High-throughput 3D droplet microfluidics system for analyzing tumor spheroids and immune cell interaction

Abstract

Microfluidics technology incorporating 3D gel droplets is provided for analysis of tumor cells and their interactions with immune cells, such as natural killer cells. The gel droplets include the presence of extracellular matrix components, which alter cell functions including viability and mobility. The system allows cells co-incorporated into 3D gel droplets to be observed in real-time using fluorescence microscopy, with fluorescence-based sorting of droplets for recovery of individual cells based on their function. High-quality RNA can be extracted from the sorted droplets for transcription profiling.

Description

[0001] TITLE

[0002] High-Throughput 3D Droplet Microfluidics System for Analyzing Tumor Spheroids and Immune Cell Interaction

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the priority of U.S. Provisional Application No. 63 / 693,469 filed 11 September 2024 and entitled “High-Throughput 3D Droplet Microfluidics System for Analyzing Tumor Spheroids and Immune Cell Interaction”, the whole of which is hereby incorporated by reference.

[0005] BACKGROUND

[0006] Droplet microfluidics technology enables high-throughput encapsulation of individual cancer cells for analyzing cell-cell interactions and heterogeneity at the single cell level1 -3. Individual cells within a tumor can interact differently with their extracellular matrix (ECM) environment and surrounding cells, influencing tumor growth, invasion, and metastasis45. This heterogeneity means that some cells may resist treatment while others may not, leading to varied therapeutic responses. Investigating at the single-cell resolution unravels these complex interactions. Singlecell droplet microfluidics platforms have mostly been implemented for blood cancers. Recent advancements in droplet microfluidics use non-adherent lymphoma and leukemia cell lines that are encapsulated in aqueous droplets to investigate tumor- immune interactions and cancer heterogeneity6-9. However, aqueous droplet microfluidics models are not suitable for studying solid tumors because they lack an extracellular matrix (ECM), which is critical for facilitating cancer cell motility as part of tumor escape strategies10. Therefore, there is a significant technological need for droplet microfluidics platforms specifically designed for solid tumors.

[0007] One of the most significant challenges in studying solid tumors, especially when exploring immunotherapy, is the cellular and molecular heterogeneity present within solid tumors5 11. Heterogeneity exists with how cancer cells interact with its ECM as well as with surrounding immune cells11’12. ECM also facilitates cell-cell interactions between tumor cells and immune cells13’15. Such interactions are vital for understanding the immune response to solid tumor cells and developing effective immunotherapies. The ECM is also essential in solid tumor development, particularly through biochemical crosslinks that promote cell mobility, migration and invasion16. MATRIGEL, a preparation derived from solubilized basement membrane and other ECM components secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, has been used to mimic the in vivo extracellular matrix environment of cells in vitro7~20’23. The use of MATRIGEL poses challenges due to its temperature-dependent polymerization.

[0008] SUMMARY

[0009] The present technology provides the use of a 3D gel droplet microfluidics model, which overcomes challenges associated with studying solid tumors. The technology allows for high-throughput formation and real-time analysis of tumor and immune cell interactions in the presence of extracellular matrix (ECM) or components thereof. The present integration of ECM components, such as MATRIGEL, into a droplet-based microfluidics system enables the formation of uniformly sized, physiologically relevant 3D gel droplets, which can be observed in real-time using fluorescence microscopy. Integration of cell viability fluorescent labels allows capturing of detailed images of tumor-immune cell interactions, pinpointing the exact moment of immune cell-mediated tumor cell death.

[0010] Additionally, a Fluorescence-Activated Droplet Sorting (FADS) system can be integrated within the 3D microfluidics platform to isolate and characterize subpopulations of immune cells based on their functional activities. In the examples described herein, enhanced prostate cancer cell (PC3) viability and NK cell mobility were achieved in a 3D gel droplet configuration. In addition, immune cell cytotoxicity was decreased in the presence of ECM. Droplet sorting based on the functional activity of immune cells was achieved with both an NK cell line as well as primary NK cells. Further, high-quality RNA from the sorted droplets was obtained, which can be used to perform downstream sequencing for transcription profiling of the sorted immune cells.

[0011] The technology also can be summarized with the following list of features. 1 . A method of analyzing tumor cell interactions with immune cells, the method comprising

[0012] (a) providing (i) a microfluidic device comprising a droplet generation module, a droplet docking array, and a fluorescence microscope, (ii) a suspension of single tumor cells, (iii) a suspension of immune cells, and (iv) a temperature-sensitive polymer;

[0013] (b) co-encapsulating one or more of the single tumor cells with one or more of the immune cells in an aqueous droplet comprising the temperature-sensitive polymer using the droplet generation module to form a gel droplet containing one or more single tumor cells and one or more immune cells;

[0014] (c) transferring the gel droplet to the droplet docking array;

[0015] (d) optionally repeating steps (b) and (c) to fill or partially fill the droplet docking array; and

[0016] (e) analyzing the docked gel droplets using the fluorescence microscope.

[0017] 2. The method of feature 1 , wherein the temperature-sensitive polymer comprises an extracellular matrix extract comprising solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, and wherein steps (b), (c), and optionally (d) are performed at about 4°C and the method further comprises step (eO), prior to step (e), of warming the device to about 37°C, whereby the extracellular matrix extract forms a gel.

[0018] 3. The method of feature 1 or feature 2, wherein the step of analyzing comprises recording images using the fluorescence microscope of one or more gel droplets over time and determining for each gel droplet one or more of cell viability, cell mobility, or cell-cell interactions.

[0019] 4. The method of any of the preceding features, further comprising

[0020] (f) harvesting one or more gel droplets from the docking array; and

[0021] (g) sorting the one or more gel droplets using a fluorescence-activated droplet sorting device based on a fluorescence signal from one or more cells in each gel droplet and collecting the sorted gel droplets in distinct containers.

[0022] 5. The method of feature 4, further comprising

[0023] (h) extracting and lysing cells from the sorted gel droplets; and

[0024] (j) subjecting the lysed cells to one or more of transcriptom ic analysis, genomic analysis, or proteomic analysis. 6. The method of any of the preceding features, wherein the single tumor cells are derived from a solid tumor or from a cell culture derived from a solid tumor.

[0025] 7. The method of any of the preceding features, wherein the immune cells are NK cells or killer T cells.

[0026] 8. The method of any of the preceding features where either or both of the single tumor cells or the immune cells are derived from a subject having or suspected of having cancer.

[0027] 9. The method of any of the preceding features, wherein step (e) comprises exposing one or more of the gel droplets to one or more test substances, and said analysis includes evaluating an ability of the one or more test substances to affect the tumor cells or the immune cells.

[0028] 10. The method of feature 9, wherein the test substance is an antitumor agent.

[0029] 11 . The method of any of the preceding features, wherein the single tumor cells and / or the immune cells are obtained from two or more individuals of a population.

[0030] 12. A method of encapsulating a biomolecule, cells, or a therapeutic agent, the method comprising

[0031] (a) providing (i) a microfluidic device comprising a droplet generation module and a droplet docking array, (ii) a suspension of the biomolecule, cells, or therapeutic agent, and (iii) a temperature-sensitive polymer that polymerizes when warmed from below ambient temperature to ambient temperature or above;

[0032] (b) co-encapsulating the biomolecule, cells, or therapeutic agent with the depolymerized temperature-sensitive polymer in a plurality of aqueous droplets using the droplet generation module;

[0033] (c) transferring the droplets to the droplet docking array while maintaining the temperature-sensitive polymer in a depolymerized state; and

[0034] (d) raising the temperature of the docking array, whereby the temperaturesensitive polymer polymerizes, thereby forming a plurality of polymer microspheres comprising the biomolecule, cells, or therapeutic agent.

[0035] 13. The method of feature 12, wherein the temperature-sensitive polymer comprises solubilized basement membrane matrix secreted by Engelbreth-Holm- Swarm (EHS) mouse sarcoma cells.

[0036] 14. The method of feature 12 or feature 13, wherein the microsphere- encapsulated biopolymer, cells, or therapeutic agent is rendered more stable as a result of said method. 15. A polymer-encapsulated biomolecule, cell, or therapeutic agent produced by a method comprising the method of any of the preceding features.

[0037] 16. A kit for use in performing the method of any of the preceding features, the kit comprising: a microfluidic device, chip, or module for encapsulating two or more components in aqueous droplets in an oil stream; a microfluidic device, chip, or module for sorting aqueous droplets based on fluorescence of droplet contents; instructions for performing said method; and optionally, one or more temperature-sensitive polymers, extracellular matrix extracts, cells, or reagents.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figs. 1A-1 B show microfluidic device design and workflow schematics. Fig. 1A represents a microfluidics device for droplet formation with merging of two different cell types and docking of droplets in a docking array. The device has separate inlets for a stream of single tumor cells (such as PC3 cells) and a stream of immune cells (such as NK cells), as well as for an extracellular matrix extract (such as a MATRIGEL solution). Fig. 1 B (upper part) depicts how tumor and immune cells are coencapsulated in aqueous droplets in an oil stream, droplets being a liquid phase containing the two types of cells and the extracellular matrix extract. The docking site array (lower part of Fig. 1 B) is filled droplets containing gelled extracellular matrix-and the two cell types, and imaging analysis including cytotoxicity / viability tests, cell-cell interactions, cell mobility were performed. Droplets were then subjected to imaging in a fluorescence microscope and fluorescent-activated droplet sorting and RNA extraction.

[0040] Figs. 2A-2B show that PC3 cell viability was enhanced in 3D MATRIGEL spheroids. Fig. 2A. Representative PC3 cell images by fluorescence microscopy merged with differential interference contrast (DIC) are shown in aqueous droplets (top) and MATRIGEL droplets (i.e., spheroids) (bottom) at 6 hour intervals over 24 hours. Fig. 2B. PC3 viability in aqueous droplets (black) versus gel droplets (red) over 24 hours. n=75 droplets analyzed in each droplet type.

[0041] Figs. 3A-3D demonstrate enhanced NK cell mobility in 3D gel droplets. Fig. 3A shows NK92 cell viability in aqueous and MATRIGEL droplets. Fig. 3B shows data distribution for NK cell mobility in MATRIGEL droplets and aqueous droplets over 24 hours. Fig. 3C shows the average total distance NK cells moved in MATRIGEL vs. in aqueous medium droplets every 6 hours. Fig. 3D shows representative images of NK cell location in the aqueous and MATRIGEL droplets over 24 hours.

[0042] Figs. 4A-4C show enhanced PC3 cell viability in 3D gel droplets coencapsulated with an NK92 cell. Fig. 4A shows PC3 cell viability in droplets containing 1 :1 co-encapsulated tumor and effector cells for both aqueous droplets and 3D MATRIGEL droplets (n=75 droplets). Fig. 4B shows NK92 cell viability at 1 :1 ratio of tumor and effector cells co-encapsulated in aqueous and 3D MATRIGEL droplets (n=75 droplets). Fig. 4C shows representative images (fluorescence + DIC) of cellcell interactions between PC3 and NK92 cells over 24 hours.

[0043] Figs. 5A-5E show high quality RNA extraction from sorted droplets based on immune cell function. Fig. 5A shows a schematic illustration of the sorter system set up including amplifier, PMT (photomultiplier tube), and 488 nm laser. Fig. 5B shows the sorter device design (PDMS microfluidics device) featuring droplet inlet and a fluorescence-activated droplet sorter with 3D electrodes. Fig. 5C shows a high sensitivity RNA Tapestation peak graph showing 18S and 28S RNA peaks and size distribution. Fig. 5D show the average RNA integrity number (RIN) of NK92 (n=4) and primary NK (n=3) cells. Fig. 5E shows representative images of electrophoretic bands for total RNA for a set of NK92 sorted samples.

[0044] DETAILED DESCRIPTION

[0045] The present technology overcomes the challenges associated with studying solid tumors through the use of a 3D gel droplet microfluidics model. The technology incorporates components of the extracellular matrix (ECM) into gel droplets which serve as 3D models for solid tumors, and enable the study and analysis of immune cell-tumor cell interactions at the level of pairs of single cells or small groups of cells in a more natural environment than previously possible using microfluidics technology.

[0046] While a variety of different extracts of biological material or mixtures of ECM components can be used in the present technology, one such gellable ECM composition is MATRIGEL (see Passaniti, et. al.23, also U.S. Patent No. 4829000, which are hereby incorporated by reference). Although MATRIGEL has been widely used in 3D culture systems, its application in droplet microfluidics combined with immune cells as herein is novel. By integrating MATRIGEL or other gellable ECM compositions into a droplet-mode microfluidics device, it is possible to generate uniformly sized 3D biomatrix droplets with high-throughput, that are physiologically suitable tumor-mimetic environments. In addition, the microfluidics platform is compatible with fluorescence microscopy imaging systems that capture real-time imaging of cell dynamics and mobility within the 3D gel droplets. Using various fluorescent labels, such as a live / dead label, tumor cell-immune cell interactions can be evaluated frame by frame over 24 hours, allowing the exact moment an immune cell kills a tumor cell to be captured. Thus, temperature-control of polymerization within microfluidic droplets must be provided. The present technology also allows the viability of tumor cells to be maintained and facilitates detailed studies of cell-cell interactions and mobility over time.

[0047] ECM extracts such as MATRIGEL can be made by extracting solubilized ECM proteins from mammalian tissue, such as tumors that express high levels of ECM proteins. For example, MATRIGEL is produced using an extract from the Engelbreth- Holm-Swarm (EHS) mouse sarcoma; similar materials also can be prepared using human or other mammalian placenta. Insoluble ECM components can be extracted, for example, using denaturing agents or chaotropic agents such as guanidine or urea. The denaturant or chaotrope can be removed, such as by dialysis or centrifugation, leaving a solution that remains soluble in the cold (e.g., 4°C) but gels at physiological temperatures for mammalian tissues (e.g., 37°C). Molecular components of MATRIGEL, for example, include the basement membrane proteins laminin, collagen IV, and entactin, as well as proteoglycans (e.g., heparan sulfate) and growth factors (e.g., TGF-beta, EGF, and FGF). Additional proteins or molecular components of extracellular matrix (e.g., collagens, elastin, fibronectin, glycoproteins, glycosaminoglycans, proteoglycans) can be present or added to an ECM extract used to form a 3D gel droplet in a microfluidic platform.

[0048] An alternative to using materials derived from mammalian tissue, such as MATRIGEL, is to use one or more temperature dependent polymers that exist as a gel at a temperature suitable for promoting cell function, such as about 37°C, but exist in water-soluble form at lower temperatures, such as about 4°C, so as to allow their manipulation in a microfluidic device in soluble form to prepare aqueous droplets in an oil stream and docking of the formed droplets in a docking array, prior to gelation by increasing the temperature while the droplets are docked in the array. Examples of such polymers include examples include poly(N-isopropylacrylamide) (PNIPAM), poly(vinylmethylether), and polyethylene oxide) (PEO); however, any polymers with suitable temperature dependence of polymerization can be used in the present technology. Suitable polymers will have a lower critical solution temperature (LCST) in a range suitable for use in a droplet-based microfluidic system, such as between about 4°C and 37°C. In such polymers, polymerization is typically entropy driven, so that an increase in temperature strengthens hydrophobic interactions that promote polymerization above the LCST. For use in the present technology, a single temperature sensitive polymer may be used, or a combination of two or more such polymers may be used. Optionally, a temperature-sensitive polymer or mixture of such polymers can be supplemented with one or more biomolecules or other agents that promote cell viability and mobility, or exert effects for analysis such as stimulation of immune cell activity (e.g., killing of target cells) or anti-tumor effects. For example, one or more components of extracellular matrix or basement membranes, such as proteins or glycoproteins or fragments thereof (including laminin, collagen IV, entactin, other collagens, elastin, fibronectin), peptides, antibodies or aptamers, peptidoglycans or glycosaminoglycans (e.g., heparan sulfate), or growth factors (e.g., TGF-beta, EGF, and FGF) can be included in the polymer solution.

[0049] Another significant component of the present technology is the use of a Fluorescence-Activated Droplet Sorting (FADS) system. A FADS system provides the ability to sort individual cells, particularly immune cells, based on their functional phenotypes as assessed using fluorescent labels. This also permits a “function-to- omics” analysis, allowing cell function to be linked to transcriptom ics, proteomics, genomics, or epigenetics at the single cell level, or based on analysis of a pool of sorted cells having similar fluorescent labeling. Thus, subpopulations of immune cells and tumor cells can be isolated based on their functional characteristics, such as the ability of immune cells to kill tumor cells or the susceptibility of tumor cells to immune- mediated killing. A FADS systems suitable for use in sorting droplets containing immune cells is described in WO 2025 / 050133 A1 , which is hereby incorporated by reference.

[0050] In the examples described below, enhanced viability is demonstrated for prostate cancer cells within the gel droplet matrix. In addition, natural killer (NK) cells exhibited higher mobility and interaction with the prostate cells in the 3D gel droplet configuration. Successful functional sorting was achieved based on the functional activity of both NK92 cell line and primary NK cells. Most notably, high-quality RNA was extracted from the sorted droplets and downstream sequencing was performed for transcription profiling of the sorted immune cells. The present technology overcomes limitations of aqueous droplet microfluidics models and better replicates solid tumor ECM, thereby enabling detailed functional analysis of tumor-immune interactions. These advantages offer new options for studying solid tumors, understanding cancer cell heterogeneity, and developing targeted therapies for the treatment of solid tumors.

[0051] Enhanced cell viability of PC3 cells in 3D gel droplets with ECM compared to aqueous droplets without ECM

[0052] The microfluidic device used to co-encapsulate prostate cancer cells (PC3 cells), NK cells, and MATRIGEL into 3D gel droplets is shown in Fig. 1 A. To observe cancer cell behavior and test cell viability in the MATRIGEL droplets compared to aqueous droplets, PC3 cells were incorporated into either droplets containing only RPMI culture medium or similar droplets also containing MATRIGEL in a gel state. The cells were treated with Calcein AM as a live cell marker. PC3 cells with the green fluorescent dye were imaged in the docking array using an inverted fluorescence microscope every 30 minutes over 24 hours to evaluate cell viability. It was observed that the presence of ECM gel helped the 3D droplets maintain their overall size during the 24-hour incubation period, without the shrinkage due to evaporation seen in the aqueous droplets (Fig. 2A). PC3 cell viability was enhanced in MATRIGEL droplets. In aqueous droplets, cell viability decreased to about 50% after 9 hours, while in MATRIGEL droplets it took about 24 hours for cell viability to decline to the same level (Fig. 2B). The findings suggest that incorporating cancer cells in ECM-containing gel droplets maintains Calcein AM fluorescence (i.e., viability) and a healthy cell morphology much longer than the use of aqueous droplets alone, due to the presence of ECM components.

[0053] ECM-enriched 3D droplets improved NK92 cell viability and mobility

[0054] Cell mobility is key in cancer migration and metastasis, as it allows tumor cells to invade surrounding tissues10. In addition, mobility is essential in immune cells for locating and attacking cancer cells as a part of the body’s defense against tumor growth and spread21. Thus, in addition to evaluating cell viability of tumor cells in the MATRIGEL droplets, the cell mobility of NK92 natural killer cells also was evaluated in the 3D gel droplet environment. Similar to the results with PC3 cells, NK92 cell viability in the MATRIGEL droplets was enhanced. In aqueous droplets lacking ECM, cell viability declined to 50% after 10 hours, while in MATRIGEL droplets, more than 70% of the NK92 cells were still alive at 24 hours (Fig. 3A). Images from the 24-hour incubation demonstrated enhanced NK92 cell mobility in the 3D configuration compared to the aqueous droplets (Fig. 3D). The distance travelled by NK92 cells was quantified under both the matrix and aqueous conditions, and the findings indicated that NK92 cells moved continuously around the MATRIGEL droplets throughout the 24-hour incubation period, whereas movement was observed only during the first 2 hours in the aqueous droplets (Fig. 3B). Additionally, the data were further stratified into 6-hour intervals, showing that NK92 cell movement was consistently higher in the 3D gel droplets across all time periods, with a notable increase during 6-12 hours, when they were the most active (Fig. 3C). Altogether, the results revealed that NK92 cell viability was enhanced in the 3D gel environment due to the improved cell mobility in the presence of ECM components.

[0055] Enhanced tumor-immune cell interactions in MATRIGEL droplets

[0056] Tumor cell-immune cell interactions and immune cell mediated cytotoxicity were investigated using PC3 and NK92 cells in both MATRIGEL and aqueous droplets, using at a 1 :1 cell ratio. PC3 cell death following contact with NK92 was evaluated, and the time required for PC3 cells to undergo apoptosis was recorded. As expected, downregulation of immune cell cytotoxicity was observed in the MATRIGEL droplets. In aqueous droplets, 50% cell viability was observed after 9 hours, whereas in the MATRIGEL droplets, it took 17 hours to reach the same level. Images from the 24-hour incubation period showed that in aqueous droplets, the two cell types came into contact easily to allow for immune cell killing of the tumor cell, as gravity pulled them together at the bottom of the droplet. In contrast, in the 3D gel environment, NK92 cells actively traveled around the droplet to locate PC3 cells. Upon contact, the active NK92 proceeded to kill the tumor cell (Fig. 4C). As expected, NK92 cell viability was enhanced in the MATRIGEL droplet when co-encapsulated with PC3 (Fig. 4B). In the aqueous droplets, 50% NK92 cell viability was reached after 10 hours, while in the MATRIGEL droplets, more than 50% of the NK92 cells were alive after 24 hours (Fig. 4B). Taken together, these findings highlight the advantage of using 3D ECM gel droplets to study tumor and immune cell interactions. Function-to-Omics analysis using FADS and Next Generation Sequencing

[0057] It is well known that cellular and molecular heterogeneity exist in both tumor and immune cells12’22Individual tumor cells and immune cells interact differently with each other and with the surrounding ECM15’16Therefore, it is important to identify transcriptom ic profiles that are associated with functional heterogeneity and in turn affect NK cell killing. Using the microfluidics-based approach of the present technology, droplets can be sorted based on the functional activity of immune cells, specifically focusing on tumor cell killing, such as by NK cells. ECM gel-containing droplets containing active NK cells can be isolated from those containing non-killing NK cells to identify and analyze the source of their functional heterogeneity, or even to alter it using genetic engineering approaches. FADS has previously been performed with aqueous droplets6, but sorting of MATRIGEL-containing droplets is a novel feature of the present technology.

[0058] PC3 cells were treated with CellEvent Caspase-3 / 7 detection reagent in green to label cells that died from NK killing. After 16 hours of incubation, droplets were sorted using a previously described sorter system6. Each droplet was excited by a 488nm laser beam at 70mW as it passed through the sorter junction (Figs. 5A and 5B). Droplets with a positive signal (active NK cells) were collected separately from those with a negative signal (non-killing NK cells). RNA was extracted from both cell populations and assessed for quality. Four individual sorts were prepared with the NK92 cell line and three sorts with primary NKs. Data from Agilent high sensitivity RNA Tapestation showed clear 18S and 28S bands, and intensity was visualized by a peak graph showing RNA fragment distribution (Figs. 5C, 5E). Both the average RNA integrity number and concentration from NK92 killers (7.45±0.50; 8.08±1 .96) and non-killers (7.30±0.53; 14.12±4.65), and primary NK killers (4.87±1 .57; 6.48±4.37) and non-killers (4.36±1.08; 6.75±3.07) were sufficient for library prep for next generation sequencing (Fig. 5D). Altogether, it was demonstrated that the sorter system is able to sort MATRIGEL droplets based on a fluorescent signal that indicates a killing event. Additionally, high-quality RNA was successfully extracted and found to be compatible with downstream sequencing for both the NK92 cell line and primary NK cells.

[0059] EXAMPLES

[0060] Example 1. Materials. Prostate cancer cell line (PC3) was cultured in RPMI-1640 media (Gibco, Waltham, MA) with 10% fetal bovine serum (FBS) (Gibco, Waltham, MA) and 1 % penicillin streptomycin solution (Coming, NY). Natural killer cell line (NK92) was cultured in MyeloCult H5100 (Stemcell technologies, Cambridge, MA) with 10% horse serum (Gibco, Waltham, MA) and 500U / mL lnterleukin-2 human recombinant (Prospec protein, Israel).

[0061] Example 2. Device fabrication and array filling.

[0062] Standard soft lithography techniques with PDMS were used to fabricate the droplet and sorter devices as previously described6. Negative photo resist Sll-82050 was spincoated on a dust free silicon wafer to a thickness of 150pm. The wafer was subsequently UV crosslinked using transparent photomask. Devices were made with 20:2 PDMS and its curing agent bonded to glass slides using a Harrick Plasma Cleaner (Harrick Plasma Inc, Ithaca, NY). AQUAPEL (PPG Industries, Pittsburg, PA) was used to make the devices hydrophobic to enhance droplet generation efficiency. The 3D droplets are formed at the droplet generation junction, where the oil phase intersect the cell-matrix phase at a flow-focusing junction. The shearing forces at this junction results in the formation of uniform MATRIGEL droplets that encapsulates the cells and MATRIGEL within the continuous oil phase. The formed MATRIGEL droplets will flow into the 1000-droplet docking site microarray for high resolution imaging of tumor cell and immune cell functional properties in the 3D configuration, including cytotoxicity / viability assessment, cell-cell interactions and cell mobility (Figure 1 ).

[0063] MATRIGEL basement membrane matrix was purchased from Corning (Corning, NY) and thawed on ice prior to withdrawn into a 1 -mL syringe. All droplet generation experiments were conducted in 4C cold room to keep MATRIGEL from crosslinking during the preparation steps. PC3 and NK92 were separately resuspended in RMP1 1640 complete media and withdrawn into a 1 -mL syringe. Novak 7500 oil with 2% v / v span-80 surfactant (MilliporeSigma, St. Louis, MO) was also withdrawn into a 1 -mL syringe. The individual Novak 7500 w / 2% surfactant, PC3, NK92 and MATRIGEL solution were perfused into the 3D droplet microfluidics device with Tygon microbore tubing (Saint-Gobain Company, Courbevoie, France) using Harvard Apparatus syringe pumps (Holland, MA). The filled devices were kept in 37C incubation chamber to allow MATRIGEL to crosslink. Real-time images of the array compartment were captured for 24 hours at 30 minute intervals. A total of 48 images were captured for an individual droplet over a 24 hour period.

[0064] Example 3. Cell mobility analysis.

[0065] Cell mobility was analyzed using Image J (NIH). The X and Y coordinates of the NK cell was recorded for each frame in an image sequence of a 24-hour experiment. The distance travelled from frame to frame was calculated using the Pythagorean Theorem using the X and Y coordinates from each interval. The collected distance data was processed in Prism 10 (California, USA) using Multiple Mann- Whitney tests.

[0066] Example 4. Fluorescence activated droplet sorting (FADS) and RNA extraction.

[0067] To sort NK cells based on immune cell killing events, PC3 and NK cells were prepared in RPMI at 4.5 million cells / mL. PC3 cells were treated with CellEvent Caspase-3 / 7 detection reagent in green (Invitrogen, Waltham, MA) to identify activated caspase-3 / 7 in the event of NK-mediated cytotoxicity. The droplets were generated using the microfluidics device (Figure 1A) with 1 :1 ratio of both cell types. Droplets were incubated for 16 hours to allow for immune cell-mediated cytotoxicity. After incubation, the droplets were injected into the sorting device, where they are excited by a 488nm laser beam at 70mW and the droplets’ optical signal were detected using a photo-multiplier tube (PMT) (Hamamatsu Photonics, Hamamatsu City, Japan). Positive and negative 3D droplet sorts are collected in separate syringes. The sorted droplets are treated with Cell Recovery Solution from Coming (Coming, NY) to recovery cells from the MATRIGEL. RNA from NK92 cells was extracted using RNeasy Mini Kit from Qiagen (Hilden, Germany) following the protocol provided by the manufacturer. RNA quality and concentration were analyzed using Agilent 2200 TapeStation system for high sensitivity RNA analysis, performed by the Biopolymer Facility at Harvard Medical School (Boston, MA). Eluted RNA from primary NK cells were sent to Novogene (Beijing, China) for ultra-low RNA input RNA-seq.

[0068] Example 5. Cell treatment and array imaging.

[0069] PC3 were treated with Calcein AM (Invitrogen, Waltham, MA) fluorescent label in media for 30 minutes at 37C to mark live cells. PC3 and NK92 cells were loaded at a 1 :1 ratio with 4.5 million / mL starting concentration. 8uM of ethidium homodimer (Biotium , Freemont, CA) was added in the cell mixture to detect dead cells over the course of the experiment. Droplets were imaged for 24 hours in 37C and 5% CO2 incubation chamber. All images were captured at 20X magnification.

[0070] As used herein, "consisting essentially of" allows the inclusion of materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term "comprising", particularly in a listing of components of a composition or elements of a device, constitutes inclusion of alternative embodiments in which “comprising” is replaced with "consisting essentially of" or "consisting of'.

[0071] While the present invention has been described in conjunction with certain preferred embodiments, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein.

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Claims

CLAIMSWhat is claimed is:1 . A method of analyzing tumor cell interactions with immune cells, the method comprising(a) providing (i) a microfluidic device comprising a droplet generation module, a droplet docking array, and a fluorescence microscope, (ii) a suspension of single tumor cells, (iii) a suspension of immune cells, and (iv) a temperature-sensitive polymer;(b) co-encapsulating one or more of the single tumor cells with one or more of the immune cells in an aqueous droplet comprising the temperature-sensitive polymer using the droplet generation module to form a gel droplet containing one or more single tumor cells and one or more immune cells;(c) transferring the gel droplet to the droplet docking array;(d) optionally repeating steps (b) and (c) to fill or partially fill the droplet docking array; and(e) analyzing the docked gel droplets using the fluorescence microscope.

2. The method of claim 1 , wherein the temperature-sensitive polymer comprises an extracellular matrix extract comprising solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, and wherein steps (b), (c), and optionally (d) are performed at about 4°C and the method further comprises step (eO), prior to step (e), of warming the device to about 37°C, whereby the extracellular matrix extract forms a gel.

3. The method of claim 1 , wherein the step of analyzing comprises recording images using the fluorescence microscope of one or more gel droplets over time and determining for each gel droplet one or more of cell viability, cell mobility, or cell-cell interactions.

4. The method of claim 1 , further comprising(f) harvesting one or more gel droplets from the docking array; and(g) sorting the one or more gel droplets using a fluorescence-activated droplet sorting device based on a fluorescence signal from one or more cells in each gel droplet and collecting the sorted gel droplets in distinct containers.

5. The method of claim 4, further comprising(h) extracting and lysing cells from the sorted gel droplets; and(j) subjecting the lysed cells to one or more of transcriptom ic analysis, genomic analysis, or proteomic analysis.

6. The method of claim 1 , wherein the single tumor cells are derived from a solid tumor or from a cell culture derived from a solid tumor.

7. The method of claim 1 , wherein the immune cells are NK cells or killer T cells.

8. The method of claim 1 where either or both of the single tumor cells or the immune cells are derived from a subject having or suspected of having cancer.

9. The method of claim 1 , wherein step (e) comprises exposing one or more of the gel droplets to one or more test substances, and said analysis includes evaluating an ability of the one or more test substances to affect the tumor cells or the immune cells.

10. The method of claim 9, wherein the test substance is an antitumor agent.11 . The method of claim 1 , wherein the single tumor cells and / or the immune cells are obtained from two or more individuals of a population.

12. A method of encapsulating a biomolecule, cells, or a therapeutic agent, the method comprising(a) providing (i) a microfluidic device comprising a droplet generation module and a droplet docking array, (ii) a suspension of the biomolecule, cells, or therapeutic agent, and (iii) a temperature-sensitive polymer that polymerizes when warmed from below ambient temperature to ambient temperature or above;(b) co-encapsulating the biomolecule, cells, or therapeutic agent with the depolymerized temperature-sensitive polymer in a plurality of aqueous droplets using the droplet generation module;(c) transferring the droplets to the droplet docking array while maintaining the temperature-sensitive polymer in a depolymerized state; and(d) raising the temperature of the docking array, whereby the temperaturesensitive polymer polymerizes, thereby forming a plurality of polymer microspheres comprising the biomolecule, cells, or therapeutic agent.

13. The method of claim 12, wherein the temperature-sensitive polymer comprises solubilized basement membrane matrix secreted by Engelbreth-Holm- Swarm (EHS) mouse sarcoma cells.

14. The method of claim 12, wherein the microsphere-encapsulated biopolymer, cells, or therapeutic agent is rendered more stable as a result of said method.

15. A polymer-encapsulated biomolecule, cell, or therapeutic agent produced by a method comprising the method of claim 12.

16. A kit for use in performing the method of claim 12, the kit comprising: a microfluidic device, chip, or module for encapsulating two or more components in aqueous droplets in an oil stream; a microfluidic device, chip, or module for sorting aqueous droplets based on fluorescence of droplet contents; instructions for performing said method; and optionally, one or more temperature-sensitive polymers, extracellular matrix extracts, cells, or reagents.

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