Device and method for analyzing living cells

Abstract

A method is provided for assessing immune cells and cancer cells by assessing cytolysis of cancer cells by effector cells, the method comprising: providing a cell-substrate impedance monitoring device operably connected to an impedance analyzer wherein the device comprises a well for receiving cells and an electrode array at a base of the well, the apparatus is further operably connected to the imaging unit; b) adding target cells characterized as cancer cells into the pores; c) disposing a layer comprising an extracellular matrix (ECM) over the target cells; d) adding effector cells over the ECM layer; and e) imaging the well and monitoring the cell-substrate impedance of the well to determine the invasion of the effector cell through the ECM layer and the effectiveness of the effector cell to kill the target cell either directly or via migration and invasion through an extracellular matrix.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 383,245, filed Nov. 10, 2022, and U.S. Provisional Application No. 63 / 580,658, filed Sep. 5, 2023. The contents of the above applications are hereby incorporated by reference in their entireties. BACKGROUND OF THE INVENTION

[0003] The tumor microenvironment (TME) of solid tumors poses a significant challenge to the immune response, and recapitulating the properties of the TME in assays can be crucial for identifying and improving cellular immunotherapies. Engineered cytotoxic natural killer (NK) cells and cytotoxic T cells (CD8 + T) must extensively infiltrate solid tumors to exert effector functions. During migration, lymphocytes must traverse cell-free spaces with a structural scaffold that constitutes the extracellular matrix (ECM). The ECM can regulate multiple cellular responses in both resident tumor cells and infiltrating lymphocytes, thereby determining the outcome. Thus, there is a need to develop methods and systems to evaluate both tumor cell and immune cell responses in the context of the ECM. SUMMARY OF THE INVENTION

[0004] In one aspect, the present disclosure provides a method for assessing the cytolysis of cancer cells by effector cells. The method includes: a) providing a cell-substrate impedance monitoring device operably connected to an impedance analyzer, wherein the device includes a well for receiving cells and an electrode array at the base of the well, and the device is further operably connected to an imaging unit; b) adding target cells characterized as cancer cells to the well; c) disposing a layer comprising extracellular matrix (ECM) above the target cells; d) adding effector cells above the ECM layer; and e) imaging the well and monitoring the cell-substrate impedance of the well to determine the invasion of the effector cells through the ECM layer and the effectiveness of the effector cells in killing the target cells directly or via migration and invasion through the extracellular matrix.

[0005] In some embodiments, the imaging unit further includes an imaging device disposed near the well.

[0006] In some embodiments, the cancer cells are from solid tumors. In some embodiments, the effector cells are immune cells. In some embodiments, the effector cells are natural killer (NK) cells. In some embodiments, the effector cells are T cells. In some embodiments, the T cells are CD8 + T cells. In some embodiments, the effector cells express a chimeric antigen receptor (CAR). In some embodiments, the NK cells are chimeric antigen receptor (CAR) CAR-NK cells. In some embodiments, the T cells are chimeric antigen receptor (CAR) CAR-T cells.

[0007] In some embodiments, the cancer cells have abnormal FGFR signaling. In some embodiments, the method further comprises adding an FGFR inhibitor to the wells. In some embodiments, the FGFR inhibitor comprises pemigatinib.

[0008] In another aspect, the present disclosure provides a system configured to perform the method of assessing the cytolysis of cancer cells by effector cells as described herein.

[0009] In yet another aspect, the present disclosure provides a method comprising: measuring the cell-substrate impedance between target cells and effector cells separated by an extracellular matrix (ECM) layer at different times during an assay; capturing images of the effector cells and the target cells at different times during the assay; and determining the effectiveness of the effector cells relative to the target cells based on changes in the cell-substrate impedance in the images over the duration of the assay.

[0010] In some embodiments, the method further comprises: applying a first dye of a first color to the target cells; and applying a second dye of a second color different from the first color to the effector cells.

[0011] In some embodiments, the method further comprises: varying the thickness of the ECM in multiple wells of a multi-well plate in which the target cells and the effector cells are disposed.

[0012] In some embodiments, the method further comprises: administering a drug compound of interest to one or more of the target cell, the ECM, and the effector cell; comparing a baseline efficacy of the effector cell relative to the target cell in the absence of the drug compound of interest with an experimental efficacy of the effector cell relative to the target cell in the presence of the drug compound of interest; and in response to the experimental efficacy meeting an efficacy threshold, using the drug compound of interest to treat or prevent a disorder associated with the target cell in a biological subject.

[0013] In some embodiments, the method further comprises: administering a first drug compound of interest to a first subset of one or more of the target cell, the ECM, and the effector cell; administering a second drug compound of interest to a second subset of one or more of the target cell, the ECM, and the effector cell, the second subset being distinct from the first subset; comparing a first efficacy of the effector cell relative to the target cell in the presence of the first drug compound of interest with a second efficacy of the effector cell relative to the target cell in the presence of the second drug compound of interest; and in response to the first drug compound of interest having greater efficacy than the second drug compound of interest, using the first drug compound of interest to treat or prevent a disorder associated with the target cell in a biological subject.

[0014] In yet another aspect, the present disclosure provides a method of treating a solid tumor, the method comprising: administering a therapeutically effective amount of an FGFR inhibitor and a chimeric antigen receptor (CAR) therapy to a subject in need thereof.

[0015] In some embodiments, the solid tumor has aberrant FGFR signaling. In some embodiments, the FGFR inhibitor is an inhibitor of one, two, three, or all of FGFR1, FGFR2, FGFR3, or FGFR4. In some embodiments, the FGFR inhibitor is an FGFR1 inhibitor. In some embodiments, the FGFR inhibitor is an FGFR2 inhibitor. In some embodiments, the FGFR inhibitor is an FGFR3 inhibitor. In some embodiments, the FGFR inhibitor is an FGFR4 inhibitor. In some embodiments, the FGFR inhibitor comprises pemigatinib.

[0016] In some embodiments, the FGFR inhibitor is administered before the administration of the CAR therapy. In some embodiments, the FGFR inhibitor is administered concurrently with the administration of the CAR therapy. In some embodiments, the FGFR inhibitor is administered after the administration of the CAR therapy.

[0017] In some embodiments, the CAR therapy is CAR T cell (CAR-T) therapy, CAR-NK cell therapy, CAR-macrophage therapy, or CAR-gd-T therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figures 1A to 1B Depicts, according to embodiments of the present disclosure, the delay in target cell killing as the invasion distance increases. Adding NK92 (E:T 3:1) at 24 hours (h) results in a decrease in cell impedance (CI) due to target cell killing ( Figure 1A ). The loss of target cells due to NK cell cytotoxicity is delayed in Matrigel and slows down as the volume representing the increasing invasion distance increases. Live cell imaging confirms the delay in the loss of target cells in the field of view ( Figure 1B ).

[0019] Figures 2A to 2B Depicts, according to embodiments of the present disclosure, a representative image showing NK cell cytotoxicity as the Matrigel volume increases. eLive Green stains NK-92 cells ( Figure 2A ), but not healthy Michigan Cancer Foundation-7 (MCF-7) cells ( Figure 2B ). The gradual loss of MCF-7 (red) targets accompanied by an increase in green fluorescence when NK-92 cells are added above the Matrigel layer indicates the accumulation of NK-92 cells and dead target cells. Morphological changes are evident at early time points (about 45 - 55 h).

[0020] Figures 3A to 3C Depicts, according to embodiments of the present disclosure, the delay in NK cell killing of target cells by inhibiting MMP. GM6001 delays but does not eliminate the decrease in impedance (CI) due to target cell killing ( Figure 3A ). A delay in the loss of the red fluorescence signal of MCF7 and an increase in green fluorescence (eLive) are observed in the presence of the MMP inhibitor ( Figure 3B ). The percentage of cell lysis calculated at 72 h [(1 - (treated / untreated)*100] shows that the rate of cell lysis slows down as the Matrigel and matrix metalloproteinase (MMP) inhibitor increase ( Figure 3C ).

[0021] Figure 4 It is a graph showing an exemplary ECM invasion and cytotoxicity assay according to an embodiment of the present disclosure.

[0022] Figures 5A to 5G It shows that according to an embodiment of the present disclosure, pemigatinib increases the efficacy of EpCAM-CAR-T in squamous cell carcinoma. The expression of EpCAM on the cell surface of different tumor cells ( Figure 5A ). The normalized cell index over time shows that the FGFR1 inhibitor reduces A431 cells ( Figure 5B ), the EpCAM CART target A431 cells in the cytotoxicity assay ( Figure 5C ); EpCAM CART (E:T is about 3.5) is ineffective against A431 cells growing in Matrigel ( Figure 5D ); in the presence of FGFRi, CAR-T cells effectively control A431 cell proliferation (although EpCAM CART (E:T is about 3.5) is ineffective against A431 cells in Matrigel) ( Figure 5E ); and the percentage of cell lysis calculated from impedance readings and live cell imaging data confirms the enhanced efficacy of EpCAM CART in the presence of pemigatinib ( Figure 5F ), which is confirmed by the representative images taken at the 100h mark ( Figure 5G ).

[0023] Figure 6A and Figure 6B It shows the experimental assay setup according to an embodiment of the present disclosure. A comparison of the instrument, the well plate, and the setup in one of the wells with a Boyden chamber is shown ( Figure 6A ). The advantages of this setup for data collection compared to traditional methods are outlined in the steps of the assay ( Figure 6B ).

[0024] Figures 7A to 7C It depicts that according to an embodiment of the present disclosure, the cytotoxicity of NK-92 is delayed as the invasion distance increases. Adding NK-92 (E:T = 3:1) at 24 hours results in a decrease in impedance (CI) due to target lysis ( Figure 7A ), and the decrease in impedance in Matrigel is delayed ( Figure 7B ). As the volume of Matrigel increases, cell lysis is further delayed ( Figure 7C ). The percentage of cell lysis is calculated with reference to untreated MCF-7 growing in Matrigel.

[0025] Figure 8A and Figure 8B It depicts that according to an embodiment of the present disclosure, MMP inhibition delays the killing of target cells by NK cells.Figure 8A It is shown that a decrease in cell lysis of GFP-NK92 (E:T = 3:1) at 24 h results in a decrease in impedance (CI) due to target cell killing. Figure 8B It is shown that live cell imaging demonstrates a reduced loss of target cells (red fluorescence) in the field of view in the presence of MMP inhibitors (2 μM and 10 μM).

[0026] Figure 9 Depicts representative images of MCF-7 clustering and a progressive loss of red fluorescence associated with cytotoxicity. Some green GFP-NK92 cells (as Figure 9 indicated by the yellow outlines in ) make multiple contacts with MCF7-red target cells in the clusters, resulting in cell death during the assay. Detailed Description

[0027] The present disclosure relates at least in part to a co-culture model for real-time assessment of immune cell invasion and tumor cell killing.

[0028] The extracellular matrix (ECM) is a collective term for various types of acellular structural components in tissues that play important roles in homeostasis. Without wishing to be bound by theory, it is believed that in some embodiments, the ECM is a complex network composed of multi-domain macromolecular arrays organized in a cell / tissue-specific manner. For example, major ECM components can include collagen, proteoglycans, elastin, and / or cell-binding glycoproteins, each of which has different physical and biochemical properties. The ECM in solid tumors is greatly altered and can contribute to regulating immune cell function in the tumor microenvironment. Lymphocytes utilized in immunotherapy, such as cytotoxic natural killer cells (NK) and cytotoxic T cells (CD8 + T), can be regulated by the ECM. As detailed herein, the Matrigel layer poses a challenge to NK cells, and an increase in invasion distance delays the kinetics of tumor cell killing. In addition, the invasion and killing of tumor targets depend on the ability of NK cells or CD8 + T cells to remodel the matrix with matrix-metalloproteinases.

[0029] To assess the effectiveness of cells attacking targets in the ECM, an experimental or assay setup where the volume of Matrigel layered above target tumor cells expressing a fluorescent protein is continuously increased (to represent an increasing invasion distance). Immune cells are seeded above the solidified Matrigel layer, and a second marker of a different color from the fluorescent protein is added to each well. Using the XCELLIGENCE RTCA available from Agilent Tech., Inc., Santa Clara, California, USA Based on impedance-based immune cell killing measurements and live cell imaging data collected on an imaging and sensing system to evaluate immune cell invasion and function.

[0030] As MCF-7 target cells adhered and proliferated, impedance increased over time; however, the addition of NK-92 cells caused impedance to decrease due to target cell killing. The total time required to reduce the impedance level to that at the time of NK-92 addition gradually increased from 46 h for 50 μL / well to 72 h for 110 μL / well. eLive stained NK cells and dead cells green, but did not stain healthy MCF-7 cells green. Consistent with the impedance data, as the Matrigel volume increased, the loss of red fluorescence from target cells and the increase in green fluorescence were gradually delayed. Similar kinetic delays were achieved with the broad-spectrum MMP inhibitor GM6001 (e.g., 2 millimolar (mM) and 10 mM), indicating that MMP plays a role in NK function. Interestingly, NK-92 cells induced significant morphological changes in MCF-7 - red target cells before fully invading through the Matrigel, indicating early distal effects.

[0031] The results suggest an effector function of NK cells, potentially involving cytokines (which is independent of the ability to invade and / or degrade the matrix), in killing susceptible target cells. The assay also demonstrated the potential of adapting the XCELLIGENCE RTCA imaging and sensing system to study various ECM-immune cell interactions.

[0032] Immune cells invade the extracellular matrix (ECM) to perform effector functions including target cell killing. Traditionally, invasion and cytotoxicity functions have been evaluated in separate endpoint assays. A potential drawback is that cytotoxicity is not easily evaluated in the presence of ECM that can modulate lymphocyte cell responses. Additionally, traditional assay systems can be cumbersome and often involve multiple steps. In some embodiments, the present disclosure provides an assay that is easy to set up (e.g., in a 96-well format on an XCELLIGENCE RTCA imaging and sensing system that combines impedance and imaging capabilities to deliver real-time readouts of invasion and cytotoxicity functions). The methods and systems described herein are relevant to cancer immunotherapy. In some embodiments, the present disclosure provides a real-time assay for simultaneously evaluating immune cell invasion and cytotoxicity. In some embodiments, the assay is used to systematically study the effects of ECM on immune cell-tumor cell interactions. In some embodiments, the methods and systems described herein avoid the drawbacks of complex setups (e.g., using microfluidic arrangements). Other benefits will be apparent to those skilled in the art upon a detailed reading of the present disclosure.

[0033] The methods and systems described herein can have the ability to assess both the invasion and killing of target tumor cells by immune cells (e.g., T cells, NK, macrophages) in the context of the ECM (e.g., the ECM of any type of solid tumor described herein). Traditionally, invasion / migration and cytotoxicity functions have been evaluated in separate assays. In some embodiments, the methods and systems described herein can simultaneously collect impedance-based cytotoxicity readings and live cell imaging data in real time, for example, in a 96-well format.

[0034] NK is a type of innate lymphocyte that eliminates infected, stressed, or transformed cells by using a series of activating and inhibitory receptors to distinguish and discriminate "altered" cells from healthy cells. NK cells and their engineered variants have emerged as attractive options for adoptive immunotherapy, along with chimeric antigen receptor (CAR) T cells.

[0035] Reasons for the lack of efficacy of adoptive cell therapy against solid tumors include, for example, limited tumor-specific antigens, the immunosuppressive environment of the tumor itself that can lead to immune cell exhaustion, the physical infiltration of immune cells into the tumor stroma, and combinations thereof. While most in vitro assays for evaluating adoptive immune cells focus on activation, proliferation, and cytotoxicity, few assays focus on the migration and extravasation of immune cells to the tumor site. In some embodiments, the present disclosure provides a real-time in vitro assay that models both the migration / invasion of immune cells to the tumor site and the propensity of immune cells to kill tumor cells.

[0036] Recreating the tumor microenvironment in solid tumors can play a key role in formulating and improving strategies for cellular immunotherapy for various cancers. The ECM can be dysregulated in the TME, and the ECM has the ability to alter cellular responses. Therefore, there is a need to evaluate both tumor cell and immune cell responses in the context of the ECM. In this regard, various components of the ECM can modulate the function of NK cells.

[0037] The ability of NK cells to invade through the extracellular space to reach target cells can depend on their ability to degrade the ECM. NK cells can express multiple MMPs, and NK-92 invasion is reduced in the presence of the MMP inhibitor GM6001 in migration assays, indicating the role of MMPs in the migration of NK cells through Matrigel. Traditional invasion / migration potential and cytotoxicity functions are evaluated separately in the workflow or using complex setups.

[0038] ECM Invasion and Cytotoxicity Assays

[0039] In some embodiments, impedance-based measurements of immune cell killing (normalized cell index, CI) and live cell imaging on the XCELLIGENCE RTCA Imaging and Sensing System (obtainable from Agilent Tech., Inc.) are used to evaluate NK invasion and function.

[0040] An exemplary protocol is described below. Briefly, different volumes of Matrigel (50 - 110 microliters (μL) / well; 6 milligrams (mg) / milliliter (mL)) representing increasing invasion distances are layered over MCF-7 human breast adenocarcinoma cells stably expressing a nuclear-localized red fluorescent protein (MCF-7) (obtainable from the American Type Culture Collection, ATCC). NK-92 cells (obtainable from Creative Bioarray, ATCC) are added at an E:T of 3:1 over the solidified Matrigel layer. 1 μL / mL of eLive Green (obtainable from Agilent Technologies, shown as green) is added. A broad-spectrum MMP inhibitor (e.g., GM6001, obtainable from Selleck Chemicals) is used to evaluate the role of matrix metalloproteinases (MMPs). Figure 4 A schematic diagram is shown in.

[0041] Certain terms are used throughout the specification and claims to refer to particular features or components. As those skilled in the art will appreciate, different people may refer to the same feature or component by different names. This document is not intended to distinguish components or features that have the same function but different names. Additionally, although the various embodiments presented herein describe specific analytical systems, reagents, culture media, therapeutic agents, cell lines, etc. for purposes of illustration of the underlying inventive concepts from a practical perspective, this disclosure contemplates that when assays for determining the effectiveness of a potential therapeutic agent or the interaction between the potential therapeutic agent and various cell lines are performed using different imaging and sensing systems, various sizes / brands of well plates, software for controlling the relevant equipment, etc., those of ordinary skill in the relevant art will be able to substitute various different elements suitable for their purposes without undue experimentation.

[0042] As used herein, the term "optimization" and its variants are used in the sense understood by data scientists to refer to actions taken to continuously improve a system relative to a goal. An optimized value will be understood to represent a "near-optimal" value for a given reward framework, which value may oscillate around a "best" value or a local or global maximum of a set of values, and may change as the goal changes or as input conditions change. Thus, the best solution for a first goal at a given time may be sub-optimal for a second goal at that time, or may also be sub-optimal for the first goal at a later time.

[0043] As used herein, various chemical compounds are represented using the relevant element abbreviations set by the International Union of Pure and Applied Chemistry (IUPAC), which are familiar to those of ordinary skill in the relevant art. Similarly, various units of measure may be used herein, which are represented in the relevant abbreviated forms set by the International System of Unit (SI), which are familiar to those of ordinary skill in the relevant art.

[0044] As used herein, "about", "approximately" and "substantially" shall be understood to refer to a number within the range of the number mentioned, for example, in the range of -10% to +10% of the number mentioned, preferably in the range of -5% to +5% of the number mentioned, more preferably in the range of -1% to +1% of the number mentioned, and most preferably in the range of -0.1% to +0.1% of the number mentioned.

[0045] In addition, all numerical ranges herein shall be understood to include all integers, whole numbers or fractions within that range. In addition, these numerical ranges shall be interpreted as providing support for claims directed to any numerical value or subset of numerical values within that range. For example, the disclosure of from 1 to 10 shall be interpreted as supporting ranges from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so on.

[0046] As used in this disclosure, the phrase "at least one of" a list of items refers to any collection of those items, including a collection having a single member and every possible combination thereof. For example, when referring to "at least one of A, B, or C" or "at least one of A, B, and C", the phrase is intended to cover the following collections: A, B, C, A - B, B - C, and A - B - C, where the collections may include one or more instances of a given member (e.g., A - A, A - A - A, A - A - B, A - A - B - B - C - C - C, etc.) and any order thereof. To avoid doubt, the phrase "at least one of A, B, and C" should not be construed to mean "at least one of A, at least one of B, and at least one of C".

[0047] As used in this disclosure, the term "determine" encompasses a variety of actions, which may include calculating, computing, processing, deriving, investigating, looking up (e.g., via a table, database, or other data structure), judging, receiving (e.g., receiving information), accessing (e.g., accessing data in a memory), retrieving, parsing, selecting, choosing, establishing, etc.

[0048] Without further elaboration, it is believed that one of ordinary skill in the art can make the most of the claimed invention using the foregoing description. The various examples and aspects disclosed herein should be construed as merely illustrative and in no way limiting the scope of the disclosure. It will be apparent to those of ordinary skill in the art that changes may be made to the details of the above examples without departing from the underlying principles discussed. In other words, various modifications and improvements to the examples specifically disclosed in the above description are within the scope of the appended claims. For example, any suitable combination of the features of the various examples described is contemplated.

[0049] In the claims, the recitation of an element in the singular is not intended to mean "one and only one" unless specifically so stated, but rather "one or more" or "at least one". Unless otherwise expressly stated, the term "some" means one or more. No claim element is to be construed under the provisions of 35 U.S.C. § 112(f) unless the element is expressly recited using the phrase "means for" or "step for". All structural and functional equivalents of the elements of the various embodiments described in this disclosure that are known or later come to be known to those of ordinary skill in the relevant art are expressly incorporated herein by reference and are intended to be covered by the claims. Moreover, nothing disclosed in this disclosure is intended to be dedicated to the public, whether or not such disclosure is expressly recited in the claims.

[0050] Enumerated embodiments

[0051] 1. A method for assessing the cytolysis of cancer cells by effector cells, the method comprising:

[0052] Providing a cell-substrate impedance monitoring device operably connected to an impedance analyzer, wherein the device comprises a well for receiving cells and an electrode array at the base of the well, and the device is further operably connected to an imaging unit;

[0053] Adding target cells characterized as cancer cells to the well;

[0054] Providing a layer comprising an extracellular matrix (ECM) over the target cells;

[0055] Adding effector cells over the ECM layer; and

[0056] Imaging the well and monitoring the cell-substrate impedance of the well to determine the invasion of the effector cells through the ECM layer and the effectiveness of the effector cells in killing the target cells directly or via migration and invasion through the extracellular matrix.

[0057] 2. The method according to embodiment 1, the method further comprising providing an imaging device near the well.

[0058] 3. The method according to embodiment 1 or 2, wherein the cancer cells are from a solid tumor or its metastatic lesions, optionally wherein the solid tumor is a malignant tumor, or prostate cancer, breast cancer, lung cancer and bronchial cancer, colon and rectal cancer, bladder cancer, thyroid cancer, renal cancer and renal pelvic cancer, uterine body cancer, oral cancer or ovarian cancer.

[0059] 4. The method according to any one of embodiments 1-3, wherein the effector cells comprise immune cells (e.g., the immune cells described herein), such as natural killer (NK) cells, T cells (e.g., CD8+ T cells, cytotoxic T cells), macrophages or combinations thereof.

[0060] 5. The method according to any one of embodiments 1-3, wherein the effector cells comprise NK cells.

[0061] 6. The method according to any one of embodiments 1-3, wherein the effector cells comprise T cells.

[0062] 7. The method according to any one of embodiments 1-3, wherein the effector cells comprise immune cells, such as CAR-T cells, CAR-NK cells, CAR-macrophages, γ-δ T cells (e.g., CAR-gd-T cells) or combinations thereof.

[0063] 8. The method according to any one of Embodiments 1-7, wherein the effector cells are obtained from a subject.

[0064] 9. The method according to Embodiment 8, wherein the subject has cancer.

[0065] 10. The method according to Embodiment 9, wherein the cancer is a solid tumor.

[0066] 11. A method comprising:

[0067] Measuring the cell-substrate impedance between a target cell and an effector cell separated by an extracellular matrix (ECM) layer at different times during an assay;

[0068] Capturing images of the effector cells and the target cells at different times during the assay; and

[0069] Determining the efficacy of the effector cells relative to the target cells based on changes in the cell-substrate impedance in the images during the course of the assay.

[0070] 12. The method according to Embodiment 11, the method further comprising:

[0071] Applying a first dye of a first color to the target cells; and

[0072] Applying a second dye of a second color different from the first color to the effector cells.

[0073] 13. The method according to Embodiment 11 or 12, the method further comprising changing the thickness of the ECM in a plurality of wells of a multi-well plate provided with the target cells and the effector cells.

[0074] 14. The method according to any one of Embodiments 11-13, the method further comprising:

[0075] Applying a drug compound of interest to one or more of the target cells, the ECM, and the effector cells;

[0076] Comparing a baseline efficacy of the effector cells relative to the target cells in the absence of the drug compound of interest with an experimental efficacy of the effector cells relative to the target cells in the presence of the drug compound of interest; and

[0077] In response to the experimental efficacy meeting an efficacy threshold, using the drug compound of interest to treat or prevent a disorder associated with the target cells in a biological subject.

[0078] 15. The method according to any one of embodiments 11-13, the method further comprising:

[0079] Applying a first drug compound of interest to one or more first subsets of the target cells, the ECM, and the effector cells;

[0080] Applying a second drug compound of interest to one or more second subsets of the target cells, the ECM, and the effector cells, the second subset being separate from the first subset;

[0081] Comparing a first efficacy of the effector cells relative to the target cells in the presence of the first drug compound of interest with a second efficacy of the effector cells relative to the target cells in the presence of the second drug compound of interest; and

[0082] In response to the first drug compound of interest having greater efficacy than the second drug compound of interest, using the first drug compound of interest to treat or prevent a disorder associated with the target cells in a biological subject.

[0083] 16. The method according to any one of embodiments 11-15, wherein the target cells are cancer cells.

[0084] 17. The method according to embodiment 16, wherein the cancer cells are from a solid tumor or a metastatic lesion thereof, optionally wherein the solid tumor is a malignant tumor, or prostate cancer, breast cancer, lung cancer and bronchial cancer, colon and rectal cancer, bladder cancer, thyroid cancer, kidney and renal pelvis cancer, uterine body cancer, oral cancer or ovarian cancer.

[0085] 18. The method according to any one of embodiments 11-17, wherein the effector cells include immune cells (e.g., the immune cells described herein), such as natural killer (NK) cells, T cells (e.g., CD8+ T cells, cytotoxic T cells), macrophages, or combinations thereof.

[0086] 19. The method according to any one of embodiments 11-17, wherein the effector cells include NK cells.

[0087] 20. The method according to any one of embodiments 11-17, wherein the effector cells include T cells.

[0088] 21. The method according to any one of embodiments 11-17, wherein the effector cells include immune cells, such as CAR-T cells, CAR-NK cells, CAR-macrophages, γ-δ T cells (e.g., CAR-gd-T cells), or combinations thereof.

[0089] 22. The method according to any one of embodiments 11-21, wherein the effector cells are obtained from a subject.

[0090] 23. The method according to embodiment 22, wherein the subject has cancer.

[0091] 24. The method according to embodiment 23, wherein the cancer is a solid tumor.

[0092] 25. A method of treating a solid tumor having or identified as having abnormal FGFR signaling, the method comprising administering to a subject in need thereof a therapeutically effective amount of an FGFR inhibitor (e.g., an inhibitor of FGFR1, FGFR2, FGFR3, FGFR4 or any combination thereof) and CAR therapy.

[0093] 26. The method according to embodiment 25, wherein the FGFR inhibitor comprises pemigatinib.

[0094] 27. The method according to embodiment 25 or 26, wherein the FGFR inhibitor is administered before the CAR therapy.

[0095] 28. The method according to any one of embodiments 25-27, wherein the FGFR inhibitor is administered concomitantly with the administration of the CAR therapy.

[0096] 29. The method according to any one of embodiments 25-28, wherein the FGFR inhibitor is administered after the CAR therapy.

[0097] 30. The method according to any one of embodiments 25-29, wherein the CAR therapy comprises immune cells expressing a chimeric antigen receptor, the chimeric antigen receptor comprising an antigen recognition domain, a hinge region, a transmembrane domain, and an intracellular cell signaling domain.

[0098] 31. The method according to embodiment 30, wherein the antigen recognition domain binds to a tumor antigen, optionally wherein the solid tumor is a malignant tumor, or prostate cancer, breast cancer, lung cancer and bronchial cancer, colon and rectal cancer, bladder cancer, thyroid cancer, renal cancer and renal pelvic cancer, uterine body cancer, oral cancer or ovarian cancer.

[0099] 32. The method according to any one of embodiments 25-31, wherein the CAR therapy is CAR-T therapy.

[0100] 33. An FGFR inhibitor (e.g., an inhibitor of FGFR1, FGFR2, FGFR3, FGFR4, or any combination thereof), which is used for the combined treatment with CAR therapy of solid tumors in a subject having or identified as having abnormal FGFR signaling.

[0101] 34. The FGFR inhibitor for use according to embodiment 33, wherein the FGFR inhibitor comprises pemigatinib.

[0102] 35. The FGFR inhibitor for use according to embodiment 33 or 34, wherein the FGFR inhibitor is administered before the administration of the CAR therapy.

[0103] 36. The FGFR inhibitor for use according to any one of embodiments 33 - 35, wherein the FGFR inhibitor is administered simultaneously with the administration of the CAR therapy.

[0104] 37. The FGFR inhibitor for use according to any one of embodiments 33 - 36, wherein the FGFR inhibitor is administered after the administration of the CAR therapy.

[0105] 38. The FGFR inhibitor for use according to any one of embodiments 33 - 37, wherein the CAR therapy comprises immune cells expressing a chimeric antigen receptor, and the chimeric antigen receptor comprises an antigen recognition domain, a hinge region, a transmembrane domain, and an intracellular cell signaling domain.

[0106] 39. The FGFR inhibitor for use according to embodiment 38, wherein the antigen recognition domain binds to a tumor antigen, optionally wherein the solid tumor is a malignant tumor, or prostate cancer, breast cancer, lung cancer and bronchial cancer, colon cancer and rectal cancer, bladder cancer, thyroid cancer, kidney cancer and renal pelvis cancer, uterine body cancer, oral cancer or ovarian cancer.

[0107] 40. The FGFR inhibitor for use according to any one of embodiments 33 - 39, wherein the CAR therapy is CAR-T therapy, CAR-NK cell therapy, CAR-macrophage therapy, or CAR-gd-T therapy.

[0108] Examples

[0109] Example 1. Using impedance and imaging to evaluate the invasion and cytotoxicity of immune cells

[0110] It is hypothesized that the Matrigel layer poses a challenge to NK-92 cells and that increasing the invasion distance delays tumor cell killing. Additionally, studies have shown that invasion / migration through Matrigel depends on matrix metalloproteinases (MMPs), which are components that cleave the ECM. Therefore, the effect of inhibiting MMP function was also evaluated.

[0111] The results indicate that the methods and systems described herein can be readily used to systematically study the effects of ECM components on tumor and immune cell interactions in the TME according to user requirements.

[0112] Materials and Methods

[0113] Cells

[0114] The MCF-7 human breast adenocarcinoma cell line (obtainable from ATCC, catalog number HTB-22) was transduced with eLenti Red (obtainable from Agilent Technologies, catalog number 8711011) at a multiplicity of infection of 1 and cultured for fourteen days in the presence of puromycin (2 micrograms (μg) / mL) to select MCF-7-Red cells that stably express a nuclear-localized red fluorescent protein (RFP). Both MCF-7 cells and MCF-7-Red cells were maintained in Eagle's Minimum Essential Medium (EMEM, obtainable from ATCC, 30-2003) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (obtainable from Sigma, catalog number 12106C-500ML) and 1% Pen / Strep (obtainable from Hyclone, catalog number SV30010).

[0115] NK-92 cells (obtainable from Creative Bioarray, CSC-C0499, and ATCC) were grown in MyeloCult H5100 medium (obtainable from Stemcell Technologies, catalog number 05150) supplemented with 30 mL of horse serum (obtainable from Gibco, catalog number 16050-122), 600 IU / mL of rhIL-2 (obtainable from Stemcell Technologies, catalog number 78036), and 1% Pen / Strep (obtainable from Hyclone, catalog number SV30010). All cell lines were maintained at 37 degrees Celsius (°C) and 5% carbon dioxide (CO 2 )

[0116] ECM Invasion and Cytotoxicity Assays

[0117] By using the XCELLIGENCE RTCA The ability of NK-92 cells to invade Matrigel and kill MCF-7-red target cells was evaluated using simultaneous impedance and imaging readings on an imaging and sensing system (obtained from Agilent Tech., Inc.). The background impedance signal was measured using 50 μL of EMEM medium in the wells of an E-Plate VIEW microplate (supplied by Agilent Technologies, catalog number 0030060101030). MCF-7-red target cells (30,000 in 100 μL) were added to each well. The plate was placed at room temperature for thirty minutes to promote even distribution of the cells on the bottom. The plate was then transferred to the XCELLIGENCE RTCA Imaging and Sensing System, and data acquisition was started. Impedance readings were collected every fifteen minutes, and photos were taken every sixty minutes. Images from four fields of view were acquired in each well in the bright field, red fluorescence channel, and green fluorescence channel. The exposure time was set to default in the bright field and 150 milliseconds (ms) in the red and green channels. Matrigel (obtained from Corning, catalog numbers 356234, 354234) was thawed overnight at 4 °C, diluted with Dulbecco's Modified Eagle Medium (DMEM), and supplemented with 10% FBS to a final total protein concentration of 6 mg / mL. After twenty-four hours, data collection was paused. The medium in the wells was aspirated, and different volumes of Matrigel (e.g., 50 μL, 75 μL, and 100 μL, data shown here) were layered on top of the MCF-7-red cells: eLive Green (obtained from Agilent Technologies, catalog number 8711003) was added to the Matrigel at a final concentration of 1:1000. EMEM (100 μL) containing eLive Green was added to the wells without ECM. The plate was incubated at 37 °C, 5% CO 2 2 for one hour to allow the Matrigel to solidify. Impedance and imaging data were collected for one hour, during which the NK-92 cells were suspended in EMEM medium containing eLive Green, and the cell number was adjusted to achieve an E:T of 3:1 in 100 μL. 100 μL of the NK-92 cell suspension was layered on top of the Matrigel, and the total volume in all wells was adjusted to 200 μL. The plate was loaded back into the XCELLIGENCE RTCA Imaging and Sensing System's holder, and data acquisition was resumed for seven days. The percentage of cell lysis was calculated using the formula [(1 - (treated / untreated)) * 100] based on the normalized cell impedance readings at seventy-two hours.

[0118] MMP Inhibition and NK Invasion Kinetics

[0119] The broad-spectrum MMP inhibitor Ilomostat (GM6001, available from Selleck Chemicals, catalog number S7157) was dissolved in Matrigel and culture medium at final concentrations of 2 micromolar (μM) and 10 μM to assess the effect of MMP-dependent remodeling of the ECM on NK-92 invasion.

[0120] Results

[0121] Target cell killing was delayed as the invasion distance of NK cells increased

[0122] As MCF-7-red target cells adhered and proliferated to stabilization upon attachment, impedance increased over time ( Figure 1A ). Addition of NK-92 cells led to a decrease in impedance due to loss of adherent target cells during killing. This loss of target cells took longer in the presence of Matrigel and was delayed as the volume representing greater invasion distance increased ( Figure 1A , representative data for 50 μL, 75 μL, and 100 μL).

[0123] Image analysis confirmed that invasion and killing were delayed as the invasion distance increased

[0124] Images were acquired at the focal plane of tumor cells adhering to the bottom of the wells in the E-view plates ( Figure 1B ). Consistent with the impedance readings, a gradual decrease in red fluorescence associated with loss of MCF-7-red cells was detected after addition of NK-92 cells. Green fluorescence increased during the course measured in wells containing NK-92. Importantly, as the Matrigel volume (invasion distance) increased, the kinetics of MCF-7 (red) cell loss and green staining (eLive Green) gain were delayed after addition of NK.

[0125] MMP inhibition delays target cell killing by NK cells

[0126] MMPs are thought to play an important role in lymphocyte invasion. As indicated herein, inhibition of MMPs delays the invasion of NK-92 cells, thereby slowing target cell killing. Impedance readings ( Figure 3A ) revealed that Ilomostat (2 μM and 10 μM) delayed target cell killing. Image analysis ( Figure 3B ) confirmed that inhibition of MMP function led to a delay in loss of MCF-7 (red) and an increase in cells stained with Elive Green. The time required for the impedance to fall back to the level at the addition of NK-92 (normalized time point) after addition of NK to the wells gradually increased from 46 h for 50 μL / well to 72 h for 110 μL / well. The percentage of cell lysis at the 72 h time point in the assay ( Figure 3C)Highlights the delay in killing by NK cells in the presence of increasing invasion distances and in the presence of MMP inhibitors.

[0127] The decrease in impedance readings due to target cell killing by invading NK-92 cells slows down with increasing distance. Consistent with the role of MMPs in NK invasion, broad-spectrum MMP inhibitors slow down the kinetics of target cell killing in the assay.

[0128] Live cell imaging confirmed that target cell killing is delayed with increasing invasion distance and in the presence of MMP inhibitors. Morphological changes of target cells were detected at approximately 45 - 55 h, indicating that distal effects may be mediated by cytokines.

[0129] This assay demonstrates that the 96-well format of the XCELLIGENCE RTCA Imaging and Sensing System can be adapted to systematically study the interaction between immune cells and tumor cells in the presence of ECM.

[0130] In this example, the ability of NK-92 cells to invade different distances through Matrigel to kill tumor cells was evaluated as a proof of concept to establish that this assay format allows for the simultaneous assessment of invasion and cytotoxicity in the presence of ECM. Target cell killing is delayed with increasing invasion distance of NK cells and in the presence of broad-spectrum MMP inhibitors. The cytotoxicity outcome measured by impedance loss and live cell imaging serves as a surrogate for the migratory / invasive potential of NK-92 cells. This is relevant to studies focusing on the TME and improving immunotherapy strategies, as the ECM can modulate the cellular responses of both invading NK cells and resident tumor cells. The platform described herein can be used to collect simultaneous real-time data on extracellular matrix invasion and target cell killing by lymphocytes in a 96-well format.

[0131] Example 2. Pemigatinib enhances the efficacy of EpCAM-CAR-T against squamous cell carcinoma

[0132] Chimeric antigen receptor (CAR) T cell therapy is generally more effective against hematological malignancies than solid tumors. Given the complex tumor microenvironment that plays a role in regulating lymphocyte responses and promoting tumor growth, the response rate of CAR-T therapy in solid tumors can be improved by combination with other treatment modalities.

[0133] Aberrant fibroblast growth factor (FGF) receptor (FGFR) signaling plays a key role in the proliferation and survival of malignant epithelial cells. This study investigated the use of a combination of an FGF inhibitor and CAR-T cells to target squamous cell carcinoma (SCC) cells in the presence of the extracellular matrix (ECM). The ECM in solid tumors may pose challenges for lymphocyte invasion and act as a reservoir for FGF, which can contribute to tumor survival and proliferation.

[0134] The hypothesis was that inhibition of FGFR signaling would reduce the proliferation of tumor targets, resulting in increased killing and improved control of tumor growth by EpCAM-CAR-T cells.

[0135] Materials and Methods

[0136] Cells

[0137] The A-431 human epidermoid carcinoma cell line (ATCC, catalog number CRL-1555) was transduced with eLenti Red (Agilent Technologies, catalog number 8711011) at a multiplicity of infection of 1 and cultured for 14 days in the presence of 2 μg / mL puromycin (InvivoGen, catalog number ant-pr-1) to select for A431-Red cells that stably express nuclear-localized red fluorescent protein (RFP). The A431-Red cells were cultured in DMEM medium (Corning, 10-013-CV) supplemented with 10% heat-inactivated FBS (Sigma, catalog number 12106C-500ML) and 1% Pen / Strep (Hyclone, catalog number SV30010). This medium was referred to as c-DMEM in the protocol.

[0138] EpCAM-CAR-T cells were cultured in ImmunoCult TM -XF T cell expansion medium (Stemcell Technologies, catalog number 10981) supplemented with 200 IU / ml of rhIL-2 (Stemcell Technologies, catalog number 78036). T cells were used for assays on day 4 after resuscitation.

[0139] Brief Description of Methods

[0140] Briefly, Matrigel (6 mg / mL) was overlaid (50 μL / well) above A431-red target tumor cells expressing nuclear-localized mKate2 (red fluorescent protein). After 2 h, Epithelial cell adhesion molecule (EpCAM)-CAR-T cells were seeded on top of the Matrigel layer. Tumor cell growth and CAR-T cell cytotoxicity were determined based on real-time impedance and live cell imaging data collected on the XCELLIGENCE RTCA Imaging and Sensing System. The role of FGFR signaling was evaluated using the inhibitor pemigatinib (1 μM, 5 μM, and 10 μM) of FGFR isoforms 1 to 3 sold under the brand name Pemazyre. The percentage of cell lysis was calculated using the formula [(1 - (treated / control)*100] based on normalized cell impedance and imaging (red fluorescence) data.

[0141] ECM Invasion and Cytotoxicity Assay

[0142] The ability of EpCAM-CAR-T cells to invade Matrigel and kill A431-red target cells was evaluated using impedance and imaging readings on the XCELLIGENCE RTCA Imaging and Sensing System. The background impedance signal was measured with 50 μL of c-DMEM medium in the wells of an E-Plate VIEW microplate (Agilent Technologies, catalog number 0030060101030). A cell suspension of A431-red cells was prepared in c-DMEM at the desired cell amount as 100 μL / well aliquots. After adding the tumor cells, the E-Plate was left to stand at room temperature for 30 min to promote uniform distribution of the tumor cells at the bottom. The plate was returned to the plate holder in the XCELLIGENCE RTCA Imaging and Sensing System to acquire data. Impedance was read every 15 min and images were taken every 60 min. Images from four fields of view of each well in the bright field and red fluorescence channels were acquired. The exposure time was set to default in the bright field and 150 ms in the red channel. Data was collected for 24 h.

[0143] Matrigel (obtainable from Corning, catalog number 354234) was thawed overnight at 4 °C and kept on ice during preparation. Matrigel was prepared with pemigatinib (Selleckchem, catalog number S0088) at final concentrations of 1 μM, 5 μM, and 10 μM and dimethyl sulfoxide (D2650, Sigma Aldrich) as a control. FBS (10% v / v) was added to the Matrigel and the total protein concentration was adjusted to 6 mg / ml with DMEM.

[0144] Pause data collection and remove the plate from the holder. Aspirate the medium in the wells and overlay the Matrigel on top of the A431-Red cells. Let the plate sit at room temperature for 30 minutes and then incubate at 37 °C / 5% CO 2 to polymerize the Matrigel. Collect impedance and imaging data for one hour, during which the EpCAM-CAR-T cells are suspended in c-DMEM medium and the cell number is adjusted to achieve the desired E:T in 100 μL. Carefully disperse the T cell suspension (100 μL) on top of the Matrigel. Load the plate back into the XCELLIGENCE RTCA Imaging and Sensing System holder and resume data collection.

[0145] Data Analysis

[0146] The software generates a real-time curve of impedance data over time (X-axis) and cell index (CI, Y-axis). The cell index is normalized to the time point before the addition of CAR-T cells. The normalized cell index (NCI(t)) is calculated by dividing the CI at time t (CI(t)) by the CI at the normalized time point (CI(t normalized)). Thus, the NCI at the normalized time point is default set to 1.0. Image analysis is performed with default settings to generate the red object count per well. The percentage of lysis is plotted using the immunotherapy module of the software. The percentage of lysis is calculated according to NCI (impedance) using the formula [(1 - (CAR-T treated / Pemigatinib treated))*100]. The percentage of lysis is calculated according to the red object count / well (imaging) using the formula [(1 - (CAR-T treated / Pemigatinib treated))*100].

[0147] Results

[0148] Figures 5A to 5G It is shown that according to an embodiment of the present disclosure, pemigatinib increases the efficacy of EpCAM-CAR-T in squamous cell carcinoma, indicating that pemigatinib can be effectively combined with CAR-T therapy for solid tumors with abnormal FGFR signaling.

[0149] Figure 5A The EpCAM expression on the cell surface of different cells was plotted, and preliminary data from the invasion-cytotoxicity assay showed that EpCAM-CAR-T cells had a poor effect on A431 cells. Therefore, it was hypothesized that FGFR signaling might play a role in A431 cell proliferation and survival in the presence of Matrigel / ECM, and pemigatinib (Pemazyre, Incyte) inhibits FGFR1-3 isoforms.

[0150] Figure 5BThe normalized cell index was plotted over time, showing that the FGFR1 inhibitor reduced the proliferation of A431 cells, indicating that pemigatinib slowed the proliferation of A431 cells in natural DMEM medium in a concentration-dependent manner.

[0151] Figure 5C EpCAM CART targeting A431 cells in the cytotoxicity assay was plotted ( Figure 5C ), showing that EpCAM-CAR-T cells triggered the lysis of A431 cells cultured in DMEM medium (E:T was 3.5). In the presence of pemigatinib (5 μM and 10 μM), A431 cell lysis was accelerated. The data indicate that reduced proliferation contributed to the effective killing of target cells. E:T was calculated based on T cells expressing CAR in the transduced T cell population.

[0152] Figure 5D EpCAM CART (E:T was approximately 3.5) was ineffective against A431 cells growing in Matrigel; in the absence of pemigatinib, EpCAM-CAR-T cells were unable to kill A431 cells cultured in Matrigel (E:T was 3.5).

[0153] Figure 5E In the presence of FGFRi, CAR-T cells effectively controlled the proliferation of A431 cells (although EpCAM CART (E:T was approximately 3.5) was ineffective against A431 cells in Matrigel), and pemigatinib reduced the proliferation of A431 cells in a concentration-dependent manner in the presence of Matrigel to improve the lysis outcome caused by EpCAM-CAR-T cells (E:T was 3.5).

[0154] Figure 5F The percentage of lysis calculated from impedance readings and live cell imaging data confirmed the enhanced efficacy of EpCAM CART in the presence of pemigatinib (the upper part shows the percentage of lysis plotted according to the normalized cell index (normalizedCell Index, NCI), and the lower part shows the normalized red fluorescence count / well). Based on these results, it was shown that pemigatinib reduced the proliferation of A431 cells in a concentration-dependent manner to improve the lysis outcome caused by EpCAM-CAR-T cells (E:T was 3.5).

[0155] Figure 5G A representative image taken at the 100 h mark of the assay from which the data plotted in Figures 5B to 5F was derived is shown; the hypothesis put forward regarding Figure 5A was confirmed.

[0156] Figures 5A to 5FThe exemplary assays and their analysis shown were performed using the A431 human epidermoid carcinoma cell line, which was transduced with eLenti Red at a multiplicity of infection of 1 and cultured for fourteen days in the presence of 2 μg / mL puromycin to select A431-Red cells that stably express a nuclear-localized red fluorescent protein (RFP). The A431-Red cells were cultured in DMEM medium supplemented with 10% heat-inactivated FBS and 1% Pen / Strep (which may be referred to as c-DMEM).

[0157] As shown in assays using the methodologies described herein, a drug compound with improved therapeutic and prophylactic properties has been discovered. These methodologies can be used to identify other drug compounds with improved therapeutic and prophylactic properties for treating various disorders in biological subjects. As demonstrated herein, pemigatinib can be effectively combined with CAR-T therapy for treating solid tumors with abnormal FGFR signaling. This conclusion is based at least in part on the results showing that A431 cells express EpCAM and that EpCAM-CAR-T cells effectively eliminate these tumor cells in a cytotoxicity assay. In the presence of Matrigel, CAR-T was ineffective (effector:target, E:T = 4:1). Since FGF signaling can promote the survival and proliferation of squamous cell carcinoma cells, the effect of the potent FGFR inhibitor pemigatinib was tested. As the tested concentration increased, pemigatinib decreased the proliferation of A431 cells. The CAR-T killing assessment revealed that the percentage of lysis increased from baseline (about 6%) to about 20% in the presence of 5 μM pemigatinib and from the baseline to about about 40% in the presence of 10 μM pemigatinib. Complete elimination of A431 cells was achieved in an invasion assay with a very high E:T = 20:1 and 1 μM pemigatinib.

[0158] Example 3. Real-time co-culture assays of immune cell invasion and cytotoxicity using the XCELLIGENCE RTCA ESIGHT imaging and sensing system

[0159] Immune cells extravasate from blood vessels to infiltrate tissues and exert effector functions, which play a key role in tumor immunosurveillance. These capabilities are also exploited by engineered cytotoxic natural killer (NK) and CAR (chimeric antigen receptor)-T cells for cancer immunotherapy.

[0160] Although cellular immunotherapies have proven effective against hematological cancers, clinical responses in solid tumors still need improvement. The complex tumor microenvironment (TME) in solid tumors regulates lymphocyte recruitment and function. A major challenge for lymphocytes in solid tumors is to traverse the cell-free spaces of the extracellular matrix (ECM)-filled structural scaffolds during migration / invasion. This ECM is heterogeneous, and its components can regulate multiple cellular responses in both resident tumor cells and infiltrating lymphocytes.

[0161] Traditionally, invasion, migration, and cytotoxicity have been evaluated in different endpoint assays using a transwell assay system to predict in vivo function. The Boyden chamber is a classic transwell setup that is widely used to evaluate migration and invasion. It consists of a cylindrical cell culture insert with a porous membrane nested within the well of a standard cell culture plate. A cell suspension is added to the inner chamber of the insert, and the cell suspension is induced to migrate out through pores of different sizes (usually, 3 - 12 μm) using a chemoattractant in the outer chamber. The inner pores can be coated with ECM to evaluate invasion. The invading / migrating cells are imaged and / or collected at a predetermined time point for quantification. The Boyden chamber can be modified to evaluate the migration and cytotoxicity of effector cells in endpoint assays.

[0162] This disclosure describes a novel real-time co-culture assay that uses the XCELLIGENCE RTCA Imaging and Sensing System to interrogate immune cell invasion and cytotoxicity, which does not require collection of embedded cells for endpoint readout, as Figure 6A and Figure 6B shown). In this setup, the ECM layer is in direct contact with the target tumor cells and can regulate the responses of both the tumor target and infiltrating lymphocytes, especially when the latter exerts its corresponding cytotoxic function. In transwell-based methods, the cytotoxicity of migrating immune cells is carried out in the outer chamber lacking ECM.

[0163] This example illustrates the evaluation of NK cell invasion and target tumor cell killing as a proof of concept for the assay system. NK is an innate lymphocyte that kills infected, stressed, or transformed cells by using a series of activating and inhibitory receptors to distinguish and discriminate 'altered' cells from healthy cells. This example uses NK-92 cells as a model to study invasion and cytotoxicity. Allogeneic NK derived from the NK-92 cell line was the first NK-based cellular immunotherapy to receive investigational new drug clinical trial authorization from the US Food and Drug Administration.

[0164] Assume that the Matrigel layer poses a challenge to NK-92 cells and that increasing the invasion distance delays tumor cell killing. Also, NK relies on proteases of the matrix-metalloproteinase (MMP) family to degrade various components in the ECM during the invasion phase.

[0165] This example shows that, depending on user needs, the 96-well format can be easily used to systematically study the effects of different ECM components on tumor and immune cell interactions in the TME.

[0166] Materials and Methods

[0167] Cells

[0168] The MCF-7 human breast adenocarcinoma cell line was transduced with eLenti Red at a multiplicity of infection of 1 and cultured for fourteen days in the presence of 2 μg / mL puromycin to select MCF7-Red cells that stably express a nuclear-localized red fluorescent protein (RFP). MCF7 cells and MCF7-Red cells were cultured in EMEM medium supplemented with 10% heat-inactivated FBS and 1% Pen / Strep.

[0169] NK-92 cells were grown in MyeloCult H5100 medium supplemented with 30 ml of horse serum, 600 IU / ml of rhIL-2, and 1% Pen / Strep.

[0170] eGFP-NK92 cells were grown in X-VIVO 15 supplemented with 5% human serum, 500 IU / ml of rhIL-2, and 0.5 μg / ml of puromycin.

[0171] ECM Invasion and Cytotoxicity Assays

[0172] The ability of NK-92 cells to invade Matrigel and kill MCF7-Red target cells was evaluated by simultaneous impedance and imaging readings on an XCELLIGENCE RTCA Imaging and Sensing System, as Figure 6A outlined. The background impedance signal was measured with 50 μL of EMEM medium in the wells of an E-Plate VIEW microplate. MCF7-Red target cells (30,000 in 100 μL) were added to the wells, and the plate was placed at room temperature for 30 minutes to promote uniform distribution of the cells at the bottom. The plate was returned to the XCELLIGENCE RTCA A plate holder in the imaging and sensing system is used to acquire data. The impedance is read every fifteen minutes, and an image is taken every sixty minutes. Images from four fields of view are acquired in each well in the bright field, red fluorescence channel, and green fluorescence channel. Separately, the exposure time is set to the default in the bright field, 150 ms in the red channel, and 300 ms in the green channel. The Matrigel is thawed overnight at 4°C, diluted with DMEM, and supplemented with 10% FBS to a final total protein concentration of 6 mg / ml. After twenty-four hours, data collection is paused. The medium in the wells is aspirated, and different volumes of Matrigel (50, 75, and 100 μL, the data shown here) are layered over the MCF7-red cells and incubated at room temperature for thirty minutes, then incubated at 37°C / 5% CO 2 2 for thirty minutes to polymerize the Matrigel. Impedance and imaging data are collected for one hour, during which the NK-92 cells are suspended in EMEM medium, and the cell number is adjusted to achieve an E:T of 3:1 in 100 μL. The NK-92 cell suspension (100 μL) is layered over the Matrigel, and the total volume in all wells is adjusted to 200 μL. The plate is loaded back into the holder, and data acquisition is resumed. The percentage of cell lysis is calculated based on the normalized cell impedance readings using the following formula: [(1 - (treated / untreated)*100].

[0173] MMP Inhibition and NK Invasion Kinetics

[0174] The broad-spectrum MMP inhibitor Ilomostat is dissolved in the Matrigel and medium at final concentrations of 2 μM and 10 μM to assess the effect on MMP-dependent NK-92 invasion. The percentage of cell lysis is calculated based on the normalized cell impedance readings and red object count data using the formula: [(1 - (NK92 + MMPi / treated with MMPi)*100].

[0175] Results

[0176] As the invasion distance of NK cells increases continuously, target cell killing is delayed

[0177] The impedance increases over time and stabilizes as the MCF7-red target cells adhere and proliferate to reach confluence, as Figure 7A shown. Adding Matrigel (50 μL) modulates the impedance profile of MCF-7 cells, and importantly, the addition of NK-92 cells at twenty-four hours is delayed, resulting in cell lysis. As the volume of Matrigel increases, the impedance drop due to cell lysis is further delayed, as Figure 7B shown, which shows representative data for 50 μL, 75 μL, and 100 μL). As Figure 7CThe KT60 (time to 60% killing relative to control) shown was 67 hours, 76 hours, and 89 hours, respectively.

[0178] MMP inhibition delays target cell killing by NK cells

[0179] Since cell lysis is delayed as the invasion distance of NK increases, it is hypothesized that MMP plays an important role in lymphocyte invasion. Consistent with the function of MMP in invasion, the percentage of cell lysis calculated from the normalized impedance readings as shown in Figure 8A and live cell imaging (e.g., red fluorescence) as shown in Figure 8B confirmed that in the presence of Ilomostat (2 μM and 10 μM), the killing of target cells by GFP-NK92 cells was delayed and reduced. Image analysis was consistent with the impedance measurements, confirming the kinetics of cell killing.

[0180] Image analysis confirmed that invasion and killing were delayed as the invasion distance increased

[0181] As Figure 9 shown, representative images of MCF-7 clusters revealed increased aggregation and cell death in response to GFP-NK92. Despite the increased tumor cell death, only a small number of GFP-NK92 were detected in the imaging field. Interestingly, highly active NK cells were detected that made multiple contacts with different MCF-7 red targets in clusters, indicating continuous killing activity.

[0182] Characterizing the tumor microenvironment in solid tumors plays a key role in formulating and improving strategies for cell immunotherapy. ECM dysregulation in the tumor microenvironment of solid tumors has been extensively studied for its ability to regulate multiple cellular responses. In this regard, effective in vitro assays are needed to evaluate the responses of tumors and immune cells in the presence of ECM.

[0183] The ability of NK and other immune cells to invade the extracellular space to reach target cells can depend on their ability to degrade ECM. Various components of ECM can regulate the function of NK cells. Therefore, the ability of NK-92 cells, as described herein, to invade a Matrigel layer and kill target cells is worthy of evaluation. The cytotoxicity outcomes measured by impedance loss and live cell imaging serve as surrogates for the migratory / invasive potential of NK-92 cells. This assay format allows for the simultaneous evaluation of invasion and cytotoxicity in the presence of ECM by demonstrating that target cell killing can be delayed by altering the invasion distance through Matrigel.

[0184] NK cells can express multiple MMPs, and migration assays have shown that NK-92 invasion in Matrigel is reduced in the presence of the MMP inhibitor GM6001. Typically, studies have evaluated invasion / migration potential and cytotoxic functions separately with complex workflows, as is the case for studies evaluating the effects of MMP inhibitors. Alternatively, some studies have evaluated both migration and cytotoxicity using complex and cumbersome experimental setups. Live cell imaging and real-time impedance readings, as described herein, demonstrate the contribution of MMPs in NK-92 invasion cytotoxicity assays. Consistent with the role of MMPs in promoting invasion, broad-spectrum MMP inhibitors delay target cell killing.

[0185] Although imaging and impedance data are consistent in invasion-cytotoxicity assays, relying solely on imaging data can be challenging. Tumor cells respond differently to Matrigel, and as highlighted in the representative images, MCF-7 target cells form irregular and disorganized clumps when undergoing cell death. Surprisingly, we did not find a large number of invading NK cells but only detected a small number of NK cells within and around the MCF clusters. Nevertheless, the images and videos collected in these assays show that highly active GFP-NK92 cells make multiple contacts with different targets in the clusters, resulting in cell death of the targets contacted during the assay.

[0186] The novel real-time co-culture assay described in this disclosure demonstrates the utility of the XCELLIGENCE RTCA imaging and sensing system in simultaneously evaluating the invasion and cytotoxic functions of lymphocytes crucial for solid tumor immunotherapy.

Claims

1. A method for assessing the cytolysis of cancer cells by effector cells, the method comprising: providing a cell-substrate impedance monitoring device operably connected to an impedance analyzer, wherein the device comprises wells for receiving cells and an electrode array at the base of the wells, and the device is further operably connected to an imaging unit; adding target cells characterized as cancer cells to the wells; providing a layer comprising an extracellular matrix (ECM) over the target cells; adding effector cells over the ECM layer; and imaging the wells and monitoring the cell-substrate impedance of the wells to determine the invasion of the effector cells through the ECM layer and the effectiveness of the effector cells in killing the target cells directly or via migration and invasion through the extracellular matrix.

2. The method according to claim 1, the method further comprising providing an imaging device near the wells.

3. The method according to claim 1 or 2, wherein the cancer cells are from a solid tumor.

4. The method according to any one of claims 1-3, wherein the effector cells are natural killer (NK) cells.

5. The method according to any one of claims 1-3, wherein the effector cells are T cells.

6. A method, the method comprising: measuring the cell-substrate impedance between target cells and effector cells separated by an extracellular matrix (ECM) layer at different times during an assay; capturing images of the effector cells and the target cells at different times during the assay; and determining the effectiveness of the effector cells relative to the target cells based on changes in the cell-substrate impedance in the images over the duration of the assay.

7. The method according to claim 6, the method further comprising: applying a first dye of a first color to the target cells; and applying a second dye of a second color different from the first color to the effector cells.

8. The method according to claim 6 or 7, the method further comprising varying the thickness of the ECM in multiple wells of a multi-well plate in which the target cells and the effector cells are disposed.

9. The method according to any one of claims 6-8, the method further comprising: applying a drug compound of interest to one or more of the target cells, the ECM, and the effector cells; comparing a baseline effectiveness of the effector cells relative to the target cells in the absence of the drug compound of interest with an experimental effectiveness of the effector cells relative to the target cells in the presence of the drug compound of interest; and responding to the experimental effectiveness meeting an effectiveness threshold by using the drug compound of interest to treat or prevent a disorder associated with the target cells in a biological subject.

10. The method according to any one of claims 6-8, the method further comprising: applying a first drug compound of interest to a first subset of one or more of the target cells, the ECM, and the effector cells; Apply a second drug compound of interest to one or more second subsets of the target cells, the ECM, and the effector cells, the second subsets being distinct from the first subsets; Compare a first efficacy of the effector cells relative to the target cells in the presence of the first drug compound of interest with a second efficacy of the effector cells relative to the target cells in the presence of the second drug compound of interest; and In response to the first drug compound of interest having greater efficacy than the second drug compound of interest, use the first drug compound of interest to treat or prevent a disorder associated with the target cells in a biological subject.

11. The method according to any one of claims 6-10, wherein the target cells are characterized as cancer cells.

12. The method according to claim 11, wherein the cancer cells are from a solid tumor.

13. The method according to any one of claims 6-12, wherein the effector cells are natural killer (NK) cells.

14. The method according to any one of claims 6-12, wherein the effector cells are T cells.

15. An FGFR inhibitor for use in a method of treating a solid tumor having abnormal FGFR signaling in a subject in combination with CAR therapy.

16. The FGFR inhibitor for use according to claim 15, wherein the FGFR inhibitor comprises pemigatinib.

17. The FGFR inhibitor for use according to claim 15 or 16, wherein the FGFR inhibitor is administered before the CAR therapy is administered.

18. The FGFR inhibitor for use according to claim 15 or 16, wherein the FGFR inhibitor is administered concomitantly with the administration of the CAR therapy.

19. The FGFR inhibitor for use according to claim 15 or 16, wherein the FGFR inhibitor is administered after the CAR therapy is administered.

20. The FGFR inhibitor for use according to any one of claims 15-19, wherein the CAR therapy is CAR-T therapy.

Images