Marking method for characterizing intercellular contact and application thereof

Abstract

The invention discloses a chemical labeling system (HIM-IP) for detecting interaction between cells. According to the system, tyrosinase and phenol are respectively modified on the surfaces of two to-be-detected cells to construct capturing cells and prey cells, and then biotin or single-stranded DNA with nucleophilic groups is used as a molecular tag for labeling. The system not only can perform detection through traditional flow cytometry, but also can be compatible with a commercialized single cell transcriptome sequencing platform to realize association of intercellular interaction information and transcriptome information on a single cell level. The system does not need to carry out complex gene engineering modification on cells, the whole modification and marking process is simple, convenient and efficient, the detection sensitivity of intercellular interaction is high, and the biological mechanism of intercellular interaction regulation and control can be deeply excavated by integrating the single cell sequencing technology.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a labeling method for characterizing intercellular contact and an application thereof. Background Art

[0002] In recent years, more and more studies have found that cell-to-cell contact interactions play a vital biological role in the regulation of many life activities. For example, physical contact between neurons is the basis of electrochemical signal transduction in the central nervous system; the entire process of T cells crossing the endothelial cell barrier from the blood vessel wall, migrating to the lymphoid tissue and finally being sensitized by antigen-presenting cells is inseparable from the signal regulation mediated by cell-to-cell contact; the formation of tissue or physiological barrier structures is also inseparable from the physical contact between neighboring cells and the signal transduction triggered by such interactions. However, the regulatory mechanism of cell-to-cell interactions is extremely complex, and the biological significance behind many cell contact patterns remains unknown.

[0003] In this research context, various biotechnologies for tracing or detecting cell-to-cell interactions have emerged. Due to the complexity and dynamics of cell-to-cell interactions, it is very challenging to develop characterization methods for such biological events. Currently developed detection technologies mainly include chemical labeling and single-cell sequencing. Chemical labeling usually uses enzymatic reactions or photocatalytic reactions to label cell-to-cell interactions, which has the advantages of high sensitivity and strong specificity. For example, PengWu et al. reported a fucosyltransferase-based chemical labeling strategy FucoID to detect the contact between T cells and DC cells (Liu, Z.; Li, JP; Chen, M.; Wu, M.; Shi, Y.; Li, W.; Teijaro, JR; Wu, P. Detecting tumor antigen-specific T cells via interaction-dependent fucosyl-biotinylation. Cell, 2020, 183, 1117.); MacMillan et al. developed a photocatalytic labeling technology μMap based on Dexter energy transfer and achieved high-resolution labeling of cell-to-cell interacting proteins (Geri, JB; Oakley, JV; Reyes-Robles, T.; Wang, T.; McCarver, SJ; White, CH; Rodriguez-Rivera, FP; Parker Jr., DL; Hett, EC; Fadeyi, OO; Oslund, RC; MacMillan, DWC Microenvironment mapping via Dexter energy transfer on immune cells. Science 2020, 367, 1091). However, chemical labeling methods can usually only analyze cell-to-cell interactions at the flow cytometry level, and cannot characterize cell-to-cell interactions at the single-cell scale and gene level.

[0004] In contrast, single-cell sequencing can perform high-throughput, multi-parameter analysis of interacting cells of specific subpopulations based on the current single-cell transcriptome platform, and can deeply reveal the regulatory mechanisms behind cell-to-cell interactions at the genetic level. However, the current single-cell sequencing method cannot distinguish the level of cell-to-cell interactions, and can usually only analyze cell samples that have already adhered to each other, ignoring the biological information of weak cell-to-cell interactions, and is not as sensitive as the chemical labeling method. Therefore, how to retain the high specificity and sensitivity of chemical labeling while incorporating the advantages of single-cell sequencing technology has important scientific and application value for further understanding of cell-to-cell interactions. However, related characterization technologies have not yet been reported. Summary of the invention

[0005] The present invention aims to develop a labeling method for detecting intercellular contacts (hereinafter referred to as HIM-IP). This labeling technology can integrate a single-cell transcriptome sequencing module while being compatible with traditional flow cytometry detection methods. Compared with existing chemical labeling strategies, HIM-IP can achieve high-throughput, parameterized characterization of intercellular interactions at the single-cell level and gene level; and compared with reported single-cell sequencing technologies, HIM-IP can distinguish between cell-to-cell interactions of different intensities and has higher detection sensitivity and specificity.

[0006] First aspect

[0007] The present invention provides a system for detecting cell-to-cell contact or interaction, the system comprising:

[0008] 1) a catcher cell, wherein the surface of the catcher cell is modified with tyrosinase;

[0009] 2) a prey cell, the surface of which is modified with a phenol group;

[0010] 3) Nucleophilic molecular tags.

[0011] According to an embodiment of the present invention, 1) in the catcher cell, tyrosinase is connected to the surface of the catcher cell by coupling;

[0012] Preferably, the tyrosinase is linked to the surface of the catcher cell via a click chemistry reaction (e.g., a copper-free click chemistry reaction);

[0013] Further preferably, tyrosinase is linked to the catcher cell surface via a click chemistry reaction between an azide group and an alkyne ring.

[0014] According to an embodiment of the present invention, the alkyne ring is a 3-9 membered alkyne ring, preferably a 7-9 membered alkyne ring, such as cyclooctyne.

[0015] According to an embodiment of the present invention, 1) the preparation method of the catcher cell is as follows:

[0016] i) modifying the surface of the catcher cell with reactive groups (such as azide groups);

[0017] ii) preparing a tyrosinase modified with a reactive group (e.g., an alkyne ring);

[0018] iii) co-incubating the tyrosinase modified with the reactive group in step i) with the catcher cells modified with the reactive group in step ii) to obtain catcher cells with tyrosinase modified on the surface.

[0019] According to an embodiment of the present invention, the reactive groups on the surface of the catcher cells are capable of undergoing a coupling reaction with the reactive groups modified on the tyrosinase.

[0020] According to an embodiment of the present invention, ii) the alkyne ring modified tyrosinase is a cyclooctyne modified tyrosinase, preferably a dibenzocyclooctyne modified tyrosinase, more preferably a dibenzocyclooctyne-polyethylene glycol (DBCO-PEG) modified tyrosinase.

[0021] According to an embodiment of the present invention, iii) the concentration of the alkyne ring modified tyrosinase is 50 μg / mL-1000 μg / mL, for example 200 μg / mL.

[0022] According to an embodiment of the present invention, i) the surface of the catcher cell is modified with azide groups by any of the following methods:

[0023] a) placing the catcher cells in a culture medium to obtain catcher cells with surface azidation modification; the culture medium comprises at least one component selected from tetraacylated N-azidoacetylgalactosamine (Ac4GalNAz), tetraacylated N-azidoacetylmannosamine (Ac4ManNAz), and azidated sialic acid; preferably, the culture time is 24-72 hours;

[0024] b) reacting azide-PEG-NHS ester with the catcher cells to obtain catcher cells with surface azide modification; the reaction time is, for example, 30 minutes;

[0025] Preferably, the reaction is carried out in a buffer, such as PBS buffer;

[0026] c) reacting p-azidoethylphenol and tyrosinase with the catcher cells to obtain catcher cells with surface azidation modification; the reaction time is, for example, 10 minutes;

[0027] Preferably, the reaction is carried out in a buffer, such as PBS buffer.

[0028] According to an embodiment of the present invention, in method c), the concentration of p-azidoethylphenol is 1 μM-20 μM, for example, 10 μM.

[0029] According to an embodiment of the present invention, in method c), the concentration of tyrosinase is 5 μg / mL-80 μg / mL, for example, 20 μg / mL.

[0030] Since there are commonly used reaction sites on the surface of cells and tyrosine kinases, such as amino groups, sulfhydryl groups, etc. If bifunctional reagents are directly used to connect cells and tyrosine kinases through these commonly used reaction sites, cross-linking reactions will also occur between cells and between tyrosine kinases, which significantly reduces the controllability of the reaction. The method for preparing the catcher cells used in the present invention utilizes the coupling reaction between the reactive groups on the surface of the catcher cells and the reactive groups modified on the tyrosinase (such as the coupling reaction between the alkyne ring and the azide), thereby modifying the tyrosine kinase to the surface of the catcher cells.

[0031] According to an embodiment of the present invention, 2) the method for constructing prey cells is as follows:

[0032] reacting a reagent containing a phenol group with the prey cells to obtain prey cells with surfaces modified with phenol groups;

[0033] Preferably, the reagent containing a phenol group is selected from: sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (SHPP), 3,4-dihydroxyphenylpropionic acid-N-succinimidyl ester, hydroxyphenylethyl acid-N-succinimidyl ester and 3,4-dihydroxyphenylethyl acid-N-succinimidyl ester;

[0034] Preferably, the reaction time is, for example, 30 minutes; the reaction temperature is, for example, room temperature;

[0035] Preferably, the reaction is carried out in a buffer, such as PBS buffer.

[0036] According to an embodiment of the present invention, the nucleophilic molecular tag comprises a nucleophilic group and a tag.

[0037] According to an embodiment of the present invention, 3) the nucleophilic group in the nucleophilic molecular tag is selected from amino, thiol and aniline.

[0038] According to an embodiment of the present invention, the label in 3) the nucleophilic molecular label is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides (such as single-stranded DNA), etc.

[0039] According to an embodiment of the present invention, 3) the nucleophilic molecular tag is selected from any one of the following:

[0040]

[0041] According to an embodiment of the present invention, 3) the nucleophilic molecular tag is selected from a single-stranded DNA molecule whose 5' end is modified with an amino group, a thiol group or an aniline group.

[0042] According to an embodiment of the present invention, the base sequence of the single-stranded DNA has the following general structure:

[0043] 5'-(N)x+capture sequence-3';

[0044] Among them, N represents any of the four bases A, T, C, and G;

[0045] The value of x is any natural number between 10 and 100.

[0046] According to an embodiment of the present invention, the capture sequence is selected from any one of the following:

[0047] Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; wherein y is any natural number between 0 and 15.

[0048] Sequence 2: 5′-GCTCACCTATTAGCGGCTAAGG-3′;

[0049] Sequence 3: 5′-GCTTTAAGGCCGGTCCTAGCAA-3′.

[0050] The base sequence of single-stranded DNA has the above-mentioned general structure and can meet the compatibility requirements of current commercial single-cell transcriptome sequencing platforms.

[0051] According to different application scenarios or detection targets or detection purposes, the catcher cells and prey cells are selected from any pair of cells that need to interact with each other to be detected, such as animal cells.

[0052] According to an embodiment of the present invention, both the catcher cells and the prey cells are Jurkat cells.

[0053] According to an embodiment of the present invention, the catcher cells are CAR-T cells and the prey cells are tumor cells.

[0054] According to an embodiment of the present invention, the catcher cells are mouse bone marrow-derived dendritic cells (BMDCs), and the prey cells are CD8 + T cells.

[0055] Second aspect

[0056] The present invention also provides a method for detecting cell-cell contact or interaction, the method comprising:

[0057] 1) Construction of catcher cells: Modifying the surface of catcher cells with tyrosinase;

[0058] 2) Constructing prey cells: modifying the surface of prey cells with phenol groups;

[0059] 3) The catcher cells, prey cells, and nucleophilic molecular tags are mixed and reacted, and then detected after the reaction. According to an embodiment of the present invention, 1) in the catcher cells, tyrosinase is connected to the surface of the catcher cells by coupling;

[0060] Preferably, the tyrosinase is linked to the surface of the catcher cell via a click chemistry reaction (e.g., a copper-free click chemistry reaction);

[0061] Further preferably, tyrosinase is linked to the catcher cell surface via a click chemistry reaction between an azide group and an alkyne ring.

[0062] According to an embodiment of the present invention, the alkyne ring is a 3-9 membered alkyne ring, preferably a 7-9 membered alkyne ring, such as cyclooctyne.

[0063] According to an embodiment of the present invention, 1) the method for constructing catcher cells is as follows:

[0064] i) modifying the surface of the catcher cell with reactive groups (such as azide groups);

[0065] ii) preparing a tyrosinase modified with a reactive group (e.g., an alkyne ring);

[0066] iii) The tyrosinase modified with the reactive group in step i) is co-incubated with the catcher cells modified with the reactive group in step ii) to obtain catcher cells with tyrosinase modified on the surface.

[0067] According to an embodiment of the present invention, the reactive groups on the surface of the catcher cells are capable of undergoing a coupling reaction with the reactive groups modified on the tyrosinase.

[0068] According to an embodiment of the present invention, ii) the alkyne ring modified tyrosinase is a cyclooctyne modified tyrosinase, preferably a dibenzocyclooctyne modified tyrosinase, more preferably a dibenzocyclooctyne-polyethylene glycol (DBCO-PEG) modified tyrosinase.

[0069] According to an embodiment of the present invention, iii) the concentration of the alkyne ring modified tyrosinase is 50 μg / mL-1000 μg / mL, for example 200 μg / mL.

[0070] According to an embodiment of the present invention, i) the surface of the catcher cell is modified with azide groups by any of the following methods:

[0071] a) placing the catcher cells in a culture medium to obtain catcher cells with surface azidation modification; the culture medium comprises at least one component selected from tetraacylated N-azidoacetylgalactosamine (Ac4GalNAz), tetraacylated N-azidoacetylmannosamine (Ac4ManNAz), and azidated sialic acid; preferably, the culture time is 24-72 hours;

[0072] b) reacting azide-PEG-NHS ester with the catcher cells to obtain catcher cells with surface azide modification; the reaction time is, for example, 30 minutes;

[0073] Preferably, the reaction is carried out in a buffer, such as PBS buffer;

[0074] c) reacting p-azidoethylphenol and tyrosinase with the catcher cells to obtain catcher cells with surface azidation modification; the reaction time is, for example, 10 minutes;

[0075] Preferably, the reaction is carried out in a buffer, such as PBS buffer.

[0076] According to an embodiment of the present invention, in method c), the concentration of p-azidoethylphenol is 1 μM-20 μM, for example, 10 μM.

[0077] According to an embodiment of the present invention, in method c), the concentration of tyrosinase is 5 μg / mL-80 μg / mL, for example, 20 μg / mL.

[0078] According to an embodiment of the present invention, 2) the method for constructing prey cells is as follows:

[0079] reacting a reagent containing a phenol group with the prey cells to obtain prey cells with surfaces modified with phenol groups;

[0080] Preferably, the reagent containing a phenol group is selected from: sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (SHPP), 3,4-dihydroxyphenylpropionic acid-N-succinimidyl ester, hydroxyphenylethyl acid-N-succinimidyl ester and 3,4-dihydroxyphenylethyl acid-N-succinimidyl ester;

[0081] Preferably, the reaction time is, for example, 30 minutes; the reaction temperature is, for example, room temperature;

[0082] Preferably, the reaction is carried out in a buffer, such as PBS buffer. According to an embodiment of the present invention, the nucleophilic molecular tag comprises a nucleophilic group and a tag.

[0083] According to an embodiment of the present invention, the nucleophilic group in the nucleophilic molecular tag is selected from amino, thiol and aniline.

[0084] According to an embodiment of the present invention, the tag in the nucleophilic molecular tag is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides (such as single-stranded DNA), etc.

[0085] According to an embodiment of the present invention, the nucleophilic molecular tag is selected from any one of the following:

[0086]

[0087] According to an embodiment of the present invention, the nucleophilic molecular tag is selected from single-stranded DNA molecules whose 5' end is modified with an amino group, a thiol group or an aniline group.

[0088] According to an embodiment of the present invention, the base sequence of the single-stranded DNA has the following general structure:

[0089] 5'-(N)x+capture sequence-3';

[0090] Among them, N represents any of the four bases A, T, C, and G;

[0091] The value of x is any natural number between 10 and 100.

[0092] According to an embodiment of the present invention, the capture sequence is selected from any one of the following:

[0093] Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; wherein y is any natural number between 0 and 15.

[0094] Sequence 2: 5′-GCTCACCTATTAGCGGCTAAGG-3′;

[0095] Sequence 3: 5′-GCTTTAAGGCCGGTCCTAGCAA-3′.

[0096] The base sequence of single-stranded DNA has the above-mentioned general structure and can meet the compatibility requirements of current commercial single-cell transcriptome sequencing platforms.

[0097] According to an embodiment of the present invention, in 3), the reaction condition is incubation at 37° C. for 30 minutes.

[0098] According to an embodiment of the present invention, in 3), the concentration of the nucleophilic molecular tag is 10 μM-200 μM, for example, 100 μM.

[0099] According to an embodiment of the present invention, in 3), after the reaction, flow cytometry is used for detection or single-cell transcriptome sequencing is used.

[0100] According to different application scenarios or detection targets or detection purposes, the catcher cells and prey cells can be derived from any pair of cells that need to interact with each other to be detected, such as animal cells.

[0101] According to an embodiment of the present invention, both the catcher cells and the prey cells are Jurkat cells.

[0102] According to an embodiment of the present invention, the catcher cells are CAR-T cells and the prey cells are tumor cells.

[0103] According to an embodiment of the present invention, the catcher cells are mouse bone marrow-derived dendritic cells (BMDCs), and the prey cells are CD8 + T cells.

[0104] The third aspect

[0105] The present invention also provides a kit for detecting intercellular contact or interaction, the kit comprising:

[0106] 1) Reagents for constructing catcher cells;

[0107] 2) Reagents for constructing prey cells;

[0108] 3) labeling reagent;

[0109] Wherein, the reagent for constructing the catcher cell is a reagent for introducing tyrosinase to the surface of the catcher cell;

[0110] The reagent for constructing the prey cell is a reagent for introducing a phenol group onto the surface of the prey cell;

[0111] The labeling reagent is a reagent containing a nucleophilic group and a tag.

[0112] According to an embodiment of the present invention, the nucleophilic group captures the o-quinone produced by the phenol group catalyzed by tyrosinase on the surface of the catcher cell, thereby completing the labeling of the prey cell.

[0113] According to an embodiment of the present invention, 1) the reagents for constructing catcher cells include a reagent for introducing reactive groups to the surface of catcher cells and tyrosinase modified with reactive groups.

[0114] According to an embodiment of the present invention, the reactive groups on the surface of the catcher cells are capable of undergoing a coupling reaction with the reactive groups modified on the tyrosinase.

[0115] According to an embodiment of the present invention, the reactive groups on the surface of the catcher cells are azide groups.

[0116] According to an embodiment of the present invention, the modified reactive group on tyrosinase is an alkynyl group, such as an alkynyl ring group.

[0117] According to an embodiment of the present invention, the reagent for introducing reactive groups into the surface of the catcher cell is selected from a reagent containing an azide group, for example, any one or more of tetraacylated N-azidoacetylgalactosamine (Ac4GalNAz), tetraacylated N-azidoacetylmannosamine (Ac4ManNAz), azidated sialic acid, azide-PEG-NHS ester, and p-azidoethylphenol.

[0118] According to an embodiment of the present invention, the reactive group-modified tyrosinase is an alkyne ring-modified tyrosinase, preferably a dibenzocyclooctyne-modified tyrosinase, such as a dibenzocyclooctyne-polyethylene glycol (DBCO-PEG)-modified tyrosinase.

[0119] According to an embodiment of the present invention, the cycloalkyne is a 3-9 membered alkyne ring, preferably a 7-9 membered alkyne ring, such as cyclooctyne.

[0120] According to an embodiment of the present invention, 2) the reagent for constructing prey cells is selected from sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (SHPP), 3,4-dihydroxyphenylpropionic acid-N-succinimide ester, hydroxyphenylethyl acid-N-succinimide ester and 3,4-dihydroxyphenylethyl acid-N-succinimide ester.

[0121] According to an embodiment of the present invention, 3) the nucleophilic group in the labeling agent is selected from amino, thiol and aniline.

[0122] According to an embodiment of the present invention, 3) the label in the labeling reagent is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides (such as single-stranded DNA), etc.

[0123] According to an embodiment of the present invention, 3) the labeling agent is selected from any one of the following:

[0124]

[0125] According to an embodiment of the present invention, 3) the labeling agent is selected from a single-stranded DNA molecule whose 5' end is modified with an amino group, a thiol group or an aniline group.

[0126] According to an embodiment of the present invention, the base sequence of the single-stranded DNA has the following general structure:

[0127] 5'-(N)x+capture sequence-3';

[0128] Among them, N represents any of the four bases A, T, C, and G;

[0129] The value of x is any natural number between 10 and 100.

[0130] According to an embodiment of the present invention, the capture sequence is selected from any one of the following:

[0131] Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; wherein y is any natural number between 0 and 15.

[0132] Sequence 2: 5′-GCTCACCTATTAGCGGCTAAGG-3′;

[0133] Sequence 3: 5′-GCTTTAAGGCCGGTCCTAGCAA-3′.

[0134] The kit optionally further comprises:

[0135] 5) buffer, such as PBS buffer;

[0136] 6) Tyrosine kinase.

[0137] The present invention also provides the use of the above kit in detecting intercellular contact or interaction.

[0138] Beneficial Effects

[0139] The basic principle of the cell-to-cell interaction labeling involved in the present invention is as follows: the phenol group can be oxidized by dissolved oxygen molecules in the environment to a highly reactive benzoquinone group under the catalysis of tyrosinase, and the benzoquinone group can be further covalently coupled with a nucleophilic group (such as an amino group, a thiol group or an aniline). In the HIM-IP system designed by the present invention, tyrosinase and phenol groups are modified on the surface of the catcher cell and the prey cell, respectively. Only when the two cells are in close contact can tyrosinase recognize and catalyze the oxidation of the phenol group, thereby coupling the nucleophilic label to the reaction site. Therefore, only the prey cells that come into contact with the catcher cell will be chemically labeled.

[0140] The HIM-IP designed by the present invention has the following advantages:

[0141] 1. The engineering transformation of cells involved in the present invention adopts chemical modification method, and there is no need to transform cells at the genetic level. The entire modification and labeling process only takes about 2 hours, which has the technical advantages of being fast and convenient. It is very suitable for primary cells or cell lines that are difficult to genetically engineer, and has strong universality.

[0142] 2. All chemical reagents involved in the HIM-IP technical solution (including tyrosinase, SHPP, biotin tags and DNA tags, etc.) can be obtained through commercial channels, basically without the need for self-synthesis, making it easy to promote and apply the technology.

[0143] 3. The biggest advantage of the technical solution involved in the present invention is that it introduces single-stranded DNA as a molecular label for cell-to-cell interactions, which can associate information about cell-to-cell interactions with transcriptomic information at the single-cell level. This technical feature makes up for the limitation that current cell-to-cell interaction detection based on chemical markers can only be analyzed at the cell population level, and effectively integrates the powerful advantages of single-cell sequencing technology in revealing the mechanism of cell-to-cell interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0144] Figure 1 Figure 1 is a schematic diagram of the HIM-IP system.

[0145] Figure 2 This is the imaging result of the azidated Jurkat cells in Example 1 after being stained with DBCO-Cy5 and Hoechst33342.

[0146] Figure 3 This is the confocal imaging result of Jurkat cells with tyrosinase modified on the surface in Example 1.

[0147] Figure 4 This is the cell surface azidation modification level in Example 2 in dependence on the azidoethylphenol concentration.

[0148] Figure 5 This is the cell surface azide modification level in Example 3 that depends on the tyrosinase concentration.

[0149] Figure 6 The modification level of tyrosinase in Jurkat cells after treatment with different concentrations of Ac4GaINAz in Example 4.

[0150] Figure 7 The figure shows the effect of tyrosinase and SHPP modification on biotin labeling efficiency in the HIM-IP system of Example 5.

[0151] Figure 8 This is a correlation diagram between the proportion of catcher cells and the proportion of biotin-labeled prey cells in the Jurkat cell interaction system in Example 5.

[0152] Fig. 9 This is a correlation diagram between the proportion of catcher cells and the proportion of biotin-labeled prey cells in the interaction system of CAR-T and Ramos or K562 cells in Example 6.

[0153] Fig.10 The flow cytometry results of HIM-IP for T cell labeling after BMDCs presented with different polypeptides in Example 7 were co-cultured with T cells.

[0154] Fig.11 This is the single-cell sequencing result after CAR-T and Ramos were labeled by single-stranded DNA interaction in Example 8. DETAILED DESCRIPTION

[0155] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0156] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0157] HIM-IP:

[0158] Ac4GalNAz: tetraacylated N-azidoacetylgalactosamine.

[0159] Ac4ManNAz: tetraacylated N-azidoacetylmannosamine.

[0160] SHPP: sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate.

[0161] Azide-PEG-NHS:

[0162] 4-(2-Azidoethyl)phenol: 4-(2-Azidoethyl)phenol, CAS: 74447-34-0.

[0163] Example 1

[0164] Construction of catcher cells based on Jurkat cells

[0165] (1) Collect 2 million Jurkat cells by centrifugation, wash twice with PBS, and resuspend in 490 μL PBS. Add 5 μL of p-azidoethylphenol solution (1 mM) and 5 μL of tyrosinase solution (2 mg / mL) to the cell suspension to make the final concentrations of p-azidoethylphenol and tyrosinase 10 μM and 20 μg / mL, respectively. Vortex gently and shake at room temperature for 10 minutes.

[0166] (2) After the reaction is completed, the cells are collected by centrifugation, washed three times with PBS and resuspended. In this step, a small amount of cells can be taken out for Hoechst 33342 and dibenzocyclooctyne-Cy5 (DBCO-Cy5) staining, and fluorescence confocal imaging can be used to detect the azide modification of the cell surface.

[0167] (3) The cell suspension washed in the previous step was mixed with the DBCO-PEG-modified tyrosinase (also labeled with FITC) solution to make the final concentration of tyrosinase in the system 200 μg / mL. After gently mixing the cells, shake the reaction at room temperature for 30 minutes.

[0168] (4) After the reaction is completed, the cells are collected by centrifugation and washed 2-3 times. After staining with Hoechst33342, the modification of tyrosinase on the cell surface is observed under a fluorescence confocal microscope.

[0169] Conclusion: Figure 2 It can be seen that the surface of Jurkat cells modified by azidation is significantly labeled with red fluorescence by DBCO-Cy5, proving that the azidation modification is successful. Figure 3 It can be seen that FITC-labeled tyrosinase is distributed on the surface of Jurkat cells, proving that tyrosinase modification on the Jurkat cell membrane is successful.

[0170] Example 2

[0171] Optimization of azide modification conditions in catcher cell construction:

[0172] The operation steps were the same as steps (1) and (2) in Example 1, except that the final concentration of p-azidoethylphenol in step (1) was set to 1 μM, 2 μM, 5 μM, 10 μM and 20 μM. The fluorescence signal intensity of DBCO-Cy5 on the surface of Jurkat cells was counted by flow cytometry.

[0173] Conclusion: Figure 4 It can be seen that the degree of azidation modification on the surface of Jurkat cells can be increased by increasing the modification concentration of p-azidoethylphenol.

[0174] Example 3

[0175] Optimization of azide modification conditions in catcher cell construction:

[0176] The operation steps were the same as steps (1) and (2) in Example 1, except that the final concentration of tyrosinase in step (1) was set to 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL and 80 μg / mL. The fluorescence signal intensity of DBCO-Cy5 on the surface of Jurkat cells was counted by flow cytometry.

[0177] Conclusion: Figure 5 It can be seen that the degree of azide modification on the surface of Jurkat cells can be increased by increasing the modification concentration of tyrosinase.

[0178] Example 4

[0179] Cell surface azidation modification strategy based on Ac4GalNAz:

[0180] (1) Jurkat cells were collected by centrifugation, and the cell culture medium was replaced with complete culture medium containing 0, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM Ac4GalNAz, and the culture was continued for 72 hours.

[0181] (2) After the culture was completed, Jurkat cells were collected and reacted with DBCO-PEG-modified tyrosinase (FITC-labeled), and then analyzed by flow cytometry.

[0182] Conclusion: Figure 6 It can be seen that the density of azidation modification on the surface of Jurkat cells increases with the increase of Ac4GalNAz concentration. The above data prove that Ac4GalNAz can be used to azidate the surface of Jurkat cells and used for subsequent tyrosinase labeling.

[0183] Example 5

[0184] HIM-IP was used to detect the interaction between Jurkat cells at the flow cytometry level:

[0185] Jurkat cells are prone to aggregation during culture, indicating that there is a strong contact-type interaction between these cells. The experimental steps for HIM-IP to detect the interaction between Jurkat cells are as follows:

[0186] (1) 10 million Jurkat cells were collected by centrifugation, washed twice with PBS, resuspended, and divided into two equal parts, one for constructing catcher cells and the other for constructing prey cells.

[0187] (2) To construct the catcher cells, 5 μL of p-azidoethylphenol solution (1 mM) and 5 μL of tyrosinase solution (2 mg / mL) were added to the cell suspension to make the final concentrations of p-azidoethylphenol and tyrosinase 10 μM and 20 μg / mL, respectively. After gently vortexing, the reaction was shaken at room temperature for 10 minutes. After the reaction was completed, the cells were washed three times by centrifugation, resuspended in 500 μL PBS, and then DBCO-PEG-modified tyrosinase was added to make the final concentration of tyrosinase in the system 200 μg / mL. After gently mixing the cells, the reaction was shaken at room temperature for 30 minutes. After the reaction was completed, the cells were washed three times with PBS and set aside. The catcher cells in the control group were Jurkat cells without any modification.

[0188] (3) To construct prey cells, SHPP was added to the Jurkat cell suspension to a final concentration of 4 μM. After a brief vortex, the cell suspension was placed at room temperature for 30 minutes for shaking reaction. After the reaction, the cells were washed once by centrifugation, and then 500 μL of Hoechst33342 dye solution (5 μg / mL) was added and stained at room temperature for 8 minutes. After the staining was completed, the cells were washed three times by centrifugation and set aside. The prey cells in the control group were Jurkat cells that were not modified with SHPP but were stained with Hoechst33342.

[0189] (4) Next, the catcher cells and prey cells were mixed in different quantitative ratios, and the aniline-biotin label was added to a final concentration of 100 μM. After incubation at 37°C for 30 minutes, the cells were collected by centrifugation, washed three times with PBS, and then FITC-labeled streptavidin stain (final concentration 20 μg / mL) was added. The cells were stained at room temperature for 10 minutes, washed three times with PBS, and then characterized by flow cytometry.

[0190] Conclusion: Figure 7It can be seen that the lack of tyrosinase modification in the catcher cells or the lack of SHPP modification in the prey cells will cause the HIM-IP system to be unable to detect cell-to-cell interactions normally. Only when both elements are present at the same time can the efficiency of cell-to-cell interaction labeling be the highest. Figure 8 It can be seen that by changing the proportion of catcher cells in the system, the proportion of prey cells labeled with biotin will also change accordingly. This result proves that HIM-IP has sufficient sensitivity to detect cell subpopulations that have cell-to-cell contacts in the system.

[0191] Example 6

[0192] HIM-IP detects the interaction between CAR-T and tumor cells at the flow cytometry level. The specific operation is as follows:

[0193] (1) Prepare CAR-T cells targeting CD19, Ramos cells (CD19 positive expression) and K562 cells (CD19 negative expression), collect the above cells by centrifugation, wash them twice with PBS and resuspend them. CAR-T cells are used to construct catcher cells, and Ramos and K562 cells are used to construct prey cells.

[0194] (2) To construct the catcher cells, add 5 μL of p-azidoethylphenol solution (1 mM) and 5 μL of tyrosinase solution (2 mg / mL) to the CAR-T cell suspension, so that the final concentrations of p-azidoethylphenol and tyrosinase are 10 μM and 20 μg / mL, respectively. After gently vortexing, shake the reaction at room temperature for 10 minutes. After the reaction is completed, the cells are washed three times by centrifugation, resuspended in 500 μL PBS, and then DBCO-PEG-modified tyrosinase is added to make the final concentration of tyrosinase in the system 200 μg / mL. After gently mixing the cells, shake the reaction at room temperature for 30 minutes. After the reaction is completed, wash the cells three times with PBS and set aside.

[0195] (3) To construct prey cells, SHPP was added to the Ramos cell or K562 cell suspension to a final concentration of 4 μM. After a brief vortex, the cell suspension was placed at room temperature for 30 minutes for shaking reaction. After the reaction, the cells were washed once by centrifugation, and then 500 μL of Hoechst33342 dye solution (5 μg / mL) was added and stained at room temperature for 8 minutes. After the staining was completed, the cells were washed three times by centrifugation and set aside.

[0196] (4) Next, the modified CAR-T cells were mixed with Ramos cells or K562 cells in different quantitative ratios, and aniline-biotin label was added to make the final concentration 100 μM. After incubation at 37°C for 30 minutes, the cells were collected by centrifugation, washed three times with PBS, and then FITC-labeled streptavidin stain (final concentration 20 μg / mL) was added. After staining at room temperature for 10 minutes and washing three times with PBS, flow cytometry characterization was performed.

[0197] Conclusion: Fig. 9 It can be seen that for the interaction system of CAR-T and Ramos cells, as the proportion of CAR-T cells continues to increase, the proportion of Ramos cells labeled with biotin also increases accordingly, proving that the cell-to-cell interaction is effectively labeled; in contrast, since K562 cells do not interact significantly with CAR-T cells themselves, the proportion of biotin-labeled cells is very low and does not change significantly with changes in the proportion of CAR-T cells in the system.

[0198] Example 7

[0199] HIM-IP detection of mouse BMDCs (mouse myeloid dendritic cells) and CD8 + T cell-cell contact, distinguishing cell-cell interactions of different intensities. The specific experimental operation is as follows:

[0200] (1) BMDCs are derived from the bone marrow of C57BL / 6 mice with CD45.1 and CD8 + T cells were extracted from the spleen of OT-1 mice with CD45.2. BMDCs were used to construct catcher cells, CD8 + T cells are used to build prey cells.

[0201] (2) To construct the catcher cells, 5 μL of p-azidoethylphenol solution (1 mM) and 5 μL of tyrosinase solution (2 mg / mL) were added to the cell suspension to make the final concentrations of p-azidoethylphenol and tyrosinase 10 μM and 20 μg / mL, respectively. After gently vortexing, the reaction was shaken at room temperature for 10 minutes. After the reaction, the cells were washed three times by centrifugation, resuspended in 500 μL PBS, and then DBCO-PEG-modified tyrosinase was added to make the final concentration of tyrosinase in the system 200 μg / mL. After gently mixing the cells, the reaction was shaken at room temperature for 30 minutes. After the reaction was completed, the cells were washed three times with PBS, and the cells were divided into four parts and incubated with the following peptides (37°C, 30 minutes): N4 peptide (SIINFEKL), A2 peptide (SAINFEKL), T4 peptide (SIITFEKL) and LCMV GP33 peptide (KAVYNFATM). After incubation, cells were washed three times with PBS and set aside.

[0202] (3) To construct prey cells, CD8 + SHPP was added to the T cell suspension to a final concentration of 4 μM. After a brief vortex, the cell suspension was placed at room temperature for 30 minutes for shaking reaction. After the reaction, the cells were washed by centrifugation 3 times and set aside.

[0203] (4) The treated BMDCs were mixed with CD8 + T cells were incubated at 37°C for 1.5 hours, and then aniline-biotin label was added to a final concentration of 100 μM. After further incubation for 30 minutes, the cells were washed 3 times with PBS, and then FITC-labeled streptavidin stain (final concentration 20 μg / mL), Cy5-labeled CD8 antibody, and Brilliant Violet785-labeled CD45.1 antibody were added. After staining for 10 minutes, the cells were washed 3 times with staining buffer and characterized by flow cytometry.

[0204] Conclusion: Fig.10 It can be seen that due to the different interaction strengths between BMDCs and T cells presented by different peptides, the ratio of biotin labeling of T cells mediated by HIM-IP also changes accordingly, namely N4>A2>T4>GP33. Therefore, HIM-IP can distinguish cell-to-cell interactions of different strengths.

[0205] Example 8

[0206] Detection of the interaction between CAR-T and tumor cells based on single-cell transcriptome sequencing platform

[0207] The specific experimental operations are as follows:

[0208] (1) Prepare CD19-targeting CAR-T cells and Ramos cells (CD19 positive), collect the cells by centrifugation, wash them twice with PBS and resuspend them. CAR-T cells are used to construct catcher cells, and Ramos cells are used to construct prey cells.

[0209] (2) To construct the catcher cells, add 5 μL of p-azidoethylphenol solution (1 mM) and 5 μL of tyrosinase solution (2 mg / mL) to the cell suspension, so that the final concentrations of p-azidoethylphenol and tyrosinase are 10 μM and 20 μg / mL, respectively. After gently vortexing, shake the reaction at room temperature for 10 minutes. After the reaction is completed, wash the cells three times by centrifugation, resuspend them in 500 μL PBS, and then add DBCO-PEG-modified tyrosinase to make the final concentration of tyrosinase in the system 200 μg / mL. After gently mixing the cells, shake the reaction at room temperature for 30 minutes. After the reaction is completed, wash the cells three times with PBS and set aside.

[0210] (3) To construct prey cells, SHPP was added to the Ramos cell suspension to a final concentration of 4 μM. After a brief vortex, the cell suspension was placed at room temperature for 30 minutes for shaking reaction. After the reaction was completed, the cells were washed by centrifugation 3 times and set aside.

[0211] (4) Next, the modified CAR-T cells were mixed with Ramos cells, and an amino-single-stranded DNA tag (sequence: 5'-CTACGGTGCCTTGAAGTGACATACGCTAAAAAAAAAAAAAAAAAAAAA-3') was added to a final concentration of 400 nM. After incubation at 37°C for 30 minutes, the cells were collected by centrifugation and washed three times with PBS. Next, the sequencing and subsequent library construction and sequencing analysis were performed according to the standardized 10XGenomics single-cell transcriptome sequencing process.

[0212] Conclusion: From the attached Fig.11 It can be seen that the single-stranded DNA labeled on Ramos cells can be detected by single-cell sequencing technology. And there are differences in the single-stranded DNA labeling levels of different Ramos cells, indicating that there is a large heterogeneity in the level of interaction between these cells and CAR-T cells. The above results show that the HIM-IP technology designed by the present invention can use single-stranded DNA molecular tags to mark cell-to-cell interactions and analyze cell-to-cell interactions through single-cell transcriptome sequencing.

[0213] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A system for detecting cell-cell contact or interaction, the system comprising: 1) a catcher cell, wherein the surface of the catcher cell is modified with tyrosinase; 2) a prey cell, the surface of which is modified with a phenol group; 3) Nucleophilic molecular tags.

2. The system according to claim 1, characterized in that 1) In the catcher cell, tyrosinase is connected to the surface of the catcher cell by coupling; preferably, tyrosinase is connected to the surface of the catcher cell by a click chemistry reaction (such as a copper-free click chemistry reaction); further preferably, tyrosinase is connected to the surface of the catcher cell by a click chemistry reaction between an azide group and an alkyne group (such as an alkyne ring). Preferably, the alkyne ring is a 3-9 membered alkyne ring, preferably a 7-9 membered alkyne ring, such as cyclooctyne. Preferably, 1) the preparation method of the catcher cells is as follows: i) modifying the surface of the catcher cell with reactive groups (such as azide groups); ii) preparing a tyrosinase modified with a reactive group (e.g., an alkynyl group, preferably an alkynyl ring); iii) co-incubating the tyrosinase modified with the reactive group in step i) with the catcher cells modified with the reactive group in step ii) to obtain catcher cells with tyrosinase modified on the surface. Preferably, the reactive groups on the surface of the catcher cells are capable of undergoing a coupling reaction with the reactive groups modified on the tyrosinase; Preferably, in ii), the reactive group-modified tyrosinase is an alkynyl-modified tyrosinase, preferably an alkynyl ring-modified tyrosinase, and further preferably a cyclooctyne-modified tyrosinase (e.g., dibenzocyclooctyne-modified tyrosinase, preferably dibenzocyclooctyne-polyethylene glycol (DBCO-PEG)-modified tyrosinase); Preferably, during the reaction in iii), the concentration of the alkynyl-modified tyrosinase is 50 μg / mL-1000 μg / mL, such as 200 μg / mL. Preferably, i) modifying the catcher cell surface with azide groups adopts any of the following methods: a) placing the catcher cells in a culture medium to obtain catcher cells with surface azidation modification; the culture medium comprises at least one component selected from tetraacylated N-azidoacetylgalactosamine (Ac4GalNAz), tetraacylated N-azidoacetylmannosamine (Ac4ManNAz), and azidated sialic acid; preferably, the culture time is 24-72 hours; b) reacting azide-PEG-NHS ester with the catcher cells to obtain catcher cells with surface azide modification; the reaction time is, for example, 30 minutes; preferably, the reaction is carried out in a buffer, such as PBS buffer; c) reacting p-azidoethylphenol and tyrosinase with the catcher cells to obtain catcher cells with surface azidation modification; the reaction time is, for example, 10 minutes; preferably, the reaction is carried out in a buffer, such as PBS buffer; preferably, the concentration of p-azidoethylphenol is 1 μM-20 μM, such as 10 μM; preferably, the concentration of tyrosinase is 5 μg / mL-80 μg / mL, such as 20 μg / mL.

3. The system according to claim 1 or 2, characterized in that: 2) The construction method of prey cells is as follows: reacting a reagent containing a phenol group with the prey cells to obtain prey cells with surfaces modified with phenol groups; Preferably, the reagent containing a phenol group is selected from one or more of the following: sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (SHPP), 3,4-dihydroxyphenylpropionic acid-N-succinimide ester, hydroxyphenylethyl acid-N-succinimide ester and 3,4-dihydroxyphenylethyl acid-N-succinimide ester; Preferably, the reaction time is, for example, 30 minutes; the reaction temperature is, for example, room temperature; Preferably, the reaction is carried out in a buffer, such as PBS buffer. Preferably, 3) the nucleophilic molecular tag comprises a nucleophilic group and a tag; Preferably, the nucleophilic group is selected from amino, thiol and aniline; Preferably, 3) the label in the nucleophilic molecular label is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides (such as single-stranded DNA), etc.; Preferably, 3) the nucleophilic molecular tag is selected from any one of the following: Preferably, 3) the nucleophilic molecular tag is selected from a single-stranded DNA molecule whose 5' end is modified with an amino group, a thiol group or an aniline group; Preferably, the base sequence of the single-stranded DNA has the following general structure: 5'-(N)x+capture sequence-3'; Wherein, N represents any one of the four bases A, T, C, and G; the value of x is any natural number between 10 and 100; Preferably, the capture sequence is selected from any one of the following: Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; where y is any natural number between 0 and 15; Sequence 2: 5′-GCTCACCTATTAGCGGCTAAGG-3′; Sequence 3: 5′-GCTTTAAGGCCGGTCCTAGCAA-3′. Preferably, according to different application scenarios or detection targets or detection purposes, the catcher cells and prey cells are selected from any pair of cells that need to be detected to interact with each other, such as animal cells; Preferably, both the catcher cells and the prey cells are Jurkat cells; Preferably, the catcher cells are CAR-T cells and the prey cells are tumor cells; Preferably, the catcher cells are mouse bone marrow-derived dendritic cells (BMDCs) and the prey cells are CD8 + T cells.

4. A method for detecting cell-cell contact or interaction, the method comprising: 1) Construction of catcher cells: Modifying the surface of catcher cells with tyrosinase; 2) Constructing prey cells: modifying the surface of prey cells with phenol groups; 3) The catcher cells, prey cells and nucleophilic molecule tags are mixed and reacted, and then detected after the reaction.

5. The method according to claim 4, characterized in that 1) In the catcher cell, tyrosinase is connected to the surface of the catcher cell by coupling; Preferably, the tyrosinase is linked to the surface of the catcher cell via a click chemistry reaction (e.g., a copper-free click chemistry reaction); Further preferably, the tyrosinase is linked to the catcher cell surface via a click chemistry reaction between an azide group and an alkyne group (eg an alkyne ring). Preferably, the alkyne ring is a 3-9 membered alkyne ring, preferably a 7-9 membered alkyne ring, such as cyclooctyne. Preferably, 1) the method for constructing catcher cells is as follows: i) modifying the surface of the catcher cell with reactive groups (such as azide groups); ii) preparing a tyrosinase modified with a reactive group (e.g., an alkynyl group, preferably an alkynyl ring); iii) co-incubating the tyrosinase modified with the reactive group in step i) with the catcher cell modified with the reactive group in step ii) to obtain the catcher cell with tyrosinase modified on the surface; Preferably, the reactive groups on the surface of the catcher cells are capable of undergoing a coupling reaction with the reactive groups modified on the tyrosinase. Preferably, in ii), the reactive group-modified tyrosinase is an alkynyl-modified tyrosinase, preferably an alkynyl ring-modified tyrosinase, and further preferably a cyclooctyne-modified tyrosinase (e.g., dibenzocyclooctyne-modified tyrosinase, preferably dibenzocyclooctyne-polyethylene glycol (DBCO-PEG)-modified tyrosinase); Preferably, during the reaction in iii), the concentration of the alkyne ring-modified tyrosinase is 50 μg / mL-1000 μg / mL, for example, 200 μg / mL; Preferably, i) modifying the catcher cell surface with azide groups adopts any of the following methods: a) placing the catcher cells in a culture medium to obtain catcher cells with surface azidation modification; the culture medium comprises at least one component selected from tetraacylated N-azidoacetylgalactosamine (Ac4GalNAz), tetraacylated N-azidoacetylmannosamine (Ac4ManNAz), and azidated sialic acid; preferably, the culture time is 24-72 hours; b) reacting azide-PEG-NHS ester with the catcher cells to obtain catcher cells with surface azide modification; the reaction time is, for example, 30 minutes; preferably, the reaction is carried out in a buffer, such as PBS buffer; c) reacting p-azidoethylphenol and tyrosinase with the catcher cells to obtain catcher cells with surface azidation modification; the reaction time is, for example, 10 minutes; preferably, the reaction is carried out in a buffer, such as PBS buffer; preferably, the concentration of p-azidoethylphenol is 1 μM-20 μM, such as 10 μM. Preferably, the concentration of tyrosinase is 5 μg / mL-80 μg / mL, such as 20 μg / mL.

6. The method according to claim 4 or 5, characterized in that: 2) The construction method of prey cells is as follows: reacting a reagent containing a phenol group with the prey cells to obtain prey cells with surfaces modified with phenol groups; Preferably, the reagent containing a phenol group is selected from one or more of the following: sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (SHPP), 3,4-dihydroxyphenylpropionic acid-N-succinimide ester, hydroxyphenylethyl acid-N-succinimide ester and 3,4-dihydroxyphenylethyl acid-N-succinimide ester; Preferably, the reaction time is, for example, 30 minutes; the reaction temperature is, for example, room temperature; Preferably, the reaction is carried out in a buffer, such as PBS buffer. Preferably, the nucleophilic molecular tag comprises a nucleophilic group and a tag; Preferably, the nucleophilic group in the nucleophilic molecular tag is selected from amino, thiol and aniline; Preferably, the label in the nucleophilic molecular label is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides (such as single-stranded DNA), etc.; Preferably, the nucleophilic molecular tag is selected from any one of the following: Preferably, the nucleophilic molecular tag is selected from a single-stranded DNA molecule whose 5' end is modified with an amino group, a thiol group or an aniline group; Preferably, the base sequence of the single-stranded DNA has the following general structure: 5'-(N)x+capture sequence-3'; Wherein, N represents any one of the four bases A, T, C, and G; the value of x is any natural number between 10 and 100; Preferably, the capture sequence is selected from any one of the following: Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; where y is any natural number between 0 and 15; Sequence 2: 5′-GCTCACCTATTAGCGGCTAAGG-3′; Sequence 3: 5′-GCTTTAAGGCCGGTCCTAGCAA-3′. Preferably, in 3), the reaction conditions are incubation at 37°C for 30 minutes; Preferably, in 3), the concentration of the nucleophilic molecular tag is 10 μM-200 μM, for example, 100 μM; Preferably, in 3), after the reaction, flow cytometry is used for detection or single-cell transcriptome sequencing is used. Preferably, according to different application scenarios or detection targets or detection purposes, the catcher cells and prey cells can be derived from any pair of cells that need to interact with each other to be detected, such as animal cells; Preferably, both the catcher cells and the prey cells are Jurkat cells; Preferably, the catcher cells are CAR-T cells and the prey cells are tumor cells; Preferably, the catcher cells are mouse bone marrow-derived dendritic cells (BMDCs) and the prey cells are CD8 + T cells.

7. A kit for detecting cell-cell contact or interaction, the kit comprising: 1) Reagents for constructing catcher cells; 2) Reagents for constructing prey cells; 3) labeling reagent; Wherein, the reagent for constructing the catcher cell is a reagent for introducing tyrosinase to the surface of the catcher cell; The reagent for constructing the prey cell is a reagent for introducing a phenol group onto the surface of the prey cell; The labeling reagent is a reagent containing a nucleophilic group and a tag.

8. The kit according to claim 7, characterized in that 1) Reagents for constructing catcher cells include reagents for introducing reactive groups onto the surface of catcher cells and tyrosinase modified with reactive groups; Preferably, the reactive groups on the surface of the catcher cells are capable of undergoing a coupling reaction with the reactive groups modified on the tyrosinase; Preferably, the reactive groups on the surface of the catcher cells are azide groups; Preferably, the modified reactive group on tyrosinase is an alkynyl group; for example, an alkynyl ring group; Preferably, the reagent for introducing a reactive group to the surface of the catcher cell is selected from a reagent containing an azide group, for example, any one or more selected from tetraacylated N-azidoacetylgalactosamine (Ac4GalNAz), tetraacylated N-azidoacetylmannosamine (Ac4ManNAz), azidated sialic acid, azide-PEG-NHS ester, and p-azidoethylphenol; Preferably, the reactive group-modified tyrosinase is an alkynyl-modified tyrosinase, preferably an alkynyl ring-modified tyrosinase, and further preferably a cyclooctyne-modified tyrosinase (e.g., dibenzocyclooctyne-modified tyrosinase, preferably dibenzocyclooctyne-polyethylene glycol (DBCO-PEG)-modified tyrosinase); Preferably, the cycloalkyne is a 3-9 membered alkyne ring, preferably a 7-9 membered alkyne ring, such as cyclooctyne. Preferably, 2) the reagent for constructing prey cells is selected from one or more of the following: sodium 1-((3-(4-hydroxyphenyl)propionyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate (SHPP), 3,4-dihydroxyphenylpropionic acid-N-succinimide ester, hydroxyphenylethyl acid-N-succinimide ester and 3,4-dihydroxyphenylethyl acid-N-succinimide ester. Preferably, 3) the nucleophilic group in the labeling agent is selected from amino, sulfhydryl and aniline; Preferably, 3) the label in the labeling agent is selected from fluorescent molecules, biotin or its derivatives, oligonucleotides (such as single-stranded DNA), etc.; Preferably, 3) the labeling agent is selected from any one of the following: Preferably, 3) the labeling agent is selected from a single-stranded DNA molecule whose 5' end is modified with an amino group, a thiol group or an aniline group; Preferably, the base sequence of the single-stranded DNA has the following general structure: 5'-(N)x+capture sequence-3'; Wherein, N represents any one of the four bases A, T, C, and G; the value of x is any natural number between 10 and 100; Preferably, the capture sequence is selected from any one of the following: Sequence 1: 5'-AAAAAAAAAAAAAAA(A)y-3'; where y is any natural number between 0 and 15; Sequence 2: 5′-GCTCACCTATTAGCGGCTAAGG-3′; Sequence 3: 5′-GCTTTAAGGCCGGTCCTAGCAA-3′.

9. The kit according to claim 7 or 8, characterized in that The kit optionally further comprises any one or more of the following: 5) buffer, such as PBS buffer; 6) Tyrosine kinase.

10. Use of the kit according to any one of claims 7 to 9 in detecting cell-cell contact or interaction.

Images