Methods for determining interaction between biological cells

Abstract

To provide methods of determining the propensity of cells to interact in a population of cells comprising at least two distinguishable subpopulations of cells, and to provide methods of diagnosing a disease or predisposition to a disease of a cell donor.SOLUTION: A method comprises determining a propensity of cells to interact in a population of cells obtained from a donor, where the population of cells comprises at least two distinguishable subpopulations of cells. The invention also provides methods of determining whether or not a subject suffering from or predisposed to a disease will respond or is responsive to treatment with a therapeutic agent by determining the alteration of the propensity of cells to interact in a population of cells obtained from the subject, where the population of cells comprises at least two distinguishable subpopulations of cells. The invention also relates to methods of screening for a therapeutic agent, comprising determining whether one or more test substances alter the propensity of cells to interact.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for determining the tendency of cell - cell interactions in a population of cells comprising at least two distinguishable sub - populations of cells. The present invention also provides a method for diagnosing a disease or a predisposition to a disease of a cell provider. The method comprises determining the tendency of cell - cell interactions in a population of cells obtained from said provider, wherein the population of cells comprises cells of at least two distinguishable sub - populations. The present invention also provides a method for determining whether a subject suffering from or predisposed to a disease responds or is responsive to treatment with a therapeutic agent by determining a change in the tendency of cell - cell interactions in a population of cells obtained from the subject. Here, the population of cells comprises cells of at least two distinguishable sub - populations. The present invention also relates to a method for screening a therapeutic agent. The method comprises determining whether one or more test substances change the tendency of said cell - cell interactions.

[0002] Understanding the overall behavior of cells after perturbation by various stimuli, such as biological agents and small molecules, has advanced significantly through single-cell level analysis, including the analysis of cell-cell relationships. The field of high-content image processing (a technique for simultaneously processing a large number of cells) continues to provide evidence that the diversity between cells and the cellular microenvironment are required in the detailed analysis of the phenotypes of cells in a population to determine rare events and fully understand population-level phenotypes (reviewed in Snijder et al. (2011) Nature Reviews 12, 119). The comprehensive variation in cell phenotypes is mainly determined by the inherent properties of cells in a developing population. These cells in the population form a specialized niche microenvironment that includes cell density, cell-cell contact, relative position, and cell space. Also, at the single-cell level, the heterogeneity of cell responses to the same perturbation can be characterized (Slack et al. (2008) PNAS 105(49): 19306-11). From the complexity of the heterogeneity seen between cells (with regard to investigating the degree to which cancer cells respond to anti-cancer drugs), the functional significance of the broad effects that may occur at the cell level in patients undergoing treatment is elucidated. However, studies based on population feature analysis at intracellular and single-cell resolution rely on genetically identical cell lines, which are not physiologically appropriate for human health and disease and typically lack the information exchange within the population that occurs in vivo to drive further cellular and pan-cellular processes.

[0003] The immune system is regulated by 1) soluble signaling molecules and 2) physical cell-cell interactions. Examples of soluble signaling molecules that regulate the immune system include cytokines and chemokines. This also includes small molecules such as adenosine. Examples of cell-cell interactions that regulate immune function include cell surface-bound ligands that interact with cell surface receptors. For example, the interaction between MHC and the T cell receptor, and the interaction between PD-1 and its ligand PD-L1 are both necessary to induce the desired response, i.e., the regulatory response. Furthermore, soluble factors can interact with receptors on the surfaces of two different target cells, bringing these receptors spatially closer and inducing signaling events. Such an example is an antibody that binds to an Fc receptor on one cell surface and recognizes a target antigen on the other cell surface. Specifically, this is the case when the target antigen is in proximity to an immunomodulatory agent or a biological pharmaceutical in a new field (e.g., blinatumomab (bispecific antibody; CD19 / CD3) or rituximab (anti-CD20 antibody)). Soluble factors can act at a distance, but for cell surface-bound regulatory molecules to act, the cells must come spatially closer. This latter factor, i.e., the physical interaction of immune cells through receptor-mediated signaling, is essential for inducing a strong immune response and cell elimination / programmed cell death. Furthermore, communication through cell-cell contact underlies the mechanism of action of the above biological immunomodulatory agents.

[0004] There are many established means for measuring the concentration of soluble signaling factors, such as ELISA, ELISPOT, and Alpha Screen, to evaluate immune function or activity. Tracking soluble factors involved in immune regulation is commonly used in disease diagnostics and other health-related biology. However, even if so, the ability to determine immunomodulation at the level of cell-cell contact altered by biologics, drugs, or other small molecule compounds in a high-throughput and robust manner is currently limited. Therefore, the ability to systematically measure cell-cell contact and changes in cell-cell contact upon perturbation of the system with a molecule of interest can reveal whether a particular molecule has immunomodulatory properties. Conversely, when testing the system with a molecule having a known immunomodulatory effect, information regarding the state of the model system can be revealed by observing the effect as a change in cell-cell contact.

[0005] A method for determining the interaction of intracellular organelles has been proposed by Helmuth et al. (2010) BMC Bioinformatics 2010, 11: 372. This method derives a statistical framework for analyzing the attraction between the target X and Y in two sets of cells. However, as used in this literature, intracellular attraction is biologically different from cell - cell contact as used herein. Also, Helmuth et al. define a "nearest - neighbor co - localization measure" between multiple targets called Ct, and using this, it can be generalized to analyze the nearest - neighbor distance distribution for a number of analysis targets. This method is not appropriate when applied to PBMC (peripheral blood mononuclear cells) / complex cell mixtures for the purpose of detecting cell - cell contact, as the number of interacting cells is typically a small subset of all cells. The nearest - neighbor distribution analysis used by Helmuth et al. is likely to miss a small subset of interacting cells and focuses on the overall clustering or attraction between cells of these two subsets. Further, this technical solution compares the observed nearest - neighbor distance distribution p(d) between target X and Y with the relative frequency of the distance q(d) from a possible target Y, ignoring the fact that other targets may occupy the space of its biological system. Thus, this approach does not correct for the overall clustering tendency. However, in cell - cell contact, it is important to correct for the overall clustering in the analysis of two subsets of PBMC or complex cell mixtures, as this is mainly determined by experimental factors rather than the biological factors of interest.

[0006] A further method for dealing with cell - cell interactions is disclosed in WO2016 / 046346. This method includes determining the number of cells in contact with another cell regardless of the number of cells a particular cell is in contact with.

[0007] In such a perspective, there is a need for means and methods for cost - effectively determining cell - cell interactions in biological samples, particularly samples containing PBMC or bone marrow cells, with high throughput.

[0008] Accordingly, the technical problem underlying the present invention is to provide improved means and methods for determining the tendency of cell - cell interactions.

[0009] This technical problem is solved by providing an embodiment characterized in the claims.

[0010] Accordingly, the present invention relates to a method for determining the tendency of cell - cell interactions in a population of cells, wherein the population of cells comprises cells of at least two distinguishable sub - populations. The method comprises: (a) determining the number of interactions between cells of a first distinguishable sub - population and cells of a second distinguishable sub - population; (b) randomly assigning the cells comprised in the population of cells to the cells of the first and second distinguishable sub - populations to determine the number of interactions in the population of cells (wherein the absolute numbers of cells in the first and second distinguishable sub - populations are the same as the numbers of cells in the first and second distinguishable sub - populations in (a)); and (c) dividing the result of (a) by the result of (b) to determine the tendency of cell - cell interactions (wherein the tendency increases with the resulting number). In a preferred embodiment, step (b) is repeated and the determined numbers of interactions are averaged. It is preferred to repeat step (b) at least 1000 times more, at least 2000 times, or at least 3000 times.

[0011] As shown in the attached examples, the method of the present invention provides, inter alia, a more robust, accurate, and thus improved measure of cell-cell interactions in a biological setting. Prior art methods, such as the above-mentioned WO2016 / 046346, rely on determining the proportion of cells in a subpopulation that are in contact with another cell. Thus, some prior methods involve counting each cell that is in contact with at least one other cell. This results in two states, "interacting" and "not interacting". In a further prior art method, Lachmanovich et al. (Journal of Microscopy, 212, 2003, pp. 122-131) measured the co-localization of objects in a microscopic image by determining the percentage of an object in one channel of a microscope that overlaps (i.e., has overlapping pixels) or is adjacent (i.e., has border pixels) to an object in another channel. This method is based on counting the number of objects that overlap or are in close proximity to other objects and generates a null hypothesis of random interactions using raw pixel data.

[0012] Another approach relies on nearest neighbor distances, such as the method provided by Helmuth et al. cited above.

[0013] An excellent review of prior methods is presented by Bolte et al. (Journal of Microscopy, 224, 2006, pp. 213-232). They review the methods used in co-localization analysis. For object-based co-localization analysis, they report methods based solely on nearest neighbor analysis. Thus, focusing on nearest neighbors in such an analysis is considered a common criterion that has been used conventionally.

[0014] Another way to determine the tendency of interacting cells, i.e., the subject, is to (a) compare the distance distributions between two subject types or between cells of two distinguishable populations by a suitable method (e.g., Kolmogorov-Smirnov test) and compare the observed distance distribution with the null hypothesis distribution, or (b) measure the degree of cell surface contact between cells of two distinguishable populations.

[0015] As found by the inventors herein, the method of the present invention has unexpectedly great advantages over conventional methods. In particular, the method of the present invention is superior to the methods and alternative approaches described in the prior art in that it is generally robust with respect to sample characteristics. For example, the method of the present invention can provide more reliable results in settings of high subject density (e.g., PBMCs and bone marrow monolayers) as disclosed herein. Furthermore, the method of the present invention is more robust to changes in the number of cells and thus has a high dynamic range (a wide measurable range). Further, unlike the methods described in the art, none of the methods described in the art take into account subjects (i.e., cells) other than the subjects (i.e., cells) for which the tendency of interaction is to be determined. However, in particular, these subjects (i.e., cells) in the monolayers described herein occupy space and thus must be considered to obtain more reliable results. Further, unlike the methods described in the art, the method of the present invention functions robustly and efficiently with different populations of cells in terms of size, particularly for small ones from about 100 to 100,000 cells and for large ones over 1,000,000 cells. Finally, the method of the present invention shows improved results compared to conventional methods, but can be used more efficiently in a high-speed setting using computer-based algorithms. That is, compared to conventional methods, the method of the present invention requires less time and is thus more efficient in terms of cost.

[0016] A surprisingly large advantage of the above-described method of the present invention is obtained by determining the total number of intercellular interactions between one cell in a first subpopulation and a plurality of cells in a second subpopulation, taking into account the situation where one cell interacts with two or more cells of a second subpopulation, as compared to conventional methods. Thus, the method of the present invention is independent of the direction in which the tendency of interaction is determined (i.e., determining the cells in subpopulation A that interact with the cells of subpopulation B, or determining the cells of subpopulation B that interact with the cells of subpopulation A). Since there is no biological directionality in the physical interaction between two cells (i.e., when the first cell interacts with the second cell, the second cell interacts in the same way as the first cell), this is a more accurate numerical representation of the biological effect underlying the above technical advantage. This is particularly important when artificially representing natural states such as monolayers described herein and in WO2016 / 046346. Thus, since the method of the present invention is independent of the direction in which intercellular interactions are determined, more reliable interaction values can be obtained, particularly in single experiments. That is, determining the total number of intercellular interactions in a single instance by the method of the present invention is independent of whether the experiment is focused on the cells of the first or second distinguishable subpopulation. Thus, in addition to being improved with respect to the above characteristics, the method of the present invention provides a faster and easier method of determining intercellular interactions and / or a more cost-effective method.

[0017] Accordingly, in the method and population of cells of the present invention, the tendency of cell-cell interactions remains the same even when the first and second distinguishable subpopulations are swapped. Thus, in one particularly preferred embodiment, the present invention relates to a method for determining the tendency of cell-cell interactions in a population of cells comprising at least two distinguishable subpopulations of cells. The method comprises: (a) determining the number of interactions between cells of a first distinguishable subpopulation and cells of a second distinguishable subpopulation; (b) randomly assigning the cells contained in the population of cells to cells of the first and second distinguishable subpopulations and determining the number of interactions in the population of cells (wherein the absolute numbers of cells of the first and second distinguishable subpopulations are the same as the numbers of cells of the first and second distinguishable subpopulations in step (a)); and (c) dividing the result of (a) by the result of (b) to determine the tendency of cell-cell interactions (wherein the tendency increases with the number obtained and the tendency determined in step (c) remains the same even when the first and second distinguishable subpopulations are swapped). This tendency is independent of determining the interactions from the first distinguishable subpopulation to the second distinguishable subpopulation or vice versa, so a single experiment is sufficient. This enables a faster, easier, and / or more cost-efficient experimental setup compared to conventional methods.

[0018] The method of the present invention is independent of the method used to determine whether there is an interaction between two cells. In a preferred embodiment, the present invention utilizes a microscopic image of cells to determine whether there is an interaction between two cells. In a more preferred embodiment, the method utilizes a microscopic image of a monolayer of cells prepared by a method described in the art, such as WO2016 / 046346. Using a suitable dye such as an antibody fluorescently labeled against a cell surface marker, the cell surface can be outlined in a microscopic image by a method well known in the art, and it can be seen that the cell surfaces of the two cells are physically in contact. Alternatively, by determining the distance between cell nuclei, if the distance is less than a cut-off value having the magnitude of a typical cell diameter, it can be determined that the cells are interacting. The validity of this approximation can be inferred from the following two arguments. (i) Cells that are interacting, such as T cells and antigen-presenting cells (APCs), are typically in a certain process (also referred to as "scanning") where the interaction increases and decreases, dissociates, reassociates, and may dissociate again. Therefore, modeling the movement of cells between interactions as a random walk model, cells that associate more frequently (i.e., have a higher affinity for each other) are statistically closer to each other than other cells. (ii) When the actual cut-off value varies in the range of about 1 to 3 average cell diameters in a population of round cells with approximately equal diameters, it is shown that the interaction value does not change significantly.

[0019] Furthermore, the present invention relates to a population of cells obtained from a cell provider that is used to determine whether the cell provider has a disease or a predisposition to develop a disease. The determination includes determining a tendency of cell-cell interactions within the population, and the population includes cells of at least two distinguishable subpopulations. The method includes: (a) determining the number of interactions between cells of a first distinguishable subpopulation and cells of a second distinguishable subpopulation; (b) randomly assigning the cells included in the population of cells to cells of the first and second distinguishable subpopulations and determining the number of interactions in the population of cells (wherein the absolute numbers of cells of the first and second distinguishable subpopulations are the same as the numbers of cells of the first and second distinguishable subpopulations in (a)), (c) dividing (a) by (b) to determine the tendency of cell-cell interactions (wherein the tendency increases with the resulting number), and (d) determining based on the tendency of the cell-cell interactions whether the cell provider has a disease or a predisposition to develop a disease.

[0020] The present invention also provides a method for diagnosing a disease or a predisposition to a disease of a cell provider. The method includes determining a tendency of cell-cell interactions in a population of cells obtained from the provider, and the population of cells includes cells of at least two distinguishable subpopulations. The method includes: (a) determining the number of interactions between cells of a first distinguishable subpopulation and cells of a second distinguishable subpopulation; (b) randomly assigning the cells included in the population of cells to cells of the first and second distinguishable subpopulations and determining the number of interactions in the population of cells (wherein the absolute numbers of cells of the first and second distinguishable subpopulations are the same as the numbers of cells of the first and second distinguishable subpopulations in step (a)), (c) dividing the result of (a) by the result of (b) to determine the tendency of cell-cell interactions (wherein the tendency increases with the resulting number), and (d) determining based on the tendency of the cell-cell interactions whether the cell provider has a disease or a predisposition to develop a disease.

[0021] The present invention also relates to a method for determining whether or not a subject who has developed a disease or has a predisposition to develop a disease responds to or is responsive to treatment with a therapeutic agent by determining a change in the tendency of cell-cell interactions in a population of cells obtained from the subject. The cells of the population include cells of at least two distinguishable subpopulations. The method includes: (a) determining the number of interactions between cells of a first distinguishable subpopulation and cells of a second distinguishable subpopulation; (b) randomly assigning the cells included in the population of cells to cells of the first and second distinguishable subpopulations to determine the number of interactions in the population of cells (wherein the absolute numbers of cells of the first and second distinguishable subpopulations are the same as the numbers of cells of the first and second distinguishable subpopulations in step (a)); (c) dividing the result of (a) by the result of (b) to determine the tendency of cell-cell interactions (wherein the tendency increases with the resulting number); and (d) determining whether the subject responds to or is responsive to the therapeutic agent by comparing the tendency before addition of the therapeutic agent with the tendency after addition of the therapeutic agent, thereby determining a change in the tendency of cell-cell interactions.

[0022] Furthermore, the present invention relates to a population of cells obtained from a cell provider for use in a diagnostic method for determining whether a subject who has developed a disease or has a predisposition to develop a disease responds or is responsive to treatment with a therapeutic agent. The determination includes determining the tendency of cell-cell interactions in a monolayer, the monolayer including cells of at least two distinguishable subpopulations. The method includes: (a) determining the number of interactions between cells of a first distinguishable subpopulation and cells of a second distinguishable subpopulation; (b) randomly assigning the cells included in the cell sample to the cells of the first and second distinguishable subpopulations and determining the number of interactions in the cell sample (wherein the absolute numbers of the cells of the first and second distinguishable subpopulations are the same as the numbers of the cells of the first and second distinguishable subpopulations in step (a)); (c) dividing the result of (a) by the result of (b) to determine the tendency of cell-cell interactions (wherein the tendency increases with the resulting number); and (d) determining whether the subject responds or is responsive to the therapeutic agent by comparing the tendency before addition of the therapeutic agent with the tendency after addition of the therapeutic agent, and determining a change in the tendency of cell-cell interactions.

[0023] Furthermore, the present invention provides a method for screening a therapeutic agent by determining a change in the tendency of cell - cell interactions in a population of cells upon addition of one or more test substances. The population of cells includes cells of at least two distinguishable sub - populations, and the method comprises: (a) determining the number of interactions between cells of a first distinguishable sub - population and cells of a second distinguishable sub - population; (b) randomly assigning the cells included in the cell sample to the cells of the first and second distinguishable sub - populations and determining the number of interactions in the cell sample (wherein the absolute numbers of cells of the first and second distinguishable sub - populations are the same as the numbers of cells of the first and second distinguishable sub - populations in step (a)); (c) dividing the result of (a) by the result of (b) to determine the tendency of cell - cell interactions (wherein the tendency increases with the obtained number); and (d) comparing the tendency before addition of the one or more test substances with the tendency after addition of the one or more test substances to determine whether the one or more test substances are qualified as therapeutic agents and to determine a change in the tendency of cell - cell interactions.

[0024] Furthermore, the present invention relates to a population of cells for use in screening a therapeutic agent by determining a change in the tendency of cell - cell interactions in a population of cells upon addition of one or more test substances. The population of cells includes cells of at least two distinguishable sub - populations. The method comprises: (a) a step of determining the number of interactions between cells of a first distinguishable sub - population and cells of a second distinguishable sub - population; (b) a step of randomly assigning the cells included in the cell sample to the cells of the first and second distinguishable sub - populations and determining the number of interactions in the cell sample (wherein the absolute numbers of cells of the first and second distinguishable sub - populations are the same as the numbers of cells of the first and second distinguishable sub - populations in step (a)); (c) a step of dividing the result of (a) by the result of (b) to determine the tendency of cell - cell interactions (wherein the tendency increases with the obtained number); and (d) a step of comparing the tendency before addition of one or more test substances with the tendency after addition of one or more test substances to determine whether the one or more test substances are qualified as therapeutic agents and to determine a change in the tendency of cell - cell interactions.

[0025] In one embodiment, the first and second distinguishable subpopulations are the same.

[0026] In a preferred embodiment of the present invention, step (b) is repeated to average the number of determined interactions. It is preferred to repeat step (b) at least 1000 times more, at least 2000 times, or at least 3000 times.

[0027] In a preferred embodiment of the present invention, the cells used in the method of the present invention or the cells in the population of the present invention are peripheral blood mononuclear cells (PBMCs) or bone marrow cells.

[0028] The diseases diagnosed by the present invention are preferably myeloproliferative disorders, inflammatory disorders, latent viral infections, cell proliferation disorders, cell chemotaxis disorders, metabolic disorders or autoimmune diseases; or leukemia or lymphoma.

[0029] Accordingly, the present invention relates to the natural tendency of cell-cell interactions, namely the physical interaction of cells, and in particular to a method for quantifying peripheral blood mononuclear cells (PBMCs), bone marrow, or other multi-lineage primary mammalian materials (e.g., samples containing any mononuclear cells collected for diagnostic purposes such as detection in an image of the material). The method of the present invention is preferably carried out using a cell sample containing a monolayer of cells and a suitable image processing technique (e.g., confocal microscopy). The cells contained in the sample to be analyzed, e.g., the monolayer, may be identified and thus distinguished using a fluorescent dye or other markers known in the art. Accordingly, the term "identifiable subpopulation" as used herein refers to cells that are part of a larger population and can be identified from other cells within that population by cell markers. That is, the cells within two identifiable subpopulations may belong to the same cell type or to different cell types as long as the expression profiles of the cell markers that can be identified using an image processing technique such as confocal microscopy are different. To easily determine whether a cell belongs to a particular subpopulation of cells, it is preferred to provide the cells as a monolayer. As shown in the attached examples, the monolayer may be formed using methods known in the art, preferably the methods taught in WO2016 / 046346. Accordingly, in a preferred embodiment of the present invention, the method of the present invention further comprises forming a monolayer containing the cells of the cell sample used prior to step (a).

[0030] In the present invention, the tendency of cell-cell interaction is represented as an interaction value. Thus, with respect to exogenous factors that affect cell-cell interaction, such as cell density and amount, the expression of adhesion molecules, receptor-ligand pairs, and other cell-derived factors that mediate cell-cell interaction, or biological causes such as agents that physically crosslink cells (e.g., specific antibodies, bispecific antibodies, or biologics), the tendency of cell-cell interaction is represented as an interaction value. Thus, the present invention also relates to how the interaction value, as a measurement of the tendency of cell-cell interaction, can determine whether a sample of a disease and / or a healthy sample responds or does not respond to a therapeutic agent and / or a drug. The present invention further relates to the use of the interaction value for diagnosing a disease or a predisposition to a disease in a patient and / or a provider. The present invention also provides a method (screening method) using the interaction value to determine whether a disease responds or is responsive to treatment with a therapeutic agent, and / or to find and test the reaction mechanism and effect of a potential new therapeutic agent or drug. In this regard, the method of the present invention for testing whether a cell of a disease is responsive to a therapeutic agent / drug and / or a chemical substance as a potential new therapeutic agent / drug, i.e., whether its interaction value changes, generally relies on the use of a reference sample. For a test where a healthy cell or a diseased cell responds (i.e., the interaction value changes), this reference sample is compared to a reference symmetric stimulus, such as a stimulus with DMSO or PBS as a medium. When determining the cause of a disease or diagnosing a disease using the interaction value, the reference sample is from a healthy provider. Thus, in a preferred embodiment, the diagnostic method provided herein and the method for determining whether a provider responds to treatment use a sample obtained from a reference provider. The interaction value defined herein is used for, but not limited to, 1) the discovery of immunomodulatory drugs (i.e., drugs or other therapies that change the properties and / or behavior of immune system cells to obtain a guaranteed outcome), 2) the definition of the mechanism of action of a treatment method with a known immunomodulatory agent, 3) the analysis of the position (i.e., spatial arrangement) of cells in an image for the definition of the sensitivity or function of a drug in a patient sample in vitro.Therefore, the novel and inventive method of the present invention may be used in combination with image processing techniques, such as, for example, image processing of single-layer cells, particularly PBMCs or bone marrow cells. Also, the method of the present invention may be used to determine the relationship between cells and the change in the relationship after incubation with a chemical substance (e.g., small molecule, biological pharmaceutical, or other soluble factor). Also, the method of the present invention may be used to determine the changes in a population contained in a cell sample. Also, the method of the present invention may be used to determine the predisposition to a disease.

[0031] Accordingly, the present invention relates, inter alia, to a method for diagnosing a disease or predisposition to a disease of a cell provider. The method includes determining a tendency of cell-cell interactions in a population of cells obtained from the provider, the population of cells including cells of at least two distinguishable subpopulations. The method includes (a) determining the number of interactions between cells of a first distinguishable subpopulation and cells of a second distinguishable subpopulation, (b) randomly assigning the cells included in the population of cells to cells of the first and second distinguishable subpopulations to determine the number of interactions in the population of cells (wherein the absolute numbers of cells of the first and second distinguishable subpopulations are the same as the numbers of cells of the first and second distinguishable subpopulations in step (a)), (c) dividing the result of (a) by the result of (b) to determine the tendency of cell-cell interactions (wherein the tendency increases with the resulting number), and (d) determining whether the cell provider has a disease or has a predisposition to develop a disease based on the tendency of the cell-cell interactions. In a preferred embodiment, step (b) is repeated to average the determined numbers of interactions. It is preferred to repeat step (b) at least 1000 times more, at least 2000 times, or at least 3000 times.

[0032] Furthermore, the therapeutic value of a drug or treatment may be determined and predicted by quantifying physical interactions between cells using the method of the present invention. Accordingly, the present invention also relates to a method for determining whether a subject suffering from or predisposed to a disease responds or is responsive to treatment with a therapeutic agent by determining a change in the tendency of cells to interact in a population of cells obtained from the subject. The population of cells includes cells of at least two distinguishable subpopulations, and the method includes: (a) determining the number of interactions between cells of a first distinguishable subpopulation and cells of a second distinguishable subpopulation; (b) randomly assigning the cells included in the population of cells to cells of the first and second distinguishable subpopulations to determine the number of interactions in the population of cells (wherein the absolute numbers of cells of the first and second distinguishable subpopulations are the same as the numbers of cells of the first and second distinguishable subpopulations in (a)); (c) dividing the result of (a) by the result of (b) to determine the tendency of interactions between cells (wherein the tendency increases with the obtained number); and (d) determining whether the subject responds or is responsive to the therapeutic agent by comparing the tendency before adding the therapeutic agent with the tendency after adding the therapeutic agent, and determining a change in the tendency of interactions between cells. In a preferred embodiment, step (b) is repeated to average the determined numbers of interactions. It is preferable to repeat step (b) at least more than 1000 times, at least 2000 times, or at least 3000 times.

[0033] The present invention is also illustrated by the following drawings in several aspects.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0046] As shown in the attached examples, the method of the present invention can provide a unique screening system that can be used particularly in biological, biochemical, and biophysical research. Further, the method of the present invention may be used in medical diagnostic and screening methods such as, for example, automated medical diagnosis and screening methods. Refer to Example 5. The method provided herein requires a minimal amount of material from the provider. For example, using the number of cells obtained by a normal diagnostic analysis protocol, more perturbations (such as individual test conditions, etc.) than are possible with current methods known in the art can be tested or analyzed per unit of provided material. Specifically, in a preferred embodiment, the cells of the population of the present invention used in the method of the present invention are about 100 cells / mm 2 (culture area) to about 30,000 cells / mm 2It is in the form of a single layer of cells formed using cells cultured in a culture device at the density of 2 (culture area) ~ about 20,000 cells / mm 2 (culture area), about 1,000 cells / mm 2 (culture area) ~ about 10,000 cells / mm 2 (culture area), about 1,000 cells / mm 2 (culture area) ~ about 5,000 cells / mm 2 (culture area), or about 1,000 cells / mm 2 (culture area) ~ about 3,000 cells / mm 2 (culture area). Most preferably, the cells are cultured at a density of about 2,000 cells / mm 2 (culture area). When the cells are cultured at the above-mentioned density, a single layer of cells is formed. The cells may be stained, fixed, and / or subjected to image processing before the analysis using the method of the present invention.

[0047] As used herein, the term "cell-cell interaction" particularly refers to direct interactions between cell surfaces that play important roles in the development and function of multicellular organisms. Through these interactions, cells can exchange information in response to changes in their microenvironment. Such cell-cell interactions may be stable, such as those occurring at cell junctions. These junctions are particularly involved in information exchange and organization between cells within tissues. Others are transient or temporary, such as interactions between cells of the immune system or interactions involved in tissue inflammation. This type of cell-cell interaction is distinguished from other types, such as the interaction between cells and the extracellular matrix.

[0048] In the method of the present invention and / or the cells of the population of the present invention, the naturally occurring cell-cell interactions are preferably maintained when forming the phenotype of the cells to be analyzed. That is, when forming a monolayer of cells used in a preferred embodiment of the present invention, the cell-cell interactions defined herein are maintained. Maintaining cell-cell interactions means that cells that interact with other cells in a natural environment also interact with other cells or cells of the same kind before the method of the present invention and during the formation of the monolayer provided herein. That is, the overall cell-cell interactions are maintained, but the cells do not necessarily maintain interactions with the same cells they were interacting with. Therefore, the method provided herein provides a model system that represents the physiologically appropriate state of the cells to be analyzed.

[0049] The above cell-cell interactions are preferably used in the method of the present invention and maintained when forming the phenotype of the cells formed by the cells of the population of the present invention, and thus are preferably maintained in the monolayer formed before the method of the present invention. Generally, cell-cell interactions occur naturally in samples containing, for example, PBMCs, bone marrow cells, or other cells. A person skilled in the art can determine whether cell-cell interactions occur naturally and whether they are preferably maintained by the cells in a monolayer during the formation of the sample. In particular, a person skilled in the art can use methods well known in the art. In particular, cell-cell interactions are maintained when detectable labels and / or dyes, especially viability dyes, are added. After the formation of the cell sample, preferably a monolayer, the cell-cell interactions may be interrupted, for example, by fixation of the cells, preferably the monolayer, before image processing.

[0050] The morphological form of the cells, preferably after monolayer formation, and the cell-cell interactions may be disrupted or newly formed by the addition of a chemical substance (e.g., a therapeutic agent) during the implementation of the method of the present invention. In the method of the present invention, such changes / variations may be used to determine whether a chemical substance therapeutic agent affects cell-cell interactions that may be predictive of a therapeutic response. In addition to or alternatively to this, the changes / variations may be used to screen for novel therapeutic agents / drugs from, for example, a chemical library. In a preferred embodiment of the present invention, the chemical substance / test compound / therapeutic agent is added before forming a monolayer of cells. This may be done, for example, by pre-filling in the form of supply droplets containing the chemical substance / test compound / therapeutic agent dissolved in DMSO or water using a supply system known to those skilled in the art (e.g., the Labcyte ECHO system). Also, this chemical substance / test compound / therapeutic agent may be dissolved in a small amount (preferably less than 20 μl, less than 15 μl, most preferably less than 10 μl) of culture medium and supplied to the wells of a multi-well plate, preferably a plate such as that described in WO2017 / 191203. In this preferred embodiment, cells for analysis symmetry are added to form a monolayer of cells, preferably PBMCs or bone marrow cells, before fixing the monolayer formed after the addition of the chemical substance / test compound / therapeutic agent. Then, the fixed monolayer may be stained with an appropriate dye and / or antibody and analyzed according to the present invention, for example, using an automated confocal microscope. Based on the obtained images, the tendency of cell-cell interactions may be determined as provided herein. The step of preparing a sample to be analyzed by the method of the present invention is preferably in this order and has been shown to have even better beneficial properties.

[0051] The morphological form of the cells used in the method of the present invention, preferably maintained when forming a monolayer, and the cell-cell interactions maintained before the method of the present invention include, for example, in particular, cell-cell interactions in PBMCs, bone marrow, or other materials containing mononuclear cells indicative of a specific disease and / or a healthy provider. [Table 1-1] [Table 1-2]

[0052] Thus, naturally occurring cell-cell interactions may be determined / evaluated / detected using cell markers. A cell marker is a protein expressed by a specific type of cell that, alone or in combination with other proteins, enables this cell to be distinguished from other types of cells. That is, cell markers expressed on the surface or within the cell (including within the cytoplasm or within internal membranes) of the cells contained in the population of cells of the present invention (e.g., the cells contained in a sample obtained from a provider) may be used to identify the cells contained in the population of cells. Thus, the two or more distinguishable subpopulations of cells are not limited to cells belonging to different cell types. Rather, as long as the two or more distinguishable subpopulations of cells are distinguishable by cell markers (e.g., cell markers expressed on the surface), the cells of these subpopulations may be of the same cell type. Such cells include, for example, cells of the same cell type with different disease stages. That is, the naturally occurring cell-cell interactions shown in the above table may be determined / evaluated / detected using the markers described above. For example, in particular, the cell marker pairs of CD11c and CD3, CD14 and CD3, CD11C and CD8, and CD19 and CD3 may be used to detect the naturally occurring cell-cell interactions indicative of a healthy provider. However, in one embodiment of the present invention, the first and second distinguishable subpopulations are the same.

[0053] When detecting / labeling the cell morphology of cells, e.g., the cells of the population of the present invention, preferably the cells contained in a monolayer, using the above cell markers to measure the cell-cell interaction tendency of positive cells indicative of a specific disease, the disease associated with the cell provider may be diagnosed and / or the treatment of the associated disease may be evaluated. Also, when detecting / labeling the cell morphology of cells, e.g., the cells of the population of the present invention, preferably the cells contained in a monolayer, using the markers indicative of a healthy provider, the naturally occurring cell-cell interactions of these healthy providers may be evaluated / determined.

[0054] As described above, those skilled in the art know the means and methods for determining / evaluating / tracking / confirming cell-cell interactions. In particular, those skilled in the art can distinguish between naturally occurring cell-cell interactions and those introduced during the preparation of cell samples. Thus, those skilled in the art understand that cells of the same type and / or different types interact in vivo. Also, those skilled in the art understand that cells of an identifiable subpopulation of cells contained in a population of cells interact in vivo. Thus, most of the cells contained in a cell sample maintain their naturally occurring cell-cell interactions. That is, most of the cells, particularly at least 50% of the cells contained in a cell sample, preferably 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the cells contained in a cell sample, interact with the same cells, cells of the same cell type, or cells of the same identifiable subpopulation of cells as in vivo. Cell-cell interactions may be confirmed / evaluated / determined using methods well known in the art. For example, confocal microscopy may be used to confirm / evaluate / determine whether there is a cell-cell interaction between cells that interact in a natural environment or between cells that do not interact in a natural environment. Such non-natural cell-cell interactions may occur particularly in the case of cell aggregates.

[0055] The method of the present invention controls the number and changes / variations in the observed cell-cell interactions in the cells of each distinguishable subpopulation contained in a population of cells / monolayer, thereby controlling cytotoxicity and / or cell growth at the overall cell density. The method of the present invention is not equivalent to other methods of analyzing adherent cell lines understood in the art, i.e., other methods of determining clonal density of cells, clonal expansion, or development of a cell line monolayer. Rather, the sample used in the method of the present invention may comprise a high-density culture mainly containing cells that are in direct contact with one or more other cells but are not necessarily attached to the culture surface, or may comprise a low-density culture in which only a few cells in the sample, preferably in the monolayer, are in direct physical contact with other cells in the sample, preferably in the monolayer. The method of the present invention may be used with samples containing intermediate density adherent and / or non-adherent cells. This sample has individual regions where cells are in contact with one or more cells and other regions where cells are not in contact with other cells. Thus, the method of the present invention provides a unique means that can be used with a wide variety of cell samples without further adaptation. Thus, the method of the present invention can be used for a wide range of applications. This is a significant advantage compared to methods known in the art. Since the method of the present invention requires little or no further adaptation to a particular sample, the method of the present invention is more efficient in terms of cost and time than conventional methods.

[0056] Specifically, conventional methods used to analyze cell-cell contacts in a plurality of monolayer non-adherent mononuclear cells or adherent cells and mixtures of non-adherent mononuclear cells derived from the blood or bone marrow of diseased or healthy providers using an automated microscope are described in the prior art, for example, in WO2016 / 046346. Briefly, the cells are processed to form a monolayer of non-adherent cells or a mixture of adherent cells and non-adherent mononuclear cells, exposed to a stimulus for a predetermined time, fixed, stained with a fluorescent labeling reagent, and cells with the desired properties such as fluorescently labeled antibodies are identified. Also, the novel method of the present invention described in WO2016 / 046346, which provides a monolayer of cells, particularly PBMCs or bone marrow cells, may be used in the present invention to provide a suitable image processing sample of the cells to be analyzed. However, the present invention focuses on such image analysis rather than the provision of a suitable sample. However, unlike the above method of preparing a monolayer of mononuclear cells, a physiologically appropriate cell-cell contact is maintained by using the system described in WO2016 / 046346 that maintains a stable cell environment. When using the analysis method described in WO2016 / 046346, cell-cell contacts are inferred from a microscopic image of the monolayer by defining a threshold value. If two cells are closer than the set threshold value, they are considered to be interacting, and vice versa. The number of cells in contact with other cells depends on the total cell density in the microscopic image of the mononuclear cell monolayer. Furthermore, when analyzing the interaction between two clearly distinguishable cell types A and B, the number of A cells in contact with B cells also depends on the ratio of A cells and B cells in the whole sample and their relative ratios. Therefore, little biological information can be obtained by simply counting the cells in contact with B cells in a microscopic image. To measure the inherent tendency regarding the interaction between cell types A and B (i.e., the tendency of cell-cell interaction due to the biological properties associated with the cells), it is necessary to normalize the number of cells in contact with B cells with respect to the total cell density and the fractions of A cells and B cells in the sample. The prior art has achieved this using the formula Obs = (number of A cells adjacent to at least one B cell) ÷ (number of A cells). Here, Fi = (number of cells with at least one adjacent cell) ÷ (total number of cells), Fa = (number of A cells) ÷ (total number of cells), and Fb = (number of B cells) ÷ (total number of cells).Therefore, E = Fa * Fb * Fi, and the normalized interaction value is calculated as the fraction of Obs / E. Typically, log2 of this fraction is obtained as a measure of the original trend regarding the interaction between cell types A and B. When the total cell density of the analyzed images is low (one interacting cell per cell) and the changes in Fa and Fb throughout the experiment are limited, the changes in cell density (Fi) and the fractions of the interacting cell types (Fa and Fb) are robustly normalized by this method of the prior art. However, when the cell density is high (more than one interacting cell per cell) and when large changes in Fa are predicted throughout the experiment (Examples 3 and 4), the interaction value becomes increasingly inaccurate. This drawback is overcome by providing the method of the present invention. Therefore, the method of the present invention is suitable for use with a wide range of samples, particularly samples with high cell density and / or samples that are subject to changes in the relative population of cells.

[0057] In particular, to solve this technical problem, the observed interaction I between cells of type A and cells of type B obs (A->B) is counted. A and B are two distinguishable subpopulations of cells, and the interaction is defined as two cells closer than a specific threshold. The total number of cells analyzed is the sum of the number of cells of type A, the number of cells of type B, and the number of cells that are neither type A nor type B.

[0058] Therefore, I obs (A - B) is randomly I rand divided by I (A - B), and I rand for the interaction predicted to be caused by I (A - B) obs(A - B) is normalized. Thus, in the method of the present invention, the number of interactions between A and B (i.e., the first and second distinguishable subpopulations) is determined by the method described herein. In a second step, by randomly assigning the cells of the population to either subgroup A or B, the number of interactions between A and B (i.e., the first and second distinguishable subpopulations) that is theoretically predicted based on relative occurrence is determined. This results in the total number of cells in each subgroup being the same. That is, a cell A that is at a threshold distance to cell B may or may not interact with a cell of subgroup B after being randomly assigned to either A or B depending on randomly assigning that cell and its adjacent cells. Thus, the total number of observed interactions may be more or less than that predicted based on the cells randomly assigned to subgroup A or B depending on the tendency of cell - cell interactions between A and B. To reliably determine the number of interactions by randomly assigning cells to subgroup A or B, it is preferable to repeat this step and average the results. Preferably, this step is repeated at least 1000 times, at least 2000 times, or at least 3000 times. Alternatively, I rand (A->B) is the total number of interactions I between all cells, as defined above tot may be calculated by counting, and is the fraction of A cells in all cells (analyzed F A ) multiplied by the fraction of B cells in all cells (analyzed F B ). Thus, the tendency of interactions, represented as an interaction value (IS’) measured by the present invention, may be defined as follows.

Equation

[0059] The threshold value is defined as a positive number. If the distance between two cells is less than the threshold value, they are considered to be interacting. Define the same fixed threshold value for all cells to be analyzed. Here, the selection of the threshold value is closely related to the type of interaction being investigated. The lower the threshold value, the more sensitive it is to changes in high-affinity and long-term interactions. Also, the higher the threshold value, the more sensitive it is to lower affinity and thus more transient interactions. Also, the threshold value must be set according to the overall size of the cell study. Larger cells require a higher threshold value, and smaller cells require a smaller threshold value.

[0060] Depending on the application, if a positive control that regulates the interaction in a defined direction is available, the optimal threshold value leading to the maximum measurement sensitivity may be determined experimentally. Here, for each rationally selected from among the threshold values within the limits defined above, calculate IS’ after treatment with the positive control and the negative control, and obtain a threshold value such that the difference between IS’ under the positive control condition and IS’ under the negative control condition is maximized and the selected standard deviation is minimized. Typically, in the method of the present invention, and the threshold value selected for the monolayer analysis of the present invention is within the range of about 2 times the diameter of the cells to be analyzed. Therefore, in a preferred embodiment of the present invention, the threshold value is in the range of about 5 μm to about 35 μm, more preferably about 10 μm to about 20 μm, and even more preferably about 10 μm to about 15 μm. In a particularly preferred embodiment, the threshold value is about 12 μm. That is, in a preferred embodiment, the number of interactions in each cell of the first distinguishable subpopulation increases by the number of cells of the second distinguishable subpopulation that are at a distance of less than about 12 μm from the cells of the first distinguishable subpopulation. This distance is preferably measured from the center to the center of the cells to be evaluated.

[0061] Accordingly, as shown in Examples 3 and 4, the present invention provides a method for quantifying the natural tendency of cell-cell interactions that robustly normalizes changes in cell density and / or the total number of cells in the population to be analyzed and / or the total number of cells in a subpopulation and / or the relative number of cells in the analyzed subpopulation when applied to microscopic images of cells, particularly PBMCs, bone marrow, other adherent primary mononuclear and / or non-adherent primary mononuclear cells, or other cell materials.

[0062] The cells used in the method of the present invention and / or the cells of the population of the present invention, preferably the cells used in the preparation of a sample containing a monolayer, particularly PBMCs, bone marrow, or other adherent primary mononuclear and / or non-adherent primary mononuclear cells, may be isolated from a sample obtained from a healthy (i.e., not suspected of having developed a disease or having a predisposition to a disease) subject, or may be isolated from a sample obtained from a subject known or suspected of having developed a disease. The disease state of the subject may be diagnosed by standard methods routinely performed by those skilled in the art, such as physicians. Such traditional methods may be supplemented or replaced by the method of the present invention. For example, to determine whether a subject has developed a disease or is suspected of having developed a disease, a cell-cell interaction pattern characteristic of the disease is determined using a sample from a subject known to have developed the disease. In addition to, or instead of, this, the differences that appear to be due to the disease may be determined using the cell-cell interaction pattern of a healthy provider. As used herein, a cell-cell interaction pattern refers to the tendency of interactions between one or more different cell types determined by the present invention.

[0063] Accordingly, the present invention provides a method for use in a diagnostic method for determining whether a disease responds to a therapeutic agent or is responsive. For example, it may be compared to an intercellular interaction pattern determined for a subject with a disease, using as a reference an intercellular interaction pattern derived from a healthy provider. After adding a therapeutic agent, it may be determined whether the overall intercellular interaction pattern has changed / varied relative to the intercellular interaction pattern of a healthy subject used as a reference, using the change / variation in the intercellular interaction pattern. Alternatively, or in addition, after adding a therapeutic agent, it may be determined whether the overall intercellular interaction pattern has changed / varied relative to the intercellular interaction pattern predicted from the mechanism of action of the therapeutic agent, using the change / variation in the intercellular interaction pattern (Example 10). Accordingly, the methods provided herein can quantify cell responses and activities at the single cell, multiple cell, and whole population level (e.g., changes in the entire population), and thus provide a unique system for evaluating the response of cells, particularly one or more therapeutic agents to PBMCs or bone marrow. Accordingly, the methods provided herein can be used, inter alia, as a comprehensive model that can provide, for example, an assessment of therapeutic response or the likelihood of a therapeutic response when cells, particularly PBMCs or bone marrow, are isolated from a sample obtained from a healthy subject.

[0064] The treatment response determined using the method of the invention or the cells of the population of the invention in the form of a monolayer of cells from two or more samples obtained from two or more providers who preferably have the same disease and / or a predisposition to develop the same disease (or a combination thereof) may be used to develop predictive criteria, baselines, or predicted responses representative of a population having the disease or a predisposition to develop the disease. Alternatively, or in addition thereto, the methods provided herein may be used as a diagnostic method to predict whether the provider who provided the sample from which the cells were isolated has the disease or a predisposition thereto and / or whether the cell provider will respond or be responsive to treatment with a therapeutic agent. The "disease" described in the present invention may refer to blood cancer, malignant hematological tumors, solid tumor cancers, solid tumor cancers seeded in peripheral tissues, inflammatory diseases (e.g., arthritis), atherosclerosis (collected from the site where the disease has developed or in the vicinity thereof), and any other disease mediated by physical information exchange between cells or physical information exchange caused by soluble factors during the progression of the disease.

[0065] To diagnose a disease of an individual using the method of the invention, the interactions between one or more cell types in a cell sample of the individual are measured. These interactions are different between individuals having the disease or a predisposition to develop the disease and healthy individuals. Further, to determine whether a patient will respond to a particular treatment using the method of the invention, the interactions between one or more cell types in the patient's cell sample are measured for samples of the patient's cells with and without the drug of interest added thereto. These interactions are different between individuals who respond to the treatment and those who do not.

[0066] For example, current methods may be used to diagnose diseases such as rheumatoid arthritis. Here, the tendency of the interaction between activated B cells (CD80+ and CD19+) and Th17 cells (CD3+ and CD28+) in synovial fluid or peripheral blood may be quantified to measure the diagnosis, severity, and drug effects of the disease on the disease. The closer the interaction of these activated cells, the higher the likelihood of being diagnosed positive for rheumatoid arthritis. The same may be done for diseases such as chronic reactive arthritis. This disease is classified as a secondary sterile inflammation after bacterial infection, and the interaction between monocytes (CD14+) and T cells (CD3+) in synovial fluid may be quantified and diagnosed after removing the bacterial pathogen. As a final example, the spatial interaction between resident inflammatory monocytes (CCR2−, CD16+) and the patient's endothelial cells (CCR5+) in the blood may be determined to diagnose atherosclerosis. All follow-up, severity determination, and drug effect quantification of these diseases may be performed using this method.

[0067] The analysis of the therapeutic response of cells in an image using the method provided herein predicts the response of the disease state to the treatment tested by the provider. In this regard, the method of the present invention has advantages over current methods available in the art. For example, when the method provided herein is applied to an image of a non-adherent material from a patient with blood cancer, not only the spatial resolution of normal cells but also the spatial resolution of diseased cells, such as cells with abnormal phenotypes or genotypes, or cells representing the disease state (e.g., having a high or low concentration relative to the concentration predicted in a healthy individual) is quantified.

[0068] Therefore, the relationship of cells in an image may be tracked after the addition of a therapeutic agent using the method provided herein. This is the quantification of the immunomodulatory ability of the therapeutic agent. Also, from these results, it can be determined whether the therapeutic agent has activity against immunomodulation and whether it can be transferred to the clinic for the treatment of the patient.

[0069] In the method of the present invention, most of the cells of the population of the present invention, preferably the cells contained in a monolayer, are observed in a physiologically appropriate state. This preferably means that 60%, 70%, 80%, 90%, 95%, or 100% of the cells are in a physiologically appropriate state. The above percentage of the cells of the population of the present invention, preferably the cells contained in a monolayer, used in the method of the present invention is determined / measured / evaluated using methods well known in the art. In particular, whether a cell sample contains cells observed in a physiologically appropriate state is determined by quantifying the cells contained in the cell sample / monolayer. This may be done using methods well known in the art. In particular, it may be quantified by image analysis compared to the cells in a reference sample. This reference sample is, for example, peripheral blood or bone marrow of one or more reference individuals (e.g., one or more healthy donors), and the cell sample, particularly the PBMC or bone marrow cell sample, is from a diseased donor. Quantification of cells is a standard diagnostic means. Thresholds for subpopulations of cells contained in cell samples, particularly PBMCs and / or bone marrow cells, are well established for healthy donors and diseased donors. Thus, physiological appropriateness may be determined based on differences in samples evaluated using the means and methods of the present invention. Validation of subpopulations of cells contained in hematopoietic cells can be found, for example, in Hallek et al. (2008) Blood 111(12). Thus, it is possible to determine whether a cell sample exhibits a physiologically appropriate state by quantification and further means and methods, such as determination of cell-cell interactions using a microscope.

[0070] In a preferred embodiment of the present invention, the cells are analyzed in monolayer form. In particular, the monolayer is formed prior to the method of the present invention, whereby image processing and / or microscopic analysis can be performed on a population of cells, in particular a PBMC population and / or a bone marrow population. Thus, the monolayer as used herein means a monolayer of cells that is mainly observed within the same focal plane of an image processing device (e.g., a microscope or automated camera known in the art or described herein). The term "monolayer" means that the cells within this layer mainly form a two-dimensional culture, i.e., this culture consists mainly of a monolayer of single cells. That is, within this culture, most of the cells are not observed on the surface or above other cells and are not observed within aggregates (e.g., consisting of cell groups above the single cells of this layer by including cells on the surface or above other cells). Thus, the cell monolayer according to the present invention, in particular the PBMC monolayer, preferably comprises a horizontal layer of cells, in particular PBMC cells, and this horizontal layer has a thickness of the height of a single cell, in particular a PBMC. Similarly, the bone marrow cell monolayer according to the present invention preferably comprises a horizontal layer of bone marrow cells having a thickness of the height of a single bone marrow cell. The term "monolayer" as used herein does not exclude the observation of cell aggregates or multi-layered constructs (i.e., regions where there are cell cultures with a height greater than that of a single cell, in particular each PBMC cell or a single bone marrow cell) or cell-free regions within the culture vessel. Rather, using this term, it means that the culture of the present invention is capable of being image-processed (e.g., by microscopy) or the visualized region consists of a monolayer of cells. This is easily achieved in most cases by providing a monolayer of cells on the surface of the cell culture. However, it will be apparent to those skilled in the art that other forms of cell samples may be used in the method of the present invention. That is, any cell representation may be used in the present invention as long as cell-cell interactions can be quantified.

[0071] In the case of non-adherent cells to be analyzed, such as PBMCs or bone marrow cells, it is known that such cells typically do not form strong contact with the surface of the cell culture or strong contact between cells. Therefore, the cell monolayers used in the present invention, particularly the PBMC monolayers used in various aspects of the present invention, are not necessarily regarded as equivalent to monolayer adherent cells understood in the art. That is, the monolayer cells used in the present invention include a layer of cells that adhere firmly, spread evenly, and cover most of the culture surface. Rather, depending on the embodiment, the cell monolayer, particularly the PBMC monolayer used in various aspects of the present invention, may include a high-density culture mainly containing cells that are in direct contact with one or more other cells but are not necessarily attached to the culture surface, or the cells may be in the monolayer but may include a low-density culture that is not in (physically direct) contact with any other cells in the culture. Also, the cell monolayer used in a particular aspect of the present invention may include a medium-density culture having individual regions where the cells contact one or more cells and other regions where the cells do not contact other cells.

[0072] In some embodiments, the present invention relates to a method for determining whether a subject having a disease or a predisposition to develop a disease responds or is responsive to treatment with a therapeutic agent and / or a pharmaceutical composition. The therapeutic agent and / or pharmaceutical composition includes compounds used for the treatment of a disease of an individual, particularly a malignant blood disease, and / or a malignant tumor or an inflammatory disease or an autoimmune disease affecting the immune system such as a disease of the bone marrow and / or lymphoid tissue. The therapeutic agent and / or pharmaceutical composition is selected from at least two or more test compounds, and each of the at least two or more test compounds is tested in a population of cells in an assay comprising the following steps. The assay comprises: (a) determining the number of interactions between cells of a first distinguishable subpopulation of cells and cells of a second distinguishable subpopulation of cells contained in the population of cells; (b) randomly assigning the cells contained in the population of cells to cells of the first and second distinguishable subpopulations of cells to determine the number of interactions in the population of cells (wherein the absolute numbers of cells of the first and second distinguishable subpopulations of cells are the same as the numbers of cells of the first and second distinguishable subpopulations of cells in step (a), respectively); (c) dividing the result of (a) by the result of (b) to determine the tendency of cell-cell interactions (wherein the tendency increases with the number obtained); and (d) determining whether the subject responds or is responsive to the therapeutic agent by comparing the tendency before adding the therapeutic agent with the tendency after adding the therapeutic agent, and determining a change in the tendency of cell-cell interactions. The assay is repeated for each of the at least two or more test compounds, and a compound that decreases or increases the tendency of cell-cell interactions is selected for treatment and / or as a component of the pharmaceutical composition.

[0073] The above method may also be used as a screening method for novel therapeutic agents / drugs. That is, in one embodiment of the present invention, there is provided a screening method for selecting a therapeutic agent or pharmaceutical composition by determining a change in the tendency of cell-cell interactions in a population of cells by the addition of one or more test substances. The population of cells includes cells of at least two distinguishable subpopulations, and the method comprises: (a) determining the number of interactions between cells of a first distinguishable subpopulation and cells of a second distinguishable subpopulation; (b) randomly assigning the cells included in the population of cells to cells of the first and second distinguishable subpopulations and determining the number of interactions in the population of cells (wherein the absolute numbers of cells of the first and second distinguishable subpopulations are the same as the numbers of cells of the first and second distinguishable subpopulations in (a)); (c) dividing the result of (a) by the result of (b) to determine the tendency of cell-cell interactions (wherein the tendency increases with the number obtained); and (d) comparing the tendency before and after the addition of the test compound to thereby determine whether the test compound has changed / altered the tendency of cell-cell interactions and determine the change in the tendency of cell-cell interactions. The assay is repeated for each of the at least two or more test compounds, and a test compound that decreases or increases the tendency of cell-cell interactions is selected as a therapeutic agent / pharmaceutical composition. Preferably, different fractions of the same population of cells are used to measure the change / alteration in tendency and determine the number of interactions before and after the addition of the test compound. This is particularly useful when a monolayer of the cell sample is used, and the monolayer is fixed before analysis.

[0074] Using detectable labels / markers / dyes, the viability and / or cell-cell interactions of cells contained in a monolayer can be determined and / or changes in viability and / or cell-cell interactions can be determined / evaluated / tracked / confirmed. Such labels / markers / dyes may be specific for one or more subpopulations contained in the monolayer of the present invention. When using such specific labels / markers / dyes, they may be selected for cell types that play a role in various diseases and / or are known to have biological functions in diseases, particularly malignant blood diseases, and / or malignancies of the bone marrow and / or lymphoid tissues.

[0075] Accordingly, in certain aspects, the present invention relates to a method of determining a change in the tendency of cell-cell interactions in a population of cells obtained from a subject having or at risk of developing a disease, to determine whether the subject responds or is responsive to treatment with a therapeutic agent and / or pharmaceutical composition comprising a compound for treating the disease. The population of cells comprises cells of at least two distinguishable subpopulations, and the method comprises: (a) determining the number of interactions between cells of a first distinguishable subpopulation and cells of a second distinguishable subpopulation contained in a cell sample obtained from the subject; (b) randomly assigning the cells contained in the cell sample to cells of the first and second distinguishable subpopulations and determining the number of interactions in the cell sample (wherein the absolute numbers of cells of the first and second distinguishable subpopulations are the same as the numbers of cells of the first and second distinguishable subpopulations in step (a)); (c) dividing the result of (a) by the result of (b) to determine the tendency of cell-cell interactions (wherein the tendency increases with the number obtained); and (d) determining whether the subject responds or is responsive to the therapeutic agent by comparing the tendency before addition of the therapeutic agent with the tendency after addition of the therapeutic agent, thereby determining a change in the tendency of cell-cell interactions. In one exemplary embodiment, the first and second distinguishable subpopulations are CD3-positive cells and CD34-positive cells, or CD34-positive cells and CD3-positive cells, respectively. Preferably, different fractions of the same cell sample are used to measure the change in tendency, and the number of interactions before and after addition of the test compound is determined. This is particularly useful when using a monolayer cell sample, which is fixed prior to analysis.

[0076] The therapeutic agents used in this specification or the pharmaceutical composition of the present invention include compounds selected from at least two or more test compounds. The test compounds are not particularly limited as long as they are suitable for use as pharmaceuticals. However, the test compounds are preferably selected from compounds that have been found to be effective in the treatment of diseases, particularly malignant blood diseases, and / or malignancies of the bone marrow and / or lymphoid tissue, inflammatory diseases, and autoimmune diseases. Compounds that have been found to be effective in the treatment of such diseases include chemical compounds and biological compounds (e.g., antibodies). Compounds that have been found to be effective in the treatment of such diseases include, but are not limited to, the following: alemtuzumab, anagrelide, arsenic trioxide, asparaginase, ATRA, azacitidine, bendamustine, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, busulfan, cepharanthine, chlorambucil, cladribine, clofarabine, cyclophosphamide, cytarabine, dasatinib, daunorubicin, decitabine, denileukin diftitox, dexamethasone, doxorubicin, duvelisib, EGCG = epigallocatechin gallate, etoposide, filgrastim, fludarabine, gemtuzumab ozogamicin, histamine dihydrochloride, homoharringtonine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, interferon α-2a (recombinant), interferon α-2b (recombinant), intravenous immunoglobulin, L-asparaginase, lenalidomide, masitinib, melphalan, mercaptopurine, methotrexate, midostaurin, mitoxantrone, MK-3475 = pembrolizumab, nilotinib, pegaspargase, peginterferon α-2a, prerixafor, ponatinib, prednisolone, prednisone, R115777, RAD001 (everolimus), rituximab, ruxolitinib, selinexor (Selinexor,KPT-330, sorafenib, sunitinib, thalidomide, topotecan, tretinoin, vinblastine, vincristine, vorinostat, zoledronate, ABL001, ABT-199 = venetoclax, ABT-263 = navitoclax, ABT-510, ABT-737, ABT-869 = lenifanib, AC220 = quizartinib, AE-941 = neovastat, AG-858, AGRO100, aminopterin, asparaginase Erwinia chrysanthemi, AT7519, AT9283, AVN-944, bafetinib, bevacizumab, bestatin, beta-aretin, bexarotene, BEZ235, BI 2536, buparlisib (BKM120), carfilzomib, carmustine, ceritinib, CGC-11047, CHIR-258, CHR-2797, CMC-544 = inotuzumab ozogamicin, CMLVAX100, CNF1010, CP-4055, cleforanib, crizotinib, ellagic acid, elsamitrucin, epoetin zeta, epratuzumab, FAV-201, Fabd (FavId), flavopiridol, G4544, galiximab, gallium maltolate, gallium nitrate, givinostat, GMX1777, GPI-0100, Grn163l, GTI 2040, IDM-4, interferon alphacon-1, IPH 1101, ISS-1018, ixabepilone, JQ1, lestaurtinib, mechlorethamine, MEDI4736, MGCD-0103, MLN-518 = tandutinib, motexafin gadolinium, natural alpha interferon, nelarabine, obatoclax, obinutuzumab, OSI-461, panobinostat, PF-114, PI-88, pivoxil butyrate, pixantrone, pomalidomide, PPI-2458, pralatrexate, proleukin, PU-H71, ranolazine, rebastinib, samarium (153sm) lexidronam, SGN-30, Skeletal targeted radiotherapy, tacedinaline, tamibarotene, temsirolimus, thioguanine, troxacitabine, vindesine, VNP40101M, Bortezomib, XL228, Hydroxychloroquine (Plaquenil), Leflunomide (Arava), Methotrexate (Trexall), Sulfasalazine (Azulfidine), Minocycline (Minocin), Abatacept (Orencia), Rituximab (Rituxan), Tocilizumab (Actemra), Anakinra (Kineret), Adalimumab (Humira), Etanercept (Enbrel), Infliximab (Remicade), Certolizumab Pegol (Cimzia), Golimumab (Simponi), Tofacitinib (Xeljanz, Xeljanz XR), Baricitinib, Celecoxib (Celebrex), Ibuprofen (prescription dose), Nabumetone (Relafen), Naproxen Sodium (Anaprox), Naproxen (Naprosyn), Piroxicam (Feldene), Diclofenac (Voltaren, Diclofenac Sodium XR, Cataflam, Cambia), Diflunisal, Indomethacin (Indocin), Ketoprofen (Orudis, Ketoprofen ER, Oruvail, Actron), Etodolac (Lodine), Fenoprofen (Nalfon), Flurbiprofen, Ketorolac (Toradol), Meclofenamate, Mefenamic Acid (Ponstel), Meloxicam (Mobic), Oxaprozin (Daypro), Sulindac (Clinoril), Salsalate (Disalcid, Amigesic, Marthritic, Salflex, Mono-Gesic, Anaflex, Salsitab), Tolmetin (Tolectin), Betamethasone, Prednisone (Deltasone, Sterapred, Liquid Pred), Dexamethasone (Dexpak, Taperpak, Decadron, Hexadrol), Cortisone, Hydrocortisone (Cortef, A-Hydrocort), Methylprednisolone (Medrol, Methacort, Depopred, Predacorten), Prednisolone, Cyclophosphamide (Cytoxan), Cyclosporine (Gengraf, Neoral,Sandimmune), azathioprine (Azasan, Imuran), and hydroxychloroquine (Plaquenil).

[0077] The following table includes the relationship between known drugs and diseases and cell markers that can be used to select cells of the first and second distinguishable subpopulations. This table also shows the variation / change in the tendency of cells induced by each drug. Therefore, such combinations may be directly implemented in the method of the present invention.

[0078] Table II: Influence of immunomodulatory drugs on the tendency of cell-cell interactions defined by marker 1 and marker 2 that can be used to determine whether a patient responds to a specific treatment

Table 2-1

Table 2-2

[0079] The samples to be analyzed, particularly the viability of the cells contained in each monolayer, may be determined / evaluated / confirmed by methods well known in the art. That is, those skilled in the art know methods for determining / evaluating / confirming the stage of cells, for example, whether the cells are viable, alive, dead, or undergoing a process of changing that stage (such as death by apoptosis or necrosis). Therefore, specifically in the method of the present invention, known markers / dyes for recognizing / labeling that the cells are in a specific stage may be used. This includes dyes / labels selectable for cells with damaged membranes or dyes / labels selectable for late cell death or early apoptosis. For example, Green Fixable Live / Dead Cell Stain (ThermoFisher, catalog number L-23101) may be used. This is an antibody against cytochrome C and uses a dye to determine DNA turnover or cell proliferation. Furthermore, the means and methods for determining / evaluating / confirming the viability of the cells contained in the cells of the present invention / the population of the present invention, particularly the cell samples used in the form of a monolayer, are known to those skilled in the art.

[0080] Using methods well known in the art, changes in the viability and / or cell-cell interactions of two or more distinguishable subpopulations contained in a cell sample, particularly a monolayer, particularly a PBMC monolayer or a bone marrow cell monolayer, may be determined / tracked / evaluated / confirmed. For example, changes may be determined / tracked / evaluated / confirmed by optical perception using a microscope. However, in high-throughput applications, it is preferred to use an automated method to determine / track / evaluate / confirm changes in the viability and / or cell-cell interactions of individual subpopulations contained in the monolayer. Such methods include, for example, identifying subpopulations contained in a cell sample, preferably a monolayer, by a detectable label. Thus, it may be determined whether the labeled / detected subpopulation exhibits cell-cell interactions, which include direct or indirect contact via the cell membrane (as described above). Thus, a distance parameter is introduced, i.e., a threshold between the labeled cells defined above. This threshold determines the total number of interactions, i.e., the number of cell-cell interactions observed between the labeled cells. In this procedure, one labeled cell of one distinguishable subgroup may interact with one or more cells of a second distinguishable subgroup, and each interaction is counted. The obtained number is compared with a random distribution function, i.e., the number predicted by random cell-cell interactions. Then, the tendency of the interaction may be calculated using the interaction value of the method of the present invention, i.e., determining whether the interaction is random or assigned. According to such a test protocol, changes in cell-cell interactions by one or more test compounds before and after adding one or more test substances to the cell sample of the present invention can be determined / tracked / evaluated / confirmed.

[0081] Accordingly, the present invention provides a method for determining 1)-4) at high throughput using a physiologically relevant multi-population cell sample, particularly a primary hematopoietic sample, in image processing research. 1) The effect of chemotherapy / immunotherapy / immunosuppressive therapy on the diagnosis of in vitro cell populations or other markers at the whole level based on single cell analysis, 2) The ability of this technology to provide predictive chemotherapy in vitro in a patient's sample, 3) The ability of this technology to determine the effect of many or one stimulus (e.g., drug) on immune function, and 4) The accumulation of many patient data sets over time to determine patterns in the evaluation of treatment. In principle, in the method of the present invention, any cell sample may be used, such as blood, bone marrow, pleural effusion, spleen homogenate, lymphoid tissue homogenate, mononuclear cells from skin homogenate, etc. However, it is preferred to use mononuclear cells. It will be apparent to those skilled in the art that the mononuclear cell sample used in the method of the present invention particularly includes PBMC and bone marrow cells and others. Accordingly, the cell sample provided herein and used in the method of the present invention, preferably a monolayer of primary mononuclear cells, may include PBMC and / or bone marrow cells. That is, the means and methods provided herein are described for general cells or PBMC, which will be apparent to those skilled in the art, but the same means and methods are provided for bone marrow cells and additional cells. Accordingly, a method using bone marrow cells, a method for determining whether a bone marrow cell provider has developed a disease or has a predisposition to develop a disease, a method for diagnosing a disease or a predisposing factor of a bone marrow provider, and a method for determining whether a subject who has developed a disease or has a predisposing factor responds or is responsive to treatment with a therapeutic agent including the use of bone marrow cells are provided herein. Further, a method for screening drugs provided herein for other hematopoietic cells (e.g., bone marrow cells), and other methods are also disclosed.

[0082] In this regard, bone marrow is a soft tissue inside the bone. Human red blood cells are produced in the center of the bone marrow in a process known as hematopoiesis. Depending on the bone marrow disease, including certain forms of cancer such as leukemia, bone marrow transplantation may be performed for treatment. In addition, those obtained by transforming bone marrow stem cells into functional nerve cells may also be used for the treatment of inflammatory bowel disease. Therefore, bone marrow cells are useful targets in the treatment of various diseases, such as cancerous diseases or inflammatory diseases such as inflammatory bowel disease. Therefore, the method provided in this specification using a bone marrow sample obtained from a provider is very useful for evaluating / determining whether the provider has developed a disease or has a predisposition to develop a disease. In addition, the method provided in this specification using bone marrow cells has various advantages in high-throughput drug screening and the like.

[0083] The dogma that adherent cells (such as macrophages, HeLa, etc.) are required to form a single layer that can be stained and image - processed was overcome by providing a single layer in WO2016 / 046346. Prior to the single layer described herein, for the determination of chemotherapy - induced molecular (biomarker) changes, the survival rate assessment of cancer stem cells, and cell - to - cell contact, particularly in high - throughput when the disease state appears or is reflected in non - adherent cells (e.g., diseases of the blood system or lymphoma and leukemia), multiple research groups were unable to perform single - cell screening technology by imaging on primary patient samples. To solve this problem, the inventors of WO2016 / 046346 provided, in addition to means and methods, a technique called "pharmacoscopy" and an image - analysis data - processing pathway. This enables the visualization of adherent and non - adherent cells in a single image, typically requiring 1 / 10 of the material per perturbation compared to methods known in the art, with maximum throughput and speed. Pharmacoscopy can provide the same information as that collected by known methods (e.g., flow cytometry), but also provides useful additional information such as phenotypes smaller than the cellular level (protein localization / co - localization) and cell microenvironment / neighbor relationships. Also, in the method described in WO2016 / 046346, fewer cells are required, thus less patient material and less liquid volume, and with little human intervention. This allows pharmacoscopy to greatly increase the number of molecular perturbations that can be tested in parallel and enables more detailed evaluation. Additionally, pharmacoscopy does not require separating diseased cells from a healthy population per se and can track drug - mediated biomarker changes while controlling the effects of non - targeted drugs in parallel. These important controls can be performed on the pattern of cell - to - cell interactions and the biomarker analysis of cells in the targeted population by tracking the pattern of cell - to - cell interactions of healthy cells from the same provider present in the same well in the same image - processing field of view. The method of the present invention uses the technique of WO2016 / 046346 but includes further unexpected significant advantages.In particular, it is now possible to analyze cell samples that are undergoing changes in a population of cells (e.g., if cells die during the experiment) and cell samples with a high cell density in a more reliable way.

[0084] Using the method of the present invention, it is possible to predict clinical treatment outcomes for individual patients from the analysis of biomarker changes and single-cell biomarker changes that are smaller than the drug-induced cell levels in a patient's blood sample. In addition to basic research, standardizing, perfecting, and making this technology available to medical professionals and outpatient clinics is particularly beneficial for personalized medicine, predictive pharmacology, drug screening, and evaluation of treatment.

[0085] Therefore, compared with the prior art, the methods provided herein can be used in a wider variety of applications than the method described in WO2016 / 046346. For example, personalized medicine, drug screening programs, screening of general drugs, screening of personalized drugs, evaluation of drug response, evaluation of the immunological properties of drugs, evaluation of treatment, confirmation of treatment effectiveness, prediction of treatment response, population (drug) response of cell samples with a high cell density and / or cell samples in which the number of cells contained in the cell sample is changing, and the like. Therefore, the means and methods provided herein can also perform drug screening, drug discovery, discovery or screening of personalized (i.e., related to a subject / patient / individual) drugs. For example, in general, drug discovery and / or drug screening in the cells of healthy and diseased patients provided herein, particularly PBMCs or bone marrow, may be compared. Also, in the means and methods provided herein, cell samples pooled as the starting material of the single layer of the present invention, particularly PBMC samples or bone marrow samples, may be used. For the discovery of personalized drugs, it is preferable to use individual PBMC samples of a subject / individual / patient as the starting material of the PBMC single layer of the present invention used by the present invention.

[0086] Using the methods of the present invention, a number of perturbations may be efficiently and rapidly assayed using a number of monolayers that may be derived from a single sample obtained from a patient, particularly a PBMC sample or a bone marrow sample. Typically, the effects of 1000, at least 4000, at least 8000, at least 12000, at least 16000, at least 20000, at least 24000, at least 50000, at least 75000, 90000, or more compounds may be assayed in multiple monolayers obtained from such a single sample. In certain embodiments, the monolayers provided herein may be imaged and analyzed using multiple channels of high-content data simultaneously. The number of data channels available depends only on the available staining methods, particularly in the field of image processing software, which is a rapidly evolving field. By currently available methods, image processing, processing, and analysis can be performed simultaneously in at least 2, more typically 4, 5, or 8 channels of high-content data.

[0087] Peripheral blood mononuclear cells (PBMCs) are blood cells with round nuclei (monocytes; as opposed to lobed nuclei). PBMCs include lymphocytes (B cells, T cells (CD4-positive or CD8-positive), and NK cells), monocytes (dendritic cells and macrophage precursors), macrophages, and dendritic cells. These blood cells are major components of the immune system that fight infections and adapt to invaders. Depending on the context of embodiments of the present invention, for creating the PBMC monolayer of the present invention, or for use in a cell culture device containing the PBMC monolayer or in the methods provided in some aspects of the present invention, it is preferable to use PBMCs purified by a Ficoll density gradient, preferably human PBMCs. The present invention may be used with any mononuclear cells. In a preferred embodiment, the present invention can determine interaction values within cells of a cell lineage including, but not limited to determination only, cells within the following cell groups and terminal cell states. Such cell groups include hematopoietic stem cells, including, but not limited to, lymphoid common progenitor cells, myeloid common progenitor cells, and their mature lineages and terminal states (progenitor B cells, B cells, double-negative T cells, positive T cells, plasma B cells, NK cells, monocytes (including macrophages, dendritic cells)). These may be observed in peripheral blood, bone marrow (localized flat bones), cord blood, spleen, thymus, lymphoid tissue, and any fluid that has accumulated as a result of disease, such as pleural fluid, but are not limited thereto. The cells may be in either a healthy or diseased state.

[0088] PBMC cells for use in the methods described herein may be isolated from whole blood using methods known in the art or any suitable method described herein. For example, the assay protocol described by Panda et al. may be used (Panda, S. and Ravindran, B. (2013). Isolation of Human PBMCs. Bio-protocol 3(3): e323). Density gradient centrifugation is preferably used for isolation. Such density gradient centrifugation separates whole blood into its component layers, such as the upper layer of plasma, the layer of PBMCs beneath it, and the polymorphonuclear cells (e.g., neutrophils and eosinophils) and red blood cells at the bottom. The red blood cells, i.e., the enucleated cells, may be lysed to further isolate the polymorphonuclear cells. Common density gradient solutions useful for such centrifugation include, but are not limited to, Ficoll (a hydrophilic polysaccharide, e.g., Ficoll®-Paque (GE Healthcare, Uppsala, Sweden) and SepMate® (StemCell Technologies, Inc., Cologne, Germany)).

[0089] Bone marrow cells for use in the methods described herein may be isolated from bone marrow using any suitable method known in the art. In particular, magnetic beads may be used to separate the bone marrow cells from other components of such samples. For example, MACS cell separation reagents may be used (Miltenyi Biotec, Bergisch Gladbach, Germany).

[0090] As is known in the art, such isolated cultures may contain, in a small percentage, other cell types of one or more populations (e.g., anucleated cells such as red blood cells). As is known in the art and / or as described herein, PBMCs may be further isolated from such other populations of cells. For example, methods of lysing red blood cells are commonly used to remove such cells from isolated PBMCs. However, the methods of the present invention do not rely on further purification methods and may directly use the PBMCs isolated herein. Accordingly, the methods disclosed herein may not include lysis of red blood cells derived in a sample of isolated PBMCs. However, when anucleated cells (e.g., red blood cells) are present, although generally thought to be less than PBMCs, they may precipitate under the surface of the culture or among PBMCs and may interfere with the formation of a monolayer suitable for image processing. Accordingly, the concentration of anucleated cells (e.g., red blood cells) relative to PBMCs is about 500:1, more preferably about 250:1, and most preferably about 100:1, and it is preferred that the concentration be as low as possible. That is, it is most preferred that an isolated PBMC sample by the method disclosed herein contains less than about 100 anucleated cells (e.g., red blood cells) relative to PBMCs.

[0091] According to embodiments of the method of the present invention and / or to provide a monolayer used in the present invention, after incubating the cells, particularly PBMCs, isolate them at a density of about 100 cells / mm 2 (culture area) to about 30000 cells / mm 2 (culture area). Incubate the cells, particularly PBMCs, at about 500 cells / mm 2 (culture area) to about 20000 cells / mm 2 (culture area), about 1000 cells / mm 2 (culture area) to about 10000 cells / mm 2 (culture area), about 1000 cells / mm 2 (culture area) to about 5000 cells / mm 2 (culture area), or about 1000 cells / mm 2 (culture area) to about 3000 cells / mm 2It is preferable to incubate at the density of (culture area). Most preferably, cells, especially PBMCs, are incubated at a density of about 2000 cells / mm 2 (culture area). The term "about" means within 10%, more preferably within 5% of the indicated value or range. Thus, in some embodiments, using the method of the present invention, cells, especially PBMCs, are incubated in a culture device at a density of about 100 cells / mm 2 (i.e., 90 - 110 cells / mm 2 (culture area)) to about 30000 cells / mm 2 (i.e., 27000 - 33000 cells / mm 2 (culture area)). More preferably, cells, especially PBMCs, are incubated at about 500 cells / mm 2 (i.e., 450 - 550 cells / mm 2 (culture area)) to about 20000 cells / mm 2 (i.e., 18000 - 22000 cells / mm 2 (culture area)), about 1000 cells / mm 2 (i.e., 900 - 1100 cells / mm 2 (culture area)) to about 10000 cells / mm 2 (i.e., 9000 - 11000 cells / mm 2 (culture area)), about 1000 cells / mm 2 (i.e., 900 - 1100 cells / mm 2 (culture area)) to about 5000 cells / mm 2 (i.e., 4500 - 5500 cells / mm 2 (culture area)), or about 1000 cells / mm 2 (i.e., 900 - 1100 cells / mm 2 (culture area)) to about 3000 cells / mm 2 (i.e., 2700 - 3300 cells / mm 2 (culture area)). Most preferably, cells, especially PBMCs, are incubated at a density of about 2000 cells / mm 2 (i.e., 1800 - 2200 cells / mm 2 (culture area)).

[0092] The number of cells, particularly PBMCs, may be determined using standard methods known in the art. In particular, the number of PBMCs may be determined using a hemocytometer or the method described in Chan et al. (Chan et al. (2013) J. Immunol. Methods 388 (1-2), 25-32). Also, the number of bone marrow cells may be determined using methods well known in the art. In particular, the number of bone marrow cells may be determined using hemocytometry. Also, other cells may be counted using methods well known in the art.

[0093] Incubation is carried out in a medium. Those skilled in the art know methods suitable for maintaining the viability of cells, particularly PBMCs or bone marrow cells. However, the medium used in the method of the present invention is not particularly limited. In this regard, the medium means a liquid containing nutrients and substances necessary for cell culture. Liquid media for culturing eukaryotic cells are known to those skilled in the art (for example, DMEM, RPMI 1640, etc.). An appropriate medium may be selected according to the type of cells to be cultured.

[0094] For example, PBMCs or bone marrow cells may be cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FCS). However, any suitable medium may be selected, provided that the components of the medium are chosen such that they do not artificially affect the PBMC response and / or the bone marrow cell response. A nutrient supplement is a substance (e.g., cytokine, growth factor, differentiation factor, mitogen, serum) added to the medium to induce or modify cell function. Nutrient supplements are known to those skilled in the art. An example of serum commonly used for eukaryotic cells is fetal bovine serum. Antibiotics such as penicillin, streptomycin, and ciprofloxacin may also be added to the medium. In one embodiment, the test substance and / or promoter may be added separately to individual units of viable cell material. The test substance may be a pharmaceutical drug or drug component. The promoter may include any substance that aids in the maintenance, growth, or differentiation of cells. In certain embodiments, the promoter is a substance that acts on immune cells, for example, by activating the immune cells. Promoters for activation of immune cells are known in the prior art. Such promoters may be polypeptides, peptides, antibodies, and other promoters. Examples include OKT-3, interferon-α, interferon-β, interferon-γ, oligonucleotide CPG, mitogens (e.g., PWM, PHA, LPS), and the like. The test substance and promoter may be injected into the cell culture medium. It is preferred to culture PBMCs in RPMI supplemented with 10% FBS / FCS (preferably with low endotoxin to minimize activation, but not necessarily required). The PBMC culture may further contain human serum from the PBMC donor.

[0095] The term "growth area" as used within the meaning of the present invention refers to the surface within the culture device where the cells are placed. "Density" as used within the meaning of the present invention is the amount of cells per unit area of the surface within the culture device where the cells are placed. This culture device may be made of any material that is compatible with the material tested against cell cultures, particularly non-cytotoxic cell cultures. Examples of such materials include plastic materials, such as thermoplastic or duroplastic materials. Examples of suitable plastics include polyethylene, polypropylene, polysulfone, polycarbonate, polyetheretherketone (PEEK), or polytetrafluoroethylene (PTFE). In particular, this device is suitable for the culture and / or maintenance of PBMC. Typical culture devices known in the art and used in the present invention include culture flasks, dishes, plates, multi-well plates. Particularly useful are multi-well plates, which can individually maintain multiple cultures, for example, for multiple perturbations, with minimal material requirements (e.g., minimal solvent requirements). Preferred culture devices include 96-well plates, 384-well plates, and 1536-well plates. It is particularly preferred to use black-walled plates for image processing, designed to specifically reduce background fluorescence / background light interference, minimize light scattering, and reduce crosstalk, as known in the art in relation to image processing analysis of cultures, particularly fluorescence image processing. The culture device may be sterilized. In a most preferred embodiment, a multi-well image processing plate having a plurality of wells is used. At least some of the wells include a first chamber formed by one or more first sidewalls and a bottom wall; a second chamber formed by one or more second sidewalls and including an opening for introducing liquid, disposed above the first chamber; and an intermediate floor provided between the first and second chambers and forming an obstacle-inhibiting structure. The intermediate floor includes at least one through-hole providing a liquid connection between the first and second chambers, and the through-hole constitutes the tip of a pipette. The pipette is inserted from the second chamber into the first chamber through the through-hole.

[0096] This device is particularly used in automated image processing systems and analysis. Therefore, it is preferably suitable for use in such systems. In a non-limiting example, the culture device may be transparent. For example, culture dishes and plates used in image processing such as fluorescence image processing are well known in the art and are commercially available. A non-limiting example of a commercially available culture plate used in the practice of the present invention is the Corning® 384-well tissue culture plate (treated black lid, transparent bottom) (Corning Inc., Massachusetts, USA) or Corning® 384 Well Flat Clear Bottom Black Polystyrene TC-Treated Microplates (product number 3712). Another example is the Perkin Elmer Cellcarrier®.

[0097] The cells of the population of the present invention, preferably in monolayer form, may be image processed by any method known in the art and / or any method described herein, and the methods provided herein may use any image processing technique known in the art. The specific image processing method is not critical and may be determined by the knowledge of those skilled in the art. Image processing may or may not use dyes or stains, may include image processing of both stained and unstained components, and / or may be performed under conditions where the stain is visible or not visible (e.g., image processing in bright field (fluorescent stains are not visible), under UV light (fluorescent stains are visible), or a combination thereof. Image processing under bright field conditions is a standard well known in the art and may be performed by standard methods and / or the methods described herein. In addition to or in place of this, other label-free image processing may be used in accordance with the present invention. Such label-free methods are known and include, for example, PhaseFocus image processing (Phase Focus Ltd, Sheffield, UK).

[0098] Also, the practice of the present invention may include adding a detectable label to the cells of the population, preferably a monolayer, particularly a PBMC monolayer (either in relation to or independently of the label-free method). This label may be detected using microscopy. These detectable labels may label individual cell structures, components, or proteins known in the art. Additionally, the label may be added to an antibody for specific labeling, thereby enabling the detection of antibody-antigen. In a preferred embodiment, the label can be visualized with visible or UV light by a detectable label. Thus, the detectable label may be fluorescent. Many visible labels are known in the art and are suitable for the present invention. This label may be detectable without further action or may become detectable only after a secondary step, such as the addition of a substrate, exposure to an enzymatic reaction, or irradiation with a specific wavelength of light.

[0099] A subpopulation of cells (target cells) used herein, i.e., a distinguishable subpopulation, particularly a PBMC subpopulation or a bone marrow cell subpopulation, may be identified by a label detectable by the expression of one or more markers on the surface or within the cell of the target cells. Alternatively, or in addition thereto, the subpopulation may be defined by the non-expression of one or more markers on the surface of the target cells or within the target cells. It may be desirable to test whether one or more markers (e.g., two markers, three markers, four markers, etc.) are expressed or not, and further confirm that the cells expressing or not expressing the marker are actually target cells (e.g., members of the cells of the desired subpopulation). For example, each of a "cocktail" of multiple antibodies against different markers may be (directly or indirectly) bound to the same label or different labels. As an example, a mixture of antibodies against different markers may each contain a binding motif that binds to the same label (e.g., may each contain the same type of Fc recognized by the same secondary antibody, or may each be biotinylated and specifically bound by the same avidin-binding label). If necessary, two or more different antibodies or a mixture of multiple antibodies may be used. Preferably, cells are stained with at least two distinguishable labels, whereby cells expressing at least two different markers of the target cell type can be identified. Also, cells may be stained with at least three, four, five, or more different distinguishable labels, whereby cells expressing markers of more target cell types can be detected. If necessary, if a cell expresses a preselected number of markers or a specific preselected combination of markers, that cell may be identified as a cell of the target species. Alternatively, if a cell does not express a preselected marker, that cell may be identified as a cell of the target species. Also, as long as the target cells can be distinguished from other cells in the population, the markers of the target cell type do not necessarily have to be unique to the target cells.In the case of PBMCs, the major components of the PBMC cell population are represented by CD11C for dendritic cells, CD14 for macrophages, CD3 (CD4 or CD8 having CD3) for T cells, and CD19 for B cells. Although the above markers overlap in subsets of these major classes of PBMCs, staining with these markers to identify subpopulations of PBMCs is widely accepted in the art. Further, reference may be made to the CD marker handbook (Becton, Dickinson and Co. 2010, California, USA) for markers suitable for use in the methods of the present invention. The major cell subpopulations contained in bone marrow cells are neutrophilic metamyelocytes, neutrophilic myelocytes, segmented neutrophils, normoblasts, and lymphocytes.

[0100] In the practice of the present invention, it is preferred to use an antibody conjugated to a detectable label. Such an antibody can target individual cell structures, and thus, a mixture of such antibodies (each carrying a different label) may be used to simultaneously visualize multiple targets / cell structures / cell components. Staining must be carefully performed so as not to disrupt the monolayer. As will be apparent to those skilled in the art, this is particularly a problem when using antibody-based labels. This is because usually, its use requires one or more washing steps to remove unbound labels that can interfere with accurate visualization, i.e., become non-specific staining and / or "noise" in the assay. Accordingly, the present invention encompasses methods of staining a cell monolayer with a detectable label, particularly an antibody-based label, that minimizes or eliminates the washing requirements after staining. The method of the present invention may include adding a detectable label at a concentration that avoids generating a noise signal without washing. This concentration may be determined by methods well known in the art and / or the methods described herein. Accordingly, the present invention encompasses using an antibody labeled above or below the concentration recommended by the antibody manufacturer.

[0101] For certain exemplary cell types, if a marker exhibits a characteristic localization or pattern within the cell, the cell may be considered positive only for that marker. For example, a cell may be considered "positive" if a cytoskeletal marker is present in the cytoskeleton, and "negative" if some dispersion is seen in the cytoplasmic staining. In such cases, the cells may be cultured under suitable conditions (e.g., adherent cultures) to establish the characteristic localization or pattern within the cell. The culture conditions and times suitable for the construction of the cytoskeleton (or other processes that organize subcellular components) that may be necessary to robustly detect a given marker can be readily determined by those skilled in the art. Also, markers can be readily selected that reduce or eliminate the need for adherent cultures as a prerequisite for robust staining.

[0102] Dyes useful for labeling proteins are known in the art. Generally, a dye is a molecule, compound, or substance that can provide an optically detectable signal, such as a colorimetric signal, a luminescence signal, a bioluminescence signal, a chemiluminescence signal, a phosphorescence signal, or a fluorescence signal. In a preferred embodiment of the present invention, the dye is a fluorescent dye. Non-limiting examples of dyes include commercially available dyes such as CF dyes (Biotium, Inc.), Alexa Fluor dyes (Invitrogen), DyLight dyes (Thermo Fisher), Cy dyes (GE Healthscience), IRDyes (Li-Cor Biosciences, Inc.), and HiLyte dyes (Anaspec, Inc.). Depending on the embodiment, the excitation wavelength and / or emission wavelength of the dye is 350 nm to 900 nm, 400 nm to 700 nm, or 450 to 650 nm.

[0103] For example, staining may involve the use of multiple detectable labels such as antibodies, autoantibodies, or the serum of a patient. The staining may be observable with visible light and ultraviolet light. The staining may include a colored reagent or an antibody directly or indirectly conjugated to an enzyme capable of generating a colored reagent. When an antibody is used as a component of the staining, a marker may be directly or indirectly conjugated to the antibody. Examples of indirect conjugation include avidin / biotin binding, binding via a secondary antibody, and combinations thereof. For example, cells may be stained with a primary antibody that binds to an antigen specific to the target and a secondary antibody that binds to the primary antibody or a molecule conjugated thereto and is capable of binding to a detectable marker. Using indirect conjugation can improve the signal-to-noise ratio, for example, by reducing background binding and / or amplifying the signal.

[0104] Alternatively, the staining may include a primary antibody or a secondary antibody (as described above) that is directly or indirectly conjugated to a fluorescent label.The fluorescent label may be selected from the group consisting of: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750 and Alexa Fluor 790, fluorescein isothiocyanate (FITC), Texas Red, SYBR Green, DyLight Fluors, green fluorescent protein (GFP), TRIT (tetramethylrhodamine isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine, biotin, digoxigenin, 5-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluorescein, TET (6-carboxy-2’,4,7,7’-tetrachlorofluorescein), HEX (6-carboxy-2’,4,4’,5’,7,7’-hexachlorofluorescein), Joe (6-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluorescein)-5-carboxy-2’,4’,5’,7’-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, Tamra (tetramethylrhodamine), 6-carboxyrhodamine, Rox (carboxy-X-rhodamine), R6G (rhodamine 6G), phthalocyanine, azomethine, cyanine (e.g., Cy3, Cy3.5, Cy5), xanthine, succinylfluorescein, N,N-diethyl-4-(5’-azobenzotriazolyl)-phenylamine, aminoacridine, and quantum dots.

[0105] Furthermore, exemplary embodiments of the present method use antibodies directly or indirectly conjugated to fluorescent molecules. Such fluorescent molecules include ethidium bromide, SYBR Green, fluorescein isothiocyanate (FITC), DyLight Fluors, green fluorescent protein (GFP), TRIT (tetramethylrhodamine isothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine, biotin, digoxigenin, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, TET (6-carboxy-2′,4,7,7′-tetrachlorofluorescein), HEX (6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein), Joe (6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein)-5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, Tamra (tetramethylrhodamine), 6-carboxyrhodamine, Rox (carboxy-X-rhodamine), R6G (rhodamine 6G), phthalocyanine, azomethine, cyanine (e.g., Cy3, Cy3.5, Cy5), xanthine, succinylfluorescein, N,N-diethyl-4-(5′-azobenzotriazolyl)-phenylamine, and aminoacridine. Other exemplary fluorescent molecules are described in the following patent documents [see, for example, U.S. Patent Nos. 6,207,299, 6,322,901, 6,576,291, 6,649,138 (a surface modification method for binding a mixed hydrophobic / hydrophilic polymer mobilizer to the surface of quantum dots), U.S. Patent Nos. 6,682,596, 6,815,064 (in the case of alloy or mixed shells).These patent documents are each incorporated herein by reference, and examples of quantum dots are described in technical documents such as "Alternative Routes toward High Quality CdSe Nanocrystals," (Qu et al., Nano Lett., 1(6):333-337 (2001)). Quantum dots with various chemical surface properties and fluorescence characteristics are commercially available from, among others, Invitrogen Corporation, Eugene, Oreg., Evident Technologies (Troy, N.Y.), and Quantum Dot Corporation (Hayward, Calif.). Quantum dots also include alloy quantum dots such as ZnSSe, ZnSeTe, ZnSTe, CdSSe, CdSeTe, ScSTe, HgSSe, HgSeTe, HgSTe, ZnCdS, ZnCdSe, ZnCdTe, ZnHgS, ZnHgSe, ZnHgTe, CdHgS, CdHgSe, CdHgTe, ZnCdSSe, ZnHgSSe, ZnCdSeTe, ZnHgSeTe, CdHgSSe, CdHgSeTe, InGaAs, GaAlAs, and InGaN. Alloy quantum dots and methods for making them are disclosed, for example, in U.S. Patent Application Publication No. 2005 / 0012182 and PCT Publication WO2005 / 001889.

[0106] After labeling the cells of the population of the present invention, preferably in monolayer form, the method may further include detecting a signal of a detectable label. The detection method may be appropriately adapted according to the type of signal emitted from the detectable label. It is preferable to use a detection method suitable for detecting a label that emits fluorescence. Also, the detection method may be automated by standard methods known in the art. For example, there are various computer-based methods that enable the analysis and interpretation of microscopic images of cells by those skilled in the art, or that can establish an automated test protocol for such analysis. Primary image analysis includes correction of irradiation bias in microscopic images, identification of individual cells from microscopic images, and measurement of marker intensity and texture, nuclear and cell diameter and shape, and position parameters, and open-source software CellProfiler (for example, version 2.1.1) may be used. Machine learning using open-source software CellProfiler Analyst (for example, version 2.0) may be used to identify marker-positive cells (for example, CD34+ progenitor cells or viability dye-positive cells), and double or triple positive cells may be identified by a sequential gating strategy. Similarly, CellProfiler Analyst may be used to create a plate overview for further analysis and hit selection (selecting a desired compound in high-throughput screening).

[0107] For the data analysis after primary image analysis, the Bioconductor cellHTS package (for example, version 2.14) or Pipeline Pilot (for example, version 9.0, Accelrys) may be used respectively. This data analysis includes plate effect normalization, control-based normalization, and hit selection.

[0108] In addition, for the implementation of the present invention, a commercially available automated microscope system such as the PerkinElmer Operetta automated microscope (PerkinElmer Technologies GmbH & Co. KG, Walldorf, Germany) may be used. This system may include corresponding image analysis software, for example, PerkinElmer's Harmony software (e.g., version 3.1.1). Using such an automated system and / or a commercially available system, primary image analysis, positive cell selection, and hit selection may be performed on microscope images by the method of the present invention.

[0109] After this primary analysis, the method of the present invention for determining the tendency of cell-cell interactions in a population of cells containing at least two distinguishable subpopulations of cells, the method of the present invention for determining the disease or disease predisposition of a cell provider, or the method of the present invention for screening for a new therapeutic agent is performed. The method includes determining the tendency of cell-cell interactions in a cell sample obtained from a provider, wherein the provider's cell sample contains at least two distinguishable subpopulations of cells. Alternatively, the method of the present invention for determining whether a subject who has developed or is predisposed to develop a disease responds or is responsive to treatment with a therapeutic agent is performed. This method includes determining the variation of cell-cell interactions in a cell sample obtained from the subject, wherein the subject's cell sample contains at least two distinguishable subpopulations of cells.

[0110] The method may further include isolating cells, particularly PBMCs or bone marrow, from a subject who has received treatment for an immune-mediated disease in the past or currently, stimulating the PBMCs or bone marrow cells, identifying subpopulations of PBMCs or bone marrow cells, comparing the tendency data of cell-cell interactions from each subpopulation of PBMCs or bone marrow cells with the subject's response to treatment, and selecting characteristic cell-cell interaction data regarding a positive response to treatment, thereby monitoring the treatment process.

[0111] Immune-mediated diseases may be divided into several categories including immunodeficiency diseases, autoimmune diseases, and hypersensitivity diseases. Immunodeficiency diseases occur when part of the immune system does not function properly. Autoimmune diseases are the result of the immune system attacking one's own body instead of pathogens. Hypersensitivity diseases occur as a result of the immune system overreacting and causing damage to the body.

[0112] The diseases diagnosed using the method of the present invention are not limited as long as they are diagnosed using cells of a population, preferably cells contained in a monolayer, and in particular, diagnosed using PBMCs or bone marrow cells, i.e., cells of a subject suffering from the disease to be diagnosed (e.g., PBMCs or bone marrow cells), and as long as the pattern of cell-cell interactions of an identifiable subpopulation of cells that may be associated with the disease is different from the pattern predicted in the cell sample of a healthy provider. For example, diseases that can be diagnosed using the means and methods provided in the present invention include, but are not limited to, hematological malignancies, and / or malignancies or immune-mediated diseases of the bone marrow and / or lymphoid tissues. In particular, diseases that can be specifically diagnosed and / or predicted by the means and methods provided in the present invention in a PBMC monolayer or a bone marrow cell monolayer include, but are not limited to, myeloproliferative disorders (i.e., hematological cancers in general), inflammatory disorders, potential viral infections, cell proliferation disorders, cell chemotaxis disorders, metabolic disorders, autoimmune diseases (e.g., staining with self-ligands, patient sera against monoclonal antibodies, or self-antigen recognition). Also, (chronic and acute) leukemia, lymphoma (mature B cells, mature T cells, NK cells, Hodgkin lymphoma), HIV, gout, shock, etc. may be diagnosed using the method of the present invention. That is, it may be diagnosed or determined whether a subject responds or is responsive to the treatment of a disease with the following ICD-10 codes using the means and methods of the present invention.Such diseases include, but are not limited to, A00 - B99: Certain infectious and parasitic diseases; C00 - C97: Malignant neoplasms; D70 - 77: Other diseases of the hematopoietic system; D80 - 89: Certain disorders involving immune response mechanisms not elsewhere classified; D82: Immunodeficiency associated with other major defects; D83: Immunodeficiency, unspecified type; D84: Other immunodeficiencies; G35 - 37: Central nervous system diseases; I00 - I03: Acute rheumatic fever; I05 - I09: Chronic rheumatic heart diseases; I01: Rheumatic fever with heart complications; I06: Rheumatic aortic stenosis; I09: Rheumatic myocarditis; I70: Atherosclerosis; K50: Crohn's disease; K51: Colitis; K52: Other non - infectious gastroenteritis and colitis; M00 - M19: Arthropathies; M05: Seropositive rheumatoid arthritis; M06: Other rheumatoid arthritis; M10: Gout; M11: Other crystalline arthropathies; M35: Sjogren's syndrome; M32: Systemic lupus erythematosus; N70 - 77: Inflammatory diseases of the pelvic organs in females; P35 - 39: Infections specific to the perinatal period; P50 - P61: Hemorrhagic and blood diseases of the fetus and newborn; Z22: Carriers of infectious diseases; Z23: Need for immunization against a single bacterial disease; and / or Z24: Need for immunization against a specific single viral disease.

[0113] "Treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventive measures. The aim here is to prevent, ameliorate, or delay (reduce) a target condition or disease, or one or more symptoms associated therewith. Similarly, "responsive" or "responding" and terms similar thereto refer to a target condition or one or more symptoms associated therewith being prevented, ameliorated, or reduced. Also, these terms as used herein refer to inhibition (e.g., reduction or arrest of growth), mitigation of the effects, and prolongation of life of a patient suffering from a disease, particularly a myeloproliferative disease, or the onset of signs indicated by such markers. Subjects in need of treatment include subjects diagnosed with a disease, subjects suspected of having developed a disease, subjects having a predisposition to develop a disease, and subjects seeking to prevent a disease. Thus, mammals treated herein may be diagnosed as having developed a disease, or may have a predisposition to, or be suspected of having, a disease.

[0114] "Responding" or "responsive" refers to a PBMC or subject in which at least one characteristic has changed following treatment. The changed characteristic of the subject may be that the target condition or disease has been alleviated or delayed.

[0115] As used herein, the terms "prevent", "preventing", and "prevention" refer to preventing the appearance and / or recurrence or onset of one or more symptoms of a cancer disease in a subject as a result of administration of a prophylactic or therapeutic agent.

[0116] The means and methods provided by the present invention are described mostly for primary hematopoietic cells, i.e., all mononuclear cells. Although it will be apparent to those skilled in the art, primary hematopoietic cells include, in particular, PBMC and bone marrow cells. Thus, the means and methods provided by the present invention described for PBMC are also disclosed for bone marrow cells and any other mononuclear cells.

[0117] The "therapeutic agent" as meant by the present invention includes, but is not limited to, polypeptides, peptides, glycoproteins, nucleic acids, synthetic drugs and natural drugs, peptoids, polyenes, macrocyclic compounds, glycosides, terpenes, terpenoids, aliphatic compounds, aromatic compounds, and molecules including derivatives thereof. In a preferred embodiment, the therapeutic agent is a compound such as a synthetic drug and a natural drug. In other preferred embodiments, the therapeutic agent brings about the alleviation and / or cure of diseases, disorders, medical conditions and / or symptoms associated therewith. One or more therapeutic agents or test compounds screened by the method of the present invention may be encapsulated in a polymer.

[0118] Suitable therapeutic agents include, but are not limited to, those shown in Goodman and Oilman's The Pharmacological Basis of Therapeutics (e.g., 9th edition) or The Merck Index (e.g., 12th edition). General examples of therapeutic agents include, but are not limited to, drugs that affect the inflammatory response, drugs that affect the composition of body fluids, drugs that affect electrolyte metabolism, chemotherapeutic agents (e.g., for hyperproliferative diseases, particularly cancer, parasitic infections, and microbial diseases), antineoplastic agents, immunosuppressive agents, drugs that affect the blood and hematopoietic organs, hormones and hormone antagonists, vitamins and nutrients, vaccines, oligonucleotides, and gene therapy. It is understood that compositions including combinations such as mixtures or blends of two or more active agents (e.g., two drugs) are also encompassed by the present invention.

[0119] In one embodiment, the therapeutic agent may be a drug, prodrug, antibody, or vaccine. Using the method of the present invention, it may be evaluated whether administration of the therapeutic agent to a patient induces a response to the therapeutic agent or components such as a delivery medium, excipient, carrier, etc. administered therewith.

[0120] The present invention is not limited by the exact nature of the therapeutic agent. In non-limiting embodiments, the methods of the present invention can be used to evaluate the response to synthetic small molecules, naturally occurring substances, naturally occurring biological agents or synthetically produced biological agents, or any combination of two or more thereof (optionally in combination with excipients, carriers, or delivery vehicles).

[0121] The term "diagnosis" (along with variant expressions such as "diagnose" or "diagnostic") means, for example, the identification of a molecular state, pathological condition, disease, or symptom, such as the identification of cancer, or the identification of a cancer patient who may benefit from a particular therapeutic dosing regimen.

[0122] The term "prognosis" (and variant expressions such as "prognose" or "prognostic") means the prediction of the likelihood of benefiting from a treatment such as cancer treatment.

[0123] As used herein, the terms "predict" or "predicting" mean the likelihood that a patient will respond well, or not, to a particular therapeutic agent. In one embodiment, predicting or predicting relates to the degree of these responses. In one embodiment, predicting or predicting relates to the probability that a patient will survive without disease progression, or experience a favorable disease state, for a particular period of time after treatment (e.g., after treatment with a particular therapeutic agent).

[0124] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials are described below. In case of conflict, the present specification, including definitions, will control. Also, these materials, methods, and examples are merely illustrative and should not be construed as limiting.

[0125] Unless otherwise specified, the general methods and techniques described herein may be performed by conventional methods well known in the art and by various general and more specific references cited and discussed herein. For example, see the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990).

[0126] Although the embodiments of the invention have been illustrated and described in detail in the drawings and the foregoing description, such illustration and description are illustrative and not restrictive. As will be apparent, changes and modifications may be made by those skilled in the art within the scope of the following claims and the spirit thereof. In particular, the invention includes within its scope further embodiments that combine the features of the different embodiments described above and below in any combination.

[0127] Also, although not described above, the invention also includes within its scope all further features shown in the individual drawings. Also, each alternative of the embodiments described in the drawings and above and each alternative of its features may be disclaimed from the subject matter of other aspects of the invention.

[0128] Furthermore, in the claims, the term "comprising" does not exclude other elements or steps, and the indefinite articles "a" or "an" do not exclude a plurality. There may be a single unit that meets the functions of several features recited in the claims. Also, terms such as "essentially", "approximately", "about" related to an attribute or a value respectively define exactly the attribute or the value. Any reference signs in the claims should not be construed as limiting the scope of the invention.

[0129] This patent or this application includes at least one color drawing. A copy of the publication of this patent or this application including a color drawing will be provided upon application to the Patent Office with the necessary fee paid.

[0130] Aspects of the present invention are further described by the following non-limiting exemplary embodiments, by which the embodiments of the present invention and many of its advantages can be better understood. The following examples are included to illustrate preferred embodiments of the present invention. Although it is obvious to those skilled in the art, the techniques disclosed in the following examples represent the techniques used in the present invention so as to function well in the implementation of the present invention, and thus can be regarded as constituting preferred modes for its implementation. However, although it is obvious to those skilled in the art, from the perspective of the present disclosure, many changes are possible in the specific embodiments disclosed, and the same or similar results can be obtained without departing from the spirit and scope of the present invention.

[0131] Unless otherwise specified, established methods of recombinant gene technology as described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001) were used. The content thereof is incorporated herein by reference.

[0132] Numerous documents are cited in this specification, including patent applications, manufacturer's instructions, and scientific literature. The disclosures of these documents are not considered relevant to the patentability of the present invention, but are incorporated herein by reference. More specifically, all reference documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference.

Example

[0133] Example 1 Experiment: Establish a test protocol for culturing PBMCs from healthy donors or patients to obtain a monolayer (single image - processable field / image - processing surface) stainable for image processing in a 384 - well plate.

[0134] Method: PBMCs were cultured according to a test protocol devised for pharmaceutical observation. In the first step, blood was collected from a healthy donor or patient. Typically, the volume is 9 - 500 ml. The blood was stored in a suitable container containing EDTA or heparin. Then, the blood sample was mixed with PBS buffer in a 1:1 ratio. For purification, 30 ml of the blood / PBS mixture was placed on top of 15 ml of lymphoprep density gradient solution (lymphocyte separation solution) in a 50 - ml tube. These tubes were rotated at 2000 rpm for 30 minutes at room temperature without interruption (do not interrupt the centrifuge). The soft membranes above and below the density gradient solution were removed and transferred to another 50 - ml tube. Usually, the volume to be removed is 10 - 15 ml. Then, the tube was filled with PBS to a final volume of 50 ml and rotated at 2000 rpm for 5 minutes without stopping the centrifuge again. The supernatant was removed and the pellet was suspended in RPMI (containing 20 ml of 10% FCS and appropriate antibiotics). The pellet should have no band of red blood cells with a thickness of 5 mm or less. Then, the cells were 4×10 5Count up to / ml and seed 50 μl into a Corning 384-well imaging plate (with black walls) at a density of 20,000 cells / well. Let the cells stand at room temperature for 10 - 15 minutes and then transfer them to a 37 °C + 5% CO₂ incubator. Then incubate the plate for a predetermined time, ideally overnight or less. When adding viability dye, carefully remove 30 μl of the supernatant using a hand or a robot, add 30 μl of an Invitrogen live / dead fixable 488 dye PBS mixture (1:1000), and leave at room temperature for 30 minutes. Remove the viability dye as the first supernatant using an automated pipette or a robot. Care must be taken not to disrupt the monolayer in this step. If the viability dye is not added or is added immediately, add 30 μl of 0.1% Triton x-114 and 2% formaldehyde and incubate the plate at room temperature for 15 minutes. Tap (the tube) with a finger to remove all of the supernatant. Since the monolayer is already fixed at this stage, this does not disrupt the monolayer. When staining, add 30 μl of antibody staining. The cocktail to be tested is a 1:300 dilution with a GFP, PE, or APC-labeled antibody used for flow cytometry. Washing steps can be avoided by dilution. Incubate the plate at room temperature for 1 hour. Tap with a finger to remove the antibody as described above and add 50 μl of a PBS (1:100) dilution of 4’,6-diamidino-2-phenylindole (DAPI). Store the plate at 4 °C until imaging. Perform imaging at room temperature using an automated confocal microscope (PerkenElmer Operetta) with four non-overlapping channels and output the data for analysis.

[0135] Results: After culturing PBMCs using the inventors' novel test protocol, adherent and non-adherent PBMCs formed a monolayer that could be imaged in a single surface field using an automated confocal microscope. This enabled automated drug screening with a minimum of 384-well plates. It was confirmed by microscopy that image processing of 20,000 cells (±5%) could be performed using a newly developed method called pharmaceutical observation method (Figure 1).

[0136] Example 2 Experiment: This example was conducted to test whether the method of the present invention produces more reliable results than the methods of the prior art, in particular the methods shown in WO2016 / 046346 or Helmuth et. al. (2010) BMC Bioinformatics.

[0137] Method: The probability that cells of type A interact with cells of type B was systematically varied while maintaining the number of A, B, and C cells in the mixture and the probability that C cells interact with B cells, to generate a synthetic dataset corresponding to a cell mixture containing A, B, and C cell types. The tendency for A cells to interact with B cells was determined by WO2016 / 046346, Helmuth et. al. (2010) BMC Bioinformatics, and the present invention.

[0138] Results: In situations where A cells interact with a maximum of one B cell or C cell, the tendency of interaction according to both WO2016 / 046346 and the present invention showed a linear relationship with the logarithm of the probability that A cells interact with B cells (Figure 3). However, when cells of type A interact with two or more B cells or C cells (i.e., a rosette is formed), the tendency of interaction determined using the method described in WO2016 / 046346 did not show a linear relationship with the logarithm of the probability that A cells interact with B cells and was flat even as the probability of interaction increased. In contrast, in the present invention, this linear relationship was maintained (Figure 4). The interaction values according to Helmuth et al. did not show a linear relationship with the logarithm of the probability that A cells interact with B cells and, in fact, had no correlation with the probability that A cells and B cells interacted.

[0139] Therefore, the measurement of the tendency of cell - cell interaction according to the present invention has a high dynamic range when cells interact with two or more other cells. This is commonly observed in cultures of primary immune cells where one type of cell often interacts with multiple other single - type cells. This phenomenon is often described as the formation of a "rosette".

[0140] Example 3 Experiment: The fractions of A, B, and C cells in the mixture were systematically varied to keep the probability of interaction between A cells and C cells with B cells constant, generating a synthetic dataset corresponding to a cell mixture containing A, B, and C cell types.

[0141] Method: The tendency of A cells to interact with B cells was determined according to WO2016 / 046346 and the present invention. In the simulation settings used, ideally, the tendency of interaction between A cells and B cells should be invariant to changes in the fractions of A cells and C cells.

[0142] Results: The tendency of interaction determined by the present invention was invariant to changes in the fractions of A cells and C cells, whereas the tendency of interaction determined by WO2016 / 046346 was not (Figure 5). In many experimental settings, for example, changes in the fractions of A, B, and C cells in response to exposure to a stimulus are not confounding factors. This is because it can be assumed that the fractions of A, B, and C cells are constant, and the stimulus mostly affects changes in the expression of cell surface molecules (e.g., upregulating MHC on the surface of DCs increases the tendency of interaction between DCs and T cells). However, in settings where the fractions of A cells and C cells can change, such as when immune effector cells attack cancer cells or pathogen-infected cells, it is important to measure a robust interaction against changes in the fraction of A cells.

[0143] Example 4: To identify small molecule chemical substances that can regulate the ability of natural killer (NK) cells to kill tumor cells, human peripheral blood mononuclear cells (PBMCs) and K562 human tumor cells were mixed at a ratio of 3:1 at approximately 4000 cells / mm in the presence of different compounds. 2Incubated at the density of. As a control, the same absolute number of K562 cells were incubated in the presence of the same set of compounds. At a predetermined set time, the cells were fixed, permeabilized by adding formaldehyde and Triton X-114, and stained with an antibody against CD56 and DAPI. Microscopic images of the cells were taken, and NK cells were identified by positive staining against CD56 using a neural network trained with 15,000 cell image examples, and K562 cells were identified by the diameter and texture of the nuclei distinguished by DAPI staining. The tendency of interaction between K562 cells and NK cells was calculated, and the survival rate of K562 in the presence and absence of PBMC (negative DMSO control) was compared. It was shown that the tendency of interaction between K562 cells and NK cells is directly related to the survival rate of K562 in the presence and absence of PBMC (negative DMSO control), and it was shown that molecules that regulate the killing of tumor cells mediated by NK cells can be identified by the tendency of interaction determined using the present invention (Figure 6).

[0144] Example 5: This example relates to the effect of biological pharmaceuticals on the interaction values measured according to the present invention.

[0145] When T cells are activated with a standard activation mixture consisting of anti-CD3, anti-CD28 antibodies, and IL2, T cells and antigen-presenting cells (APCs) (e.g., CD11c-positive cells) interact more frequently.

[0146] The PBMC monolayer prepared as described above was treated with a mixture of anti-CD3 antibody, anti-CD28 antibody, and 500 IU / mL of IL2 for 48 hours. The monolayer was fixed and stained with different combinations of anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, and anti-CD11c antibody, and the tendency of interaction between cells was measured using the present invention.

[0147] Activation of T cells increased the measured interaction values between CD3-positive cells, CD4-positive cells, or CD8-positive cells and CD11c-positive cells. Thus, this method was confirmed (Figure 7).

[0148] Example 6: This example relates to the effect of biological macromolecules on the interaction values measured according to the present invention. CD14-positive monocytes form groups during treatment with lipopolysaccharide (LPS).

[0149] The PBMC monolayer prepared as described above was treated with 10 ng / mL of LPS for 48 hours. The monolayer was fixed, stained with a CD14 antibody, and the tendency of cell-cell interactions was measured using the present invention.

[0150] Group formation of CD14-positive cells became clearly prominent during treatment with LPS. This reflects an increase in the measured interaction values between CD14-positive cells, and thus this method was confirmed (Figure 8).

[0151] Example 7: Objective: This example relates to the determination of the effect on the measured interaction tendency of biological agents known to induce interactions between two cell types.

[0152] Method: Blinatumomab is a BiTE-form bispecific antibody that crosslinks B cells and T cells and induces T cells to kill B cells.

[0153] A PBMC monolayer was prepared as described in WO2016 / 046346, treated with blinatumomab or a PBS control (0) for 48 hours, stained with fluorescently labeled CD3 and CD19 antibodies, and image processed using an automated confocal microscope. Subsequently, the interaction tendency between B cells (CD19-positive) and T cells (CD3-positive) in the prepared non-adherent cell monolayer was determined using the present invention.

[0154] Result: The addition of blinatumomab increased the interaction tendency between B cells and T cells compared to the PBS control (0). Thus, this method was confirmed. (Figure 9).

[0155] Example 8: This example relates to the diagnosis of diseases. The interaction tendency between activated B cells (CD80+ and CD19+) and Th17 T cells (CD3+ and CD28+) in synovial fluid or peripheral blood can be quantified to diagnose the severity of rheumatoid arthritis and psoriatic arthritis. The closer the interaction of these activated cells, the higher the likelihood of being diagnosed with positive rheumatoid arthritis.

[0156] Chronic reactive arthritis is a disease classified as a secondary sterile inflammation after bacterial infection, but it can be diagnosed by quantifying the interaction between monocytes (CD14+) and T cells (CD3+) in synovial fluid after the removal of bacterial pathogens.

[0157] Atherosclerosis can be diagnosed by determining the spatial interaction between resident inflammatory monocytes (CCR2−, CD16+) in the blood and the patient's endothelial cells (CCR5+).

[0158] Example 9: This example relates to the evaluation and detection of the immunomodulatory ability of small molecule drugs. Crizotinib is approved for non-small cell lung cancer, and its immunomodulatory properties are unknown.

[0159] PBMCs from healthy donors are treated with various concentrations of crizotinib or DMSO control, and the tendency of the interaction between CD11C cells and CD3 cells in the non-adherent cell monolayer obtained from the treated / untreated mixed samples according to WO2016 / 046346 is determined using the present invention.

[0160] Crizotinib has an immunomodulatory tendency to increase the interaction between DCs and T cells as indicated by the interaction value, and this interaction may be a factor not known in the clinical mechanism of action of the drug (Figure 10).

[0161] Example 10: Objective: This example relates to the determination of the effect of a biological agent known to induce interaction between two cell types on the measured interaction tendency.

[0162] Method: Rituximab is an anti-CD20 antibody that crosslinks B cells and NK cells, brings them into contact, induces NK cells, and kills B cells.

[0163] As described in WO2016 / 046346, a PBMC monolayer was prepared, treated with rituximab or a PBS control (0) for 48 hours, fixed, stained with fluorescently labeled CD56 antibody and CD19 antibody, and imaged using an automated confocal microscope. Subsequently, the interaction tendency between B cells (CD19 positive) and NK cells (CD56 positive) in the prepared non-adherent cell monolayer was determined using the present invention.

[0164] Results: The addition of blinatumomab increased the interaction tendency between B cells and NK cells compared to the PBS control (0). Therefore, this method was confirmed (Figure 11).

[0165] Example 11: Objective: This example relates to determining whether a patient diagnosed with a disease responds to treatment and whether the biological mechanism of action can be evaluated.

[0166] Method: Rituximab is an anti-CD20 antibody that crosslinks B cells and NK cells, induces NK cells, and kills B cells.

[0167] Bone marrow from a patient with B-cell acute lymphoblastic leukemia (B-ALL) was mixed with the same patient's PBMC, treated with rituximab or a PBS control, and the interaction tendency between B cells and NK cells in the non-adherent cell monolayer obtained from the treated / untreated mixed sample according to WO2016 / 046346 was determined using the present invention.

[0168] Results: The interaction tendency between B cells and NK cells increased in patients who responded to rituximab, but did not increase in patients who did not respond clinically.

[0169] Example 12: Objective: This example relates to determining whether a patient diagnosed with a disease responds to treatment and whether the biological mechanism of action can be evaluated.

[0170] Method: Blinatumomab is a BiTE - shaped bispecific antibody that cross - links B cells and T cells and induces T cells to kill B cells.

[0171] Bone marrow from a patient with B - cell acute lymphoblastic leukemia (B - ALL) was mixed with PBMCs from the same patient, treated with blinatumomab or PBS control, and the tendency of B - cell and T - cell interaction in the non - adherent cell monolayer obtained from the treated / untreated mixed sample according to WO2016 / 046346 was determined using the present invention.

[0172] Results: In patients who responded to blinatumomab, the tendency of B - cell and T - cell interaction increased, but in patients who did not respond clinically, the interaction did not increase.

[0173] Example 13: Objective: This example relates to the measurement of the tendency of cell - cell interaction between multiple different distinguishable sub - populations in the same sample to construct a fingerprint vector representing the change in the tendency of cell - cell interaction in response to treatment with biological stimuli.

[0174] Method: A 384-well plate (Perkin Elmer Cell Carrier) was filled with 15 μL of RPMI (Gibco) containing 10% FCS (Gibco) and penicillin / streptomycin, and cytokine dissolved at a concentration of 15 ng / mL. PBMCs were isolated from the leptomeninges obtained from the Red Cross using Lymphoprep according to the manufacturer's test protocol (Stem Cell), counted, and resuspended in the same RPMI solvent at a final concentration of 20,000 cells / 30 μL. According to WO2016 / 046346, 30 μL of this cell suspension was added to each well filled with the cytokine solution to form a monolayer of PBMCs. By this order of addition, a more uniform monolayer can be obtained than in the reverse order of first adding the cells and then the cytokine. The monolayer was cultured at 37 °C / 5% CO2 for 48 hours, 15 μL of a solution of 0.5% formaldehyde and 0.1% Triton X-114 was added and fixed for 15 minutes, and stained with different fluorescently labeled antibodies to identify cells of different distinguishable subpopulations. Using the said formaldehyde solution has advantages in the setting of fixing PBMC monolayers or bone marrow monolayers. This is because the desired effect of cell fixation can be obtained without damaging specific epitopes and typically reducing the brightness of the fluorescent dye molecules to the same level as that used in the art without reducing it. After image processing with an automated Perkin Elmer Opera Phenix fluorescence microscope, if the cell nuclei are separated by a tendency of interaction represented as less than 20 pixels in a magnification 10-fold image obtained with a microscope with binning doubled and as a log2 interaction value, the tendency of different distinguishable subpopulations of interacting cells was determined according to the present invention as the interacting cells.

[0175] Results: When all measured interaction values were summarized as vectors, a fingerprint normalized to PBS-treated cells was obtained. This fingerprint describes the overall change in the tendency of cell-cell interactions due to treatment with cytokines or other stimuli. Cytokines or stimuli with T cell activation properties had fingerprints that were quite similar to each other compared to stimuli without T cell activation properties (Figure 13).

[0176] Example 14: Pleural effusions were collected from patients with B-cell lymphoma developed by gradient density centrifugation. This pleural effusion consisted of a mixture of cell types found in the bone marrow and PBMC that had migrated to the pleural cavity and the associated fluid layer, and contained cancer cells and healthy cells. Also, the corresponding blood samples were placed on Lymphoprep gradient solution and purified. Cells obtained from the pleural effusion were titrated against the corresponding PBMC samples and monolayer-derived cells prepared by WO2016 / 046346. The monolayer was treated with blinatumomab at different concentrations, and pathologists determined the interaction of the tumor cell population pre-determined to be CD10 positive using effector T cells by the method described in the present invention. Blinatumomab is designed to crosslink tumor B cells and T cells as its mode of action to induce T cell-mediated killing of tumor B cells, but no such change in interaction was observed (Figure 12). Also, the fraction of tumor B cells did not change significantly with in vitro blinatumomab treatment. The treating physician decided to administer blinatumomab to the patient, but the patient responded only temporarily or slightly.

[0177] Example 15: Purpose: Diagnosis of disease

[0178] Method: Isolate peripheral blood from patients suffering from autoimmune or inflammatory diseases and healthy controls. Also, isolate peripheral blood mononuclear cells (PBMCs) using Lymphoprep (Stem Cell). Prepare a monolayer of PBMCs according to WO2016 / 046346 and treat with different cytokines including IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-15, TNFa, CXCL12, IL-33, and other in vitro perturbations for 24 hours, 48 hours, and 72 hours. Fix the monolayer and stain with fluorescently labeled antibodies against CD3, CD4, CD8, CD11c, CD14, CD19, CD20, and CD56 so that all possible combinations of marker-positive cells (e.g., CD3 and CD11c, CD3 and CD19, CD3 and CD20, and CD3 and CD56, etc.) in individual channels can be imaged using an automated fluorescence microscope. Determine the tendency of interactions between all major cell types present in PBMCs (T cells, dendritic cells, monocytes, B cells, and NK cells) characterized by the expression of the above markers according to the present invention. In particular, when the centers of mass of two cells are separated by a distance smaller than their approximate average cell diameter, these cells are considered to be interacting. Collect all interaction tendencies between all major cell types in response to all tested perturbations and quantify them as interaction values according to the method of the present invention. Overview these collected interaction values as a fingerprint vector characterizing each provider in the space of interaction tendencies (see Example 14). This analysis may be performed on a sufficient number of patient samples and control samples to determine the characteristics of the fingerprints of healthy providers and the fingerprints of different subtypes of the disease. Project the fingerprint vector of each individual tested using multidimensional scaling (MDS) or other suitable dimensionality reduction techniques onto a 2D plane and identify the individual groups corresponding to healthy and diseased individuals. Then, for new blood samples obtained from patients rather than healthy controls, classify them corresponding to specific groups by a method known in the art by evaluating the similarity of their fingerprints to the known fingerprints (e.g., Euclidean distance measurement according to the k-nearest neighbor classifier).

[0179] In addition, Example 9 of this specification provides a disease diagnosis example.

[0180] Example 16: Objective: Discovery of a novel T cell activator

[0181] Method: Isolate peripheral blood from healthy donors and isolate peripheral blood mononuclear cells with Lymphoprep (Stem Cell). Prepare a monolayer of PBMCs according to WO2016 / 046346 and treat with reagents known to induce different cytokines and T cell activation (e.g., IL-2, IL-15, CD3-binding antibody, CD28-binding antibody, superantigen, and other stimuli with low T cell activation potential (e.g., LPS, poly I:C, and others)). Fix the monolayer and stain with fluorescently labeled antibodies against CD3, CD4, CD8, CD11c, CD14, CD19, CD20, and CD56 so that all possible combinations of marker-positive cells (e.g., CD3 and CD11c, CD3 and CD19, CD3 and CD20, and CD3 and CD56, etc.) can be imaged using an automated fluorescence microscope. Determine the tendency of interactions between all major cell types present in PBMCs (T cells, dendritic cells, monocytes, B cells, and NK cells) characterized by the expression of the above markers according to the present invention, and generate a fingerprint in the interaction tendency space that characterizes each treatment condition. This analysis may be performed on a sufficient number of samples to determine the characteristics of the fingerprint of the T cell activator (i.e., a vector representing the tendency of defined cell-cell interactions, see also Example 14). Here, for the T cell activation ability of a new, i.e., unknown, stimulus, its fingerprint may be evaluated by comparison with the known fingerprints of T cell activators by methods known in the art (e.g., Euclidean distance measurement according to the k-nearest neighbor classifier).

Claims

**Claim 1** A method for determining the tendency of cell - cell interactions in a population of cells comprising cells of at least two distinguishable sub - populations, said method comprising a) determining the number of interactions between cells of a first distinguishable sub - population and cells of a second distinguishable sub - population; b) randomly assigning the cells contained in the population of cells to cells of the first and second distinguishable sub - populations and determining the number of interactions in the population of cells (wherein the absolute numbers of cells of the first and second distinguishable sub - populations are respectively the same as the numbers of cells of the first and second distinguishable sub - populations in step (a)), and c) dividing the result of (a) by the result of (b) to determine the tendency of cell - cell interactions (wherein the tendency increases with the resulting number), the method. **Claim 2** A population of cells obtained from a cell provider for use in determining whether the cell provider has developed a disease or has a predisposition to developing a disease, said determination comprising determining the tendency of cell - cell interactions within the population, said population comprising cells of at least two distinguishable sub - populations, said method comprising a) determining the number of interactions between cells of a first distinguishable sub - population and cells of a second distinguishable sub - population; b) randomly assigning the cells contained in the population of cells to cells of the first and second distinguishable sub - populations and determining the number of interactions in the population of cells (wherein the absolute numbers of cells of the first and second distinguishable sub - populations are respectively the same as the numbers of cells of the first and second distinguishable sub - populations in step (a)); c) dividing the result of (a) by the result of (b) to determine the tendency of cell - cell interactions (wherein the tendency increases with the resulting number), and d) determining whether the cell provider has developed a disease or has a predisposition to developing a disease based on the tendency of the cell - cell interactions, the cells. **Claim 3** A method for diagnosing a disease or predisposition to a disease of a cell provider, said method comprising determining the tendency of cell - cell interactions in a population of cells obtained from said provider, said population of cells comprising cells of at least two distinguishable sub - populations, said method comprising a) determining the number of interactions between cells of a first distinguishable sub - population and cells of a second distinguishable sub - population; (b) randomly assigning the cells contained in the cells of the population to the cells of the first and second distinguishable subpopulations to determine the number of interactions in the cells of the population (where the absolute numbers of the cells of the first and second distinguishable subpopulations are the same as the numbers of the cells of the first and second distinguishable subpopulations in step (a)), (c) dividing the result of (a) by the result of (b) to determine the tendency of cell - cell interactions (where the tendency increases with the obtained number), and (d) determining whether the cell provider has developed a disease or has a predisposition to develop a disease based on the tendency of the cell - cell interactions. A method comprising: **Claim 4** A method for determining whether a subject who has developed a disease or has a predisposition to develop a disease responds or is responsive to treatment with a therapeutic agent by determining a change in the tendency of cell - cell interactions in a population of cells obtained from the subject, wherein the population of cells comprises cells of at least two distinguishable subpopulations, and the method comprises (a) determining the number of interactions between the cells of the first distinguishable subpopulation and the cells of the second distinguishable subpopulation, (b) randomly assigning the cells contained in the cells of the population to the cells of the first and second distinguishable subpopulations to determine the number of interactions in the cells of the population (where the absolute numbers of the cells of the first and second distinguishable subpopulations are the same as the numbers of the cells of the first and second distinguishable subpopulations in step (a)), (c) dividing the result of (a) by the result of (b) to determine the tendency of cell - cell interactions (where the tendency increases with the obtained number), and (d) determining a change in the tendency of cell - cell interactions by comparing the tendency before adding the therapeutic agent with the tendency after adding the therapeutic agent, thereby determining whether the subject responds or is responsive to the therapeutic agent. A method comprising: **Claim 5** A population of cells obtained from a cell provider used in a diagnostic method for determining whether a subject who has developed a disease or has a predisposition to develop a disease responds or is responsive to treatment with a therapeutic agent, wherein the determination comprises determining the tendency of cell - cell interactions in a monolayer, the monolayer comprising cells of at least two distinguishable subpopulations, and the method comprises Step of determining the number of interactions between cells of a first distinguishable subpopulation and cells of a second distinguishable subpopulation Step of randomly assigning the cells included in the cell sample to the cells of the first and second distinguishable subpopulations to determine the number of interactions in the cell sample (wherein the absolute numbers of cells of the cells of the first and second distinguishable subpopulations are respectively the same as the numbers of cells of the cells of the first and second distinguishable subpopulations in step (a)), Step of dividing the result of (a) by the result of (b) to determine the tendency of cell-cell interactions (wherein the tendency increases with the obtained number), and Step of determining the change in the tendency of cell-cell interactions by comparing the tendency before adding the therapeutic agent with the tendency after adding the therapeutic agent, thereby determining whether the subject responds to the therapeutic agent or has reactivity, including cells

6. A method for screening a therapeutic agent by determining a change in the tendency of cell-cell interactions in a population of cells by adding one or more test substances, wherein the population of cells includes cells of at least two distinguishable subpopulations, and the method includes Step of determining the number of interactions between cells of a first distinguishable subpopulation and cells of a second distinguishable subpopulation Step of randomly assigning the cells included in the cell sample to the cells of the first and second distinguishable subpopulations to determine the number of interactions in the cell sample (wherein the absolute numbers of cells of the cells of the first and second distinguishable subpopulations are respectively the same as the numbers of cells of the cells of the first and second distinguishable subpopulations in step (a)), Step of dividing the result of (a) by the result of (b) to determine the tendency of cell-cell interactions (wherein the tendency increases with the obtained number), and Step of determining the change in the tendency of cell-cell interactions by comparing the tendency before adding the one or more test substances with the tendency after adding the one or more test substances, thereby determining whether the one or more test substances are qualified as a therapeutic agent, including a method

7. A population of cells for use in screening a therapeutic agent by determining a change in the tendency of cell-cell interactions in a population of cells by adding one or more test substances, wherein the population of cells includes cells of at least two distinguishable subpopulations, and the method includes Step (a) of determining the number of interactions between cells of a first distinguishable subpopulation and cells of a second distinguishable subpopulation; Step (b) of randomly assigning the cells included in the cell sample to cells of the first and second distinguishable subpopulations to determine the number of interactions in the cell sample (wherein the absolute numbers of cells of the first and second distinguishable subpopulations are the same as the numbers of cells of the first and second distinguishable subpopulations in step (a) respectively); Step (c) of dividing the result of (a) by the result of (b) to determine the tendency of cell-cell interactions (wherein the tendency increases with the obtained number), and Step (d) of determining a change in the tendency of cell-cell interactions by comparing the tendency before addition of the one or more test substances with the tendency after addition of the one or more test substances, thereby determining whether the one or more test substances are qualified as therapeutic agents, including cells.

8. The method according to any one of claims 1, 3, 4, 6, or the cells of the population according to claim 2, 5, or 7, wherein the first and second distinguishable subpopulations are the same.

9. The method according to any one of claims 1, 3, 4, 6, 8, or the cells of the population according to claim 2, 5, 7, or 8, wherein the number of interactions determined by repeating step (b) is averaged, preferably step (b) is repeated at least 1000 times.

10. The method according to any one of claims 1, 3, 4, 6, 8, 9, or the cells of the population according to claim 2, 5, 7, 8, 9, wherein the cells are (a) peripheral blood mononuclear cell (PBMC) cells or (b) bone marrow cells.

11. The method according to any one of claims 1, 3, 4, 6, 8, 9, 10, or the cells of the population according to claim 2, 5, 7, 8, 9, or 10, wherein the disease is a myeloproliferative disorder, an inflammatory disorder, a potential viral infection, a cell proliferation disorder, a cell chemotaxis disorder, a metabolic disorder, or an autoimmune disease.

12. The method according to claim 10, or the cells of the population according to claim 10, wherein the disease is leukemia or lymphoma.

13. The method according to any one of claims 1, 3, 4, 6, 8, 9, 10, or the cells of the population according to claim 2, 5, 7, 8, 9, or 10, wherein the cells exist in a monolayer form.

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