, including but not limited to a minibody, a synthetic antibody (sometimes referred to as an "antibody mimic"), a human antibody, an antibody fusion (sometimes referred to as an "antibody conjugate"), and a fragment thereof, respectively.

[0042] 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. In this configuration, 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, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although usually with lower affinity than the entire binding site. "Fab fragments" also contain the constant domain of the light chain and the first constant domain of the heavy chain (C H 1) and a heavy chain C fragment containing one or more cysteines from the antibody hinge region. H An "F(ab')2 fragment" differs from an "Fab' fragment" by having additional residues at the carboxy terminus of one domain. An "F(ab')2 fragment" originally is produced as a pair of "Fab' fragments" which have hinge cysteines between them. Methods for preparing such antibody fragments can include, for example, using papain or pepsin digestion.

[0043] Nanobodies are composed of the antigen-binding V of the IgG heavy chain. H Nanobodies are recombinantly expressed VH domains. Nanobodies typically have a molecular weight of approximately 12-15 kDa and are among the smallest naturally occurring antigen-binding fragments. These isolated VH domains are H The H domain retains the ability to bind to antigen.

[0044] For further discussion of antibody fragments, see, e.g., Bates and Power, Antibodies, 8:28, 2019. In addition, kits that can be used to attach labels (e.g., PHRODO™) to antibodies are commercially available (e.g., Thermo Fisher Scientific's PHRODO™ Deep Red Antibody Labeling Kit).

[0045] The antibody or antibody fragment used to bind the detectable label to a cell (e.g., a cancer cell) can target any suitable cell surface marker on the cell (e.g., a cell surface polypeptide or portion thereof). 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, FAPα, ASGR1, or HER2.

[0046] In some cases, the antigen-binding domain of the antibody or antibody fragment can target a cell-specific antigen such as CD33, a marker for AML, hi some cases, the antigen-binding domain of the antibody or antibody fragment can target CD31 (a marker for endothelial cells), CD19 (a marker for B-cell lymphoma and leukemia), or HER2 (a marker for breast cancer cells).

[0047] In some cases, live cells can be labeled by adding an appropriate amount of one or more antibodies or antibody fragments, each conjugated to a detectable label, to a medium containing dissociated cells from a tissue sample. For example, about 0.005 μg to about 5 μg (e.g., about 0.005 μg to about 0.05 μg, about 0.05 μg to about 0.5 μg, or about 0.5 μg to about 5 μg) of a detectable marker conjugated to one or more antibodies 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 MOS can be labeled by adding an appropriate amount of one or more antibodies or antibody fragments, each conjugated to a detectable label, to MOS that have already formed and are in culture. For example, 0.005 μg to about 5 μg (e.g., about 0.005 μg to about 0.05 μg, about 0.05 μg to about 0.5 μg, or about 0.5 μg to about 5 μg) of a detectable marker conjugated to an antibody 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). Note that when using two or more labeled antibodies or labeled antibody fragments targeting different cellular antigens, each antibody or antibody fragment can target a different antigen. In some cases, the different antigens may be present on different types of cells. Furthermore, each of the two or more antibodies or antibody fragments can be conjugated to a different detectable marker, thereby allowing differentially labeled cells to be distinguished from one another. The cells or MOS can be incubated with the detectable marker(s) for any suitable time (e.g., from about 30 minutes to about 4 hours). In some cases, the cells or MOS can then be washed to remove excess label. Once one or more antibody-conjugated labels have been applied to dissociated cells, the cells can be used to generate MOS.Whether the MOS contains labeled cells before or after MOS formation, it can then be imaged to detect the label using any suitable means (e.g., flow cytometry or fluorescence microscopy). In some cases, the MOS can be used in other assays, depending on the cells to which the detectable label is bound.

[0048] Methods for using labeled MOS MOS containing detectably labeled cells (e.g., live cells) as described herein can be used in a variety of assays. For example, specific cells (e.g., tumor cells, immune cells, fibroblasts, endothelial cells) can be labeled before or after their incorporation into the MOS, and the MOS can be imaged to observe where the cells reside, how they interact with other cells within the MOS, and so on. In some cases, MOS containing labeled cells can be used to screen various therapeutic agents to predict which therapeutic agent(s) may be effectively and safely used in a patient from whom a tissue sample has been obtained. For example, MOS can be used for toxicity screening of drugs (e.g., immuno-oncology or "IO" drugs) or other chemical compositions, and it can be determined whether one or more drug compositions will effectively treat a patient before the patient receives drug therapy, based at least in part on the response of cells within the MOS to the one or more drugs. This can enable very rapid patient screening before the patient is exposed to months of treatment that may otherwise be ineffective. Methods using MOS for screening can be performed automatically or manually. A general description of screening methods using MOS can be found in U.S. Patent Application Publication No. 2021 / 0285054, for example, paragraphs

[0190] to

[0194] and Figure 17.

[0049] In some cases, the methods and materials provided herein can include using MOS containing labeled live cells to assess phagocytosis of cells in the MOS and / or assess the effect of a candidate agent (e.g., a therapeutic agent such as a drug) on ​​phagocytosis of labeled cells. Phagocytosis is a cellular process for ingesting and removing particles at least 0.5 μm in diameter, such as apoptotic cells, microorganisms, and foreign bodies. Phagocytosis is most efficiently carried out by specialized cells (called phagocytes), including macrophages, neutrophils, monocytes, dendritic cells, and osteoclasts; these cells express receptors that activate signaling pathways that result in phagocytosis. Other types of cells, including fibroblasts, epithelial cells, and endothelial cells, can also accomplish phagocytosis, but with less efficiency. See, e.g., Uribe-Querol and Rosales, Front. Immunol., 11:1066, 2020; doi.org / 10.3389 / fimmu.2020.01066.

[0050] In some cases, PMOS (e.g., MM MOS) containing labeled cells can be used in screening assays to determine the effect of one or more potential therapeutic agents on the phagocytosis of labeled cancer cells by phagocytes within the PMOS. For example, the composition to be tested can contain a drug, a drug diluent, a drug formulation, a combination of drugs (e.g., multiple active ingredients), a drug form, a drug concentration, etc. In some cases, a drug formulation can be a formulation containing a mixture of a drug and one or more inactive ingredients. MOS containing labeled cells can also be used in longitudinal high-throughput screening, e.g., to identify additional (e.g., improved) labeling reagents for technology and assay development. For example, MOS containing labeled cells can be used in longitudinal studies to identify new functional dyes, track specific cell populations, and / or capture dynamic responses longitudinally, such as by tracking drug effects on specific cell populations over time in complex mixtures of cells from a primary sample contained within the MOS.

[0051] In some cases, immune cells contained in MOS or exogenously added to preformed MOS can be labeled using the methods described herein, and the label can be detected / measured to assess immune cell behavior. 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 MOS containing immune cells and cancer cells. In some cases, such labeled antibodies can be used to stain immune cells that are exogenously added to a culture of MOS containing cancer cells. After staining, the label can be detected and / or measured to track interactions between immune cells and cancer cells in the MOS. In some cases, the label can be detected / measured to assess the response of cells to drug treatment (e.g., treatment with an IO drug).

[0052] Performing the assays provided herein can include, for example, placing MOS containing labeled, live cells into wells of a multiwell plate (e.g., a 96-well plate) or a multiwell grid (e.g., a 10,000-microwell array formed in a 100 x 100-well grid). MOS (e.g., gel droplets) can be applied to, or in some cases, onto, a plurality of microwell arrays and incubated with culture medium. MOS can be cultured for about 1 to about 14 days. On a selected day (e.g., day 3), one or more candidate phagocytosis-stimulating agents can be administered to the wells to examine the effect of the agent(s) on phagocytosis of cancer cells within the MOS. The wells can be imaged (e.g., via fluorescence microscopy) to assess signal output from the pH-sensitive label to determine whether phagocytosis has occurred and / or whether the level of phagocytosis is affected by one or more of the candidate agents. In some cases, signal output can be measured over time to assess the rate of phagocytosis (e.g., to determine whether a drug can increase the level or rate of phagocytosis of tumor cells by phagocytes in the MOS of a cancer patient. An increase in the level or rate of phagocytosis after addition of a drug can indicate that the drug is useful as a therapeutic agent against cancer cells in the mammal from which the biological sample used to generate the MOS was obtained.

[0053] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0054] Example 1 - Labeling of MOS containing CRC Live MOS were stained according to the method illustrated in Figure 1A. Different antibodies were conjugated with different colored fluorescent dyes at ratios ranging from approximately 1:100 to approximately 1:2000. This antibody mixture was then added to wells containing MOS generated from CRC cell lines. After 1–2 hours of incubation on a rocker in an incubator, the labeled MOS were imaged using the IMAGEXPRESS® Micro Confocal High-Content Imaging System (IXM; MOLECULAR DEVICES®, San Jose, CA) or another suitable imaging platform. Representative images of MOS stained with CD45 (green) and EpCAM (magenta) are shown in Figure 1B. From left to right, the upper panels of the figure show MOS containing PBMCs, MOS containing CRC cells, and MOS containing both PBMCs and CRC cells at a 1:10 ratio. The lower panels, from left to right, show MOS containing both PBMCs and CRC cells at a 1:5 ratio, MOS containing both PBMCs and CRC cells at a 1:1 ratio, and MOS containing both PBMCs and CRC cells at a 2:1 ratio. An antibody mixture (EpCAM and CD45) was added to wells containing different types of MOS, stained for approximately 1 hour, and then imaged using IXM. These studies demonstrated that cells within the PBMC population were labeled with CD45, while CRC cells were labeled with EpCAM. Additional representative images of antibody-stained MOS are shown in Figure 1C. The upper panels, from left to right, show MOS containing CRC and tumor-infiltrating lymphocytes stained with EpCAM and CD45, MOS derived from primary lung tissue stained with EpCAM and CD45, and mixed-culture MOS containing cancer-associated fibroblasts or CRC cells stained with FAPα and EpCAM. The bottom panels, from left to right, show MOS containing both CRC and hepatocytes stained with EpCAM and ASGR1, MOS containing CRC cells, HUVECs, and cancer-associated fibroblasts stained with CD31 and EpCAM, and MOS containing primary lung-derived cells stained with EpCAM and CD45.Additionally, Figure 1D shows representative images of MOS generated from two CRC cell lines (501304 and 500040) stained with EpCAM. The fluorescent signal from EpCAM staining was captured using a CELIGO® Imaging Cytometer (Redwood City, CA). These studies further demonstrated that cells within the MOS were appropriately stained using differently labeled antibodies targeting different cell markers.

[0055] Further studies were conducted to investigate how MOS staining affects their proliferation or drug response. As shown in Figures 2A-C, no significant differences in proliferation were observed between MOS generated from three different CRC cell lines stained with or without EpCAM after 7 days of culture, as measured by the CTG assay. Furthermore, no differences in drug response were observed when MOS generated from two different CRC cell lines were stained with or without EpCAM at a 1:500 dilution and then treated with different doses of 5-FU for 3 days. Drug response was measured by the CTG assay (Figure 2D for 501304 cells, Figure 2F for 500040 cells) or by live / dead cell dye staining (Figure 2E for 501304 cells, Figure 2G for 500040 cells).

[0056] Additional images of stained live MOS are shown in Figure 3A, including representative images of live MOS containing CRC cells co-encapsulated with fibroblasts, endothelial cells, and PBMCs. MOS were stained with EpCAM and treated with vehicle or oxaliplatin (1.23 μM or 33.3 μM) for 3 days, followed by staining with calcein AM (CAM) and ethidium homodimer (EtH). Higher doses of oxaliplatin resulted in increased EtH staining and decreased calcein AM staining, resulting in greater killing. When MOS containing only CRC cells or MOS containing CRC cells, fibroblasts, endothelial cells, and PBMCs were treated with oxaliplatin, differences in response to treatment were observed between MOS encapsulated with CRC-only MOS and MOS encapsulated with CRC-only MOS, as determined by CTG assay (Figure 3B). However, based on the live cell dye (CAM):dead cell dye (EtH) ratio from EpCAM-positive cells, the drug response of CRC-only MOS versus MOS containing both CRC and non-cancer cells was much more similar (Figure 3C).

[0057] Example 2 - Use of Labeled MM MOS in Phagocytosis Assays MM MOS containing PHRODO™-labeled bone marrow cells were generated as shown in Figure 4A. Phagocytosis of the labeled bone marrow cells results in a green fluorescent signal that can be detected by flow cytometry. As shown in Figure 4A, CD38 antibody was labeled with PHRODO™, and MOS was generated from the MM sample and cultured with PHRODO™-labeled CD38 antibody to increase CD38 expression within the MOS. + Bone marrow cells were tagged and imaged after culturing PHRODO™-tagged MOS with or without PBMC-derived macrophages (Figure 4C). As a positive control, co-culture with monocyte-derived macrophages (MDM macrophages) was performed to demonstrate phagocytosis.

[0058] MM cells were also labeled with PHRODO™-zymosan to measure total phagocytosis, as shown in Figure 4B. Zymosan was labeled with PHRODO™ Green, and the PHRODO™-labeled zymosan was embedded with MM samples, after which the level of phagocytosis was assessed by flow cytometry.

[0059] The results of measurements from the methods shown in Figures 4A and 4B are listed in Figure 4C. 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 were also imaged on day 5. Representative images are shown in Figure 5A. MM MOS were cultured with PHRODO™-labeled zymosan (top) or PHRODO™-labeled anti-CD38 antibody (bottom). Figure 5B shows histograms generated by flow cytometry of MM MOS with no label (top), PHRODO™-labeled anti-CD38 antibody (middle), or PHRODO™-labeled zymosan (bottom). These studies demonstrated the presence of CD38 in MM MOS. + It was demonstrated that phagocytosis of cells was detected.

[0060] Phagocytes and CD11b + The percentage of phagocytic cells was measured and plotted in the graphs shown in Figures 6A and 6B. MOS was generated directly from frozen-thawed MM samples, and PHRODO™-labeled antibodies or PHRODO™-labeled zymosan was added to the culture medium immediately prior to MOS generation. Data were collected after 24 hours for (1) unlabeled MM samples, (2) zymosan-labeled MM samples, (3) PHRODO™-labeled MM samples, and (4) MM samples with PBMC-derived macrophages. The percentage of green cells among live cells for each condition after 24 hours of incubation is plotted in Figure 6A. The percentage of green cells among live cells for each condition after 24 hours of incubation is plotted in Figure 6A. The percentage of green cells among live cells for "tumor" and "tumor" samples was also plotted in Figure 6B. + The increased percentage of phagocytic cells observed in the "MDM" sample indicated that phagocytosis had occurred in this MM sample. +The percentage of cells is plotted in Figure 6B. Almost all of the green cells are CD11b + These studies demonstrated that phagocytosis was detectable in MM MOS on day 1, that the majority of phagocytes were CD11b positive, and that the addition of MDM increased the proportion of phagocytosis-positive events (less than 5% of total cells were macrophage-derived).

[0061] Additional studies to determine the percentage of phagocytes, more specifically CD11b-positive phagocytes, were performed after 5 days of culture, and the results are plotted in Figure 7. MOS were generated directly from frozen-thawed MM samples and cultured for 5 days. Anti-CD47 control or IgG4 control was added to the culture medium starting on day 0 (the day of MOS generation). PHRODO™-labeled antibody was added to the culture medium on day 5, and data were collected 24 hours later. The conditions used were untreated, IgG4 added on day 5 after MOS generation, and 10 μg / mL anti-CD47 added on day 5 after MOS generation. Green CD11b in live cells per condition after 24 hours of incubation. + The percentages of cells are plotted in Figure 7. All conditions were in MM samples encapsulated with PBMC-derived macrophages. The increased percentage of phagocytosis seen in the "untreated" samples demonstrated that phagocytosis occurred, while the increased percentage of phagocytosis in the anti-CD47 samples suggested that anti-CD47 induced more phagocytosis. These studies demonstrated that phagocytosis was detected in MM MOS at day 5, that the addition of MDM increased the percentage of phagocytosis-positive events, and that anti-CD47 induced phagocytosis.

[0062] The PHRODO™ anti-CD38 assay was validated using frozen MM samples from a second patient. All data were generated by flow cytometry. MOS were generated directly from frozen-thawed MM samples, and PHRODO™-labeled antibodies were added to the culture medium either immediately after MOS generation (day 0) or on day 5 of MOS culture. Data were collected after 24 hours. The following conditions were used: (1) no PHRODO™-labeled antibodies, (2) MM encapsulated with PHRODO™-labeled zymosan, and (3) untreated (no drug added). The percentage of green cells among viable cells in each condition after 24 hours of incubation is plotted in Figure 8A. The increase in the percentage of green cells observed in the "untreated" sample demonstrated that phagocytosis had occurred in this MM sample. Figure 8B shows the CD11b phagocytosis of the green cells in Figure 8A. + This graph plots cell gating, and almost all of the green cells are CD11b + Consistent with the work described above, these studies detected phagocytosis in day 1 and day 5 MM MOS and demonstrated that the majority of phagocytes were CD11b positive.

[0063] In further studies, we performed a time course assay in which we tagged MM myeloid cells in MOS with a PHRODO™-labeled CD38 antibody, treated MOS with an anti-CD47 antibody, and then performed flow cytometry at 2, 4, 6, 8, 16, and 24 hours post-treatment (Figure 9A). This assay revealed a higher percentage of CD11b MOS stained with PHRODO™ anti-CD38 than with isotype PHRODO™ stained MOS. + The cells are PHRODO™ + This was demonstrated (Figure 9B).

[0064] Other embodiments While the present invention has been described in conjunction with this Detailed Description, it should be understood that the above description is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method comprising: Labeling a population of cells within a microorganosphere (MOS) with a detectable label, wherein at least a portion of the cells within the population comprise the detectable label. A method comprising:

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

3. The method of claim 1 , wherein the labeling comprises adding the detectable label to the population of cells before the MOS is formed.

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

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

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

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

8. 8. The method of any one of claims 1 to 7, wherein the detectable label is bound to the cell via an antibody or antibody fragment, the antibody or antibody fragment comprising an antigen-binding region that interacts with an epitope on the cell.

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

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

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

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

13. 1. A method comprising: providing a MOS comprising a population of cells, wherein at least a portion of the cells within the population comprise a detectable pH-sensitive label; measuring a first signal level from the detectable pH-sensitive label, wherein the first signal level directly correlates to a level of phagocytosis of the cells within the portion of the cells; contacting the MOS with a candidate agent; measuring a second signal level from the detectable pH-sensitive label, wherein the second signal level directly correlates 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 if the second measured signal level is increased compared to the first measured signal level, identifying the candidate agent as an agent that enhances phagocytosis of the population of cells; A method comprising:

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

15. 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 label is a fluorescent dye.

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

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

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

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

21. 1. A method for treating a mammal, said method comprising: providing a MOS comprising a population of cells from the mammal, wherein at least a portion of the cells in the population comprise a detectable pH-sensitive label; measuring a first signal level from the detectable pH-sensitive label, wherein the first signal level directly correlates to a level of phagocytosis of the cells within the portion of the cells; contacting the MOS with a candidate agent; measuring a second signal level from the detectable pH-sensitive label, wherein the second signal level directly correlates 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; administering the candidate agent to the mammal; A method comprising:

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

23. 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 label is a fluorescent dye.

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

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

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

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

29. 1. A method for treating a mammal, the method comprising administering a therapeutic agent to a mammal identified as having cells that undergo an increased level of phagocytosis when contacted with the therapeutic agent, wherein the mammal: providing a MOS comprising a population of cells from the mammal, wherein at least a portion of the cells in the population comprise a detectable pH-sensitive label; measuring a first signal level from the detectable pH-sensitive label, wherein the first signal level directly correlates to a level of phagocytosis of the cells within the portion of the cells; contacting the MOS with a candidate agent; measuring a second signal level from the detectable pH-sensitive label, wherein the second signal level directly correlates to a level of phagocytosis of cells within the portion of cells after the contacting; and determining that the second measured signal level is increased compared to the first measured signal level; Identified by the method.

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

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

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

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

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

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

36. 36. The method of 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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