A method of labeling cells in proximity

Abstract

The application provides a method for marking adjacent cells, which comprises: sequentially adding a photocatalyst and a probe into cells to be marked, and realizing marking of adjacent cells under deep red light irradiation; wherein the photocatalyst comprises chlorin E6 or a derivative thereof, and the probe comprises biotin aniline or a derivative thereof. The method for marking adjacent cells has the characteristics of high efficiency, sensitivity and non-invasiveness, can effectively analyze dynamic interaction of tumor-immune cells, and provides an important experimental tool for immune surveillance and / or immunotherapy of tumors.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a method for neighboring cell labeling. Background Technology

[0002] In recent years, tumor immunotherapy has become an important area of ​​cancer treatment. Cell-cell interactions within the tumor microenvironment closely influence tumor development and patient response to immunotherapy. Therefore, a deeper understanding of the interactions between tumor cells and immune cells, especially under in situ conditions, is crucial for developing more effective immunotherapy strategies.

[0003] Photocatalytic proximity labeling allows for better spatiotemporal control of the labeling reaction, and using exogenous biotin probes for cell labeling avoids the bias problem caused by endogenous substrate limitations. However, current technologies are limited to using short-wavelength blue-green light as the excitation source to initiate the labeling reaction, leading to limitations in tissue penetration and biocompatibility. Literature reports that blue light can excite endogenous photosensitive substances (such as melanin, riboflavin, and porphyrin) leading to the generation of singlet oxygen. The oxidative damage to neighboring proteins caused by these singlet oxygen molecules can be captured by nucleophilic probes, thus creating a biotinylated background.

[0004] Therefore, there is an urgent need to develop a neighboring cell labeling method that can achieve effective in-situ labeling in tissue samples. Summary of the Invention

[0005] The purpose of this application is to provide a neighboring cell labeling method that is highly efficient, sensitive, and non-invasive, enabling effective analysis of the dynamic interaction between tumor and immune cells, and providing an important experimental tool for tumor immune surveillance and / or immunotherapy. The specific technical solution is as follows:

[0006] This application provides a method for neighboring cell labeling, comprising: sequentially adding a photocatalyst and a probe to cells to be labeled, and incubating them under deep red light irradiation to achieve neighboring cell labeling; wherein, the photocatalyst comprises dihydroporphyrin E6 (chlorin e6, Ce6) or a derivative thereof, and the probe comprises biotin aniline or a derivative thereof.

[0007] In one embodiment of this application, the photocatalyst is a Ce6 nanobody conjugate; the Ce6 nanobody conjugate includes His-tag nanobodies, a ZHER-Ce6 complex targeting the proto-oncogene human epidermal growth factor receptor 2 (HER2), a P43-Ce6 complex targeting human programmed death-ligand 1 (PD-L1), and an inactivating mutant of the P43-Ce6 complex, the dP43-Ce6 complex.

[0008] In one embodiment of this application, the synthesis steps of the Ce6 nanobody conjugate include:

[0009] Nanobodies expressing the C-terminal sequence LPETG-His-tag were linked to the GGGK(N3)GGK(N3)K(N3) peptide using the transpeptidase mgSrtA, and then subjected to an azide-alkyne cycloaddition reaction (Click reaction) with the compound shown in Formula I to obtain nanobodies Ce6 conjugates.

[0010]

[0011] In one embodiment of this application, the synthesis steps of the compound shown in Formula I include: (1) methyl esterification protection of the two carboxyl groups of Ce6; (2) carboxyl activation using benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), addition of mono-tert-butyloxycarbonyl-protected ethylenediamine, and condensation reaction using N,N-diisopropylethylamine (DIPEA) or triethylamine (TEA) as a base; (3) deprotection reaction of the tert-butyloxycarbonyl group using trifluoroacetic acid (TFA) or 1,4-dioxane hydrogen chloride; (4) condensation reaction of the amino group and the N-hydroxysuccinimide (NHS) active ester using N,N-diisopropylethylamine (DIPEA) or triethylamine (TEA) as a base; and (5) hydrolysis removal of the methyl ester protecting group using lithium hydroxide to obtain the compound shown in Formula I with bioorthogonal reactive groups.

[0012] In one embodiment of this application, the wavelength of the deep red light irradiation is 640–680 nm.

[0013] In one embodiment of this application, the concentration of the photocatalyst is 50–200 nM.

[0014] In one embodiment of this application, the concentration of the probe is 100–200 μM.

[0015] In one embodiment of this application, the incubation temperature for adding the photocatalyst is 2–8°C, and the incubation time is 100–140 min.

[0016] In one embodiment of this application, the incubation temperature for adding the probe is 2–8°C, and the incubation time is 25–40 min.

[0017] In one embodiment of this application, the method further includes: performing flow cytometry and transcriptome analysis to confirm the effect of neighboring cell labeling.

[0018] The beneficial effects of this application are as follows: This application provides a method for neighboring cell labeling, which includes: sequentially adding a photocatalyst and a probe to cells to be labeled and incubating them, thereby achieving neighboring cell labeling under deep red light irradiation; wherein, the photocatalyst comprises dihydroporphyrin E6 or a derivative thereof, and the probe comprises biotin aniline or a derivative thereof. The neighboring cell labeling method (hereinafter referred to as CAT-Tissue) of this application has the following beneficial effects: (1) High labeling efficiency and fast speed: Compared with the prior art, the CAT-Tissue method has a significant improvement in labeling efficiency and a fast labeling reaction speed. It can complete photocatalytic neighboring labeling within 1 minute and effectively capture intercellular interactions in complex tumor microenvironments; (2) Superior tissue penetration: The application of deep red light ensures effective labeling in tissue samples, increasing the applicability of the method; (3) Non-invasive analysis: The CAT-Tissue method maintains the in situ state of cells, reduces changes in cell function, and provides a reliable basis for subsequent experiments; (4) High sensitivity: The CAT-Tissue method can identify and analyze intercellular interactions in complex backgrounds, providing more accurate biological data; (5) Compatible with multiple downstream analyses: By reading the biotin signal on the cell surface, the CAT-Tissue method can easily identify and sort immune cells that interact with target cells, and is compatible with transcriptome analysis and phenotypic analysis, laying the foundation for in-depth research on target cell interacting cells. In summary, the method presented in this application, employing a deep red light-mediated photocatalytic proximity labeling strategy, possesses high efficiency, sensitivity, and non-invasiveness, enabling effective analysis of the dynamic interactions between tumor and immune cells. Its non-genetic coding nature gives this method broad application potential in clinical sample analysis, providing an important experimental tool for future research and development in tumor immunotherapy.

[0019] Of course, implementing any product or method of this application does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other embodiments can be obtained based on these accompanying drawings.

[0021] Figure 1 This is a flowchart illustrating the CAT-Tissue process.

[0022] Figures 2a to 2d This study aims to screen and kineticize proximity-labeled photocatalysts and probes mediated by deep red light; among them, Figure 2a This is a schematic diagram of the probe screening reaction using bovine serum albumin (BSA) labeling under 660nm red light irradiation. Figure 2b The chemical structures of four different biotin nucleophilic probes are shown. Figure 2c Western blot analysis of the labeling efficiency of four biotinylated nucleophilic probes (100 μM) at different Ce6 concentrations (0, 1, 10 μM); Figure 2d WB analysis and quantitative curves of the photocatalytic labeling reaction kinetics with Ce6 as photocatalyst (10 μM) and Biotin-An as probe (100 μM) were obtained, including three biological replicates.

[0023] Figures 3a to 3e Construction and characterization of Ce6 nanobody conjugates; among which, Figure 3a The steps for synthesizing Ce6 nanobody conjugates; Figure 3b The chemical structure of Ce6-DBCO; Figures 3c to 3e Mass spectrometry characterization of Ce6 nanobody conjugates ZHER-Ce6 (targeting HER2), P43-Ce6 (targeting hPD-L1), and dP43-Ce6 (mutation control), respectively. The black line is the mass spectrum of the deconvolution of the nanobody Nb-N3 after linking the peptide, and the red line is the mass spectrum of the deconvolution of the nanobody Nb-Ce6 after the click reaction.

[0024] Figures 4a to 4f Validation of Ce6 nanobody conjugate-mediated photocatalytic self-labeling of target cells; among which, Figure 4a This is a schematic diagram of the photocatalytic self-labeling of MDA-HER2-GFP cells mediated by ZHER-Ce6 under red light irradiation; Figure 4b Flow cytometry analysis of cell surface biotin signals in MDA cells expressing or not expressing HER2 under light or dark conditions; Figure 4c Representative immunofluorescence imaging images of MDA-HER2-GFP cells self-labeled, with MDA-WT cells as a negative control (scale bar, 20 μm); Figure 4d This is a schematic diagram of photocatalytic self-labeling of MC38-hPD-L1 cells mediated by P43-Ce6 under red light irradiation. Figures 4e to 4f Flow cytometry histograms and bar charts were used to analyze biotin signals on the surface of MC38 cells incubated with P43-Ce6, dP43-Ce6 and ZHER-Ce6, or cells that did not express hPD-L1.

[0025] Figures 5a to 5f This serves as validation of the cell capture results in the co-culture system; among which, Figure 5aThis diagram illustrates the intercellular labeling between CD40L-CD40-mediated interaction cells. HEK 293T cells transfected with CD40L-PD-L1 or CD40L and incubated with P43-Ce6 (50 nM) were co-incubated with HEK 293T cells transfected with CD40 or CD45 before photocatalytic labeling. All plasmids contained a fluorescent protein (tdTomato or EGFP) for tracking. Figures 5b to 5c For bait cells (tdTomato) + ) and prey cells (EGFP) + Biotin + Flow cytometry contour plots and statistical histograms of cell proportions; Figure 5d A schematic diagram of intercellular labeling in TCR-pMHC-mediated interaction; Figures 5e to 5f Flow cytometry contour plots and statistical histograms of biotinylation in JC5 cells after co-culturing with K562 cells loaded with different antigenic peptides incubated with P43-Ce6 and after photocatalytic labeling were analyzed.

[0026] Figures 6a to 6d The results validate the CAT-Tissue in situ proximity labeling strategy on mouse tumor tissue sections; among which, Figure 6a A schematic diagram of the experimental procedure for using CAT-Tissue to enrich immune cells near tumor cells; Figure 6b For tumor adjacent CD8 + T cells, NK cells, and CD4 + Representative flow cytometry contour plot analysis of T cell biotin labeling signals; Figure 6c For Biotin + Statistical histogram analysis of cell proportions, gate strategy, etc. Figure 6b As shown; Figure 6d Representative immunofluorescence imaging of frozen sections of CAT-Tissue labeled tumor tissue sections, showing that biotinylated signals are concentrated in the tumor cells and adjacent immune cells (scale bar, 50 μm).

[0027] Figures 7a to 7i For tumor-adjacent CD8 cells isolated from MC38-hPD-L1 tumors via CAT-Tissue + Characterization analysis of T cell gene expression and functional features; among which, Figure 7a For tumor adjacent CD8 + T cells (Top 30) and CD8 cells that are far from tumors + A schematic diagram of the bulk-RNA sequencing experimental workflow for T cells (Bottom30); Figure 7b For Biotin + CD8 +T cells (red) and Biotin - CD8 + Principal component analysis (PCA) of the T cell (cyan) transcriptome, with each point representing a biological replicate; Figure 7c For Biotin + vs Biotin - CD8 + Volcano plot analysis of differentially expressed genes on T cells: red dots represent genes that are significantly upregulated, while blue dots represent genes that are significantly downregulated. The threshold for significant up / down regulation of genes was set as follows: Benjamin-Hochberg FDR(p.adjust) < 0.05 and |log2(foldchange)| ≥ 1. Figure 7d For Figure 7c Genes that were significantly upregulated were subjected to GOBP pathway enrichment analysis; Figure 7e For Biotin + vs Biotin - CD8 + T cells perform CD8 + T cell activation / disabling gene set and antigen-specific CD8 + Gene set enrichment analysis (GSEA) of the gene set upregulated by T cells; Figure 7f For Biotin + and Biotin - CD8 + A comparison of the transcriptional expression of T cell activation and exhaustion marker genes; Figure 7g For Biotin + and Biotin - CD8 + Flow cytometry analysis of PD-1, TIM-3, CD39 and CD137 in T cells; Figure 7h For Biotin + and Biotin - CD8 + Comparison of the transcriptional expression of cytotoxic molecular genes in T cells; Figure 7i For Biotin + and Biotin - CD8 + Flow cytometry analysis of Granzyme B and Perforin in T cells. Detailed Implementation

[0028] The technical solutions of this application will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0029] To address the limitations of existing spatial omics technologies, such as high cost, low resolution, and the inability to obtain cells of interest for subsequent analysis, this application aims to develop in-situ proximity labeling technology for tissue imaging to capture global cell-cell interactions. To subsequently apply this technology to clinical pathological samples, this application plans to employ a photocatalytic non-genetically encoded proximity labeling strategy to acquire in-situ cellular spatial information. However, currently reported photocatalytic proximity labeling methods for capturing cell interactions are generally limited to short-wavelength light sources, exhibiting poorer tissue penetration and biocompatibility compared to longer-wavelength deep-red and near-infrared light. Therefore, this application develops a deep-red light-mediated photocatalytic proximity labeling strategy to achieve in-situ proximity labeling of tissues. The specific technical solution is as follows:

[0030] This application provides a method for neighboring cell labeling, comprising: sequentially adding a photocatalyst and a probe to cells to be labeled, and incubating them under deep red light irradiation to achieve neighboring cell labeling; wherein the photocatalyst comprises Ce6 or a derivative thereof, and the probe comprises biotin aniline or a derivative thereof.

[0031] Using light within the near-infrared window wavelength as the excitation source enables better neighbor labeling at the tissue and even in vivo levels, exhibiting stronger tissue penetration and biocompatibility. The neighbor cell labeling method of this application utilizes the superior tissue penetration of deep red light, combined with a highly efficient photocatalytic reaction (Ce6 or its derivatives exhibit excellent neighbor labeling ability under deep red light irradiation, with a superior labeling reaction rate reaching a plateau phase in approximately half a minute of illumination, making it an ideal deep red light photocatalyst; biotin aniline or its derivatives are the best nucleophilic probes), solving the problem of effective in-situ labeling in tissue samples in existing technologies. This neighbor cell labeling method possesses characteristics such as high efficiency, sensitivity, and non-invasiveness, enabling effective analysis of the dynamic interactions between tumor and immune cells. Its non-genetic coding nature makes this method widely applicable in clinical sample analysis, providing an important experimental tool for future research and development in tumor immunotherapy.

[0032] This application does not impose any particular limitation on the cells to be labeled, as long as they can achieve the purpose of this application. For example, the cells to be labeled can be cells that express nanobodies that can target specific antigens.

[0033] It should be noted that "incubation with photocatalyst and probe sequentially in the cells to be labeled" means that the photocatalyst is added to the cells for incubation first, followed by probe incubation. In one embodiment of this application, a washing step is included after incubation with the photocatalyst and before probe incubation. This application does not impose any particular limitation on the washing step, as long as it achieves the purpose of this application.

[0034] For example, a biological buffer containing a certain concentration of protein can be used to wash the sample.

[0035] In this application, the Ce6 derivative refers to an analogue derived from Ce6 based on basic chemical knowledge and having similar effects to Ce6; the biotin aniline derivative refers to an analogue derived from biotin aniline based on basic chemical knowledge and having similar effects to biotin aniline.

[0036] In one embodiment of this application, the photocatalyst is a Ce6 nanobody conjugate; the Ce6 nanobody conjugate includes His-tag nanobody, ZHER-Ce6 complex targeting HER2, P43-Ce6 complex targeting PD-L1, and dP43-Ce6 complex, an inactivating mutant of P43-Ce6 complex.

[0037] The inventors discovered in their research that, compared to antibody conjugate construction strategies involving the reaction of NHS and amino groups, the Ce6 nanobody conjugate of this application has better site selectivity and a more uniform number of catalyst conjugates, while also not conjugating to key residues at the nanobody binding site, thus not affecting the nanobody's recognition and binding to the target.

[0038] In one embodiment of this application, the synthesis steps of the Ce6 nanobody conjugate include:

[0039] A nanobody expressing the C-terminal sequence LPETG-His-tag (hereinafter referred to as Nb-LPETG) was linked to the GGGK(N3)GGK(N3)K(N3) peptide using the transpeptidase mgSrtA, and then reacted with the compound shown in Formula I (hereinafter referred to as Ce6-DBCO) to obtain the nanobody Ce6 conjugate.

[0040]

[0041] In this application, there are no particular limitations on the method of expressing nanobodies with the C-terminal sequence LPETG-His-tag, as long as it can achieve the purpose of this application. For example, Nb-LPETG can be expressed in E. coli.

[0042] In this application, a zirconia peptide is first linked to the tail of a nanobody using a mgSrtA-mediated transpeptide reaction, and then three Ce6 molecules are coupled to the nanobody using a click reaction.

[0043] In one embodiment of this application, the synthesis steps of the compound shown in Formula I include: (1) methyl esterification protection of the two carboxyl groups of Ce6; (2) carboxyl group activation using HATU, addition of mono-tert-butyloxycarbonyl-protected ethylenediamine, and condensation reaction using DIPEA or TEA as a base; (3) deprotection reaction of tert-butyloxycarbonyl using TFA or 1,4-dioxane hydrogen chloride; (4) condensation reaction of amino group and NHS active ester using DIPEA or TEA as a base; and (5) hydrolysis removal of the methyl ester protecting group using lithium hydroxide to obtain the compound shown in Formula I with bioorthogonal reactive groups.

[0044] In one embodiment of this application, the synthesis steps of the compound represented by Formula I are as follows:

[0045]

[0046] In this application, the specific method of synthesis step (1) of the compound shown in Formula I is not particularly limited, as long as it achieves the purpose of this application. For example, the two carboxyl groups of Ce6 react with methanol in the presence of a catalyst (such as sulfuric acid) to generate the corresponding methyl ester. This application does not particularly limit the reaction conditions for methyl esterification protection, as long as they achieve the purpose of this application. For example, the reaction can be carried out at room temperature for 4 to 6 hours.

[0047] In this application, the reaction conditions for step (2) of the synthesis of the compound shown in Formula I are not particularly limited, as long as they achieve the purpose of this application. For example, the reaction can be carried out at room temperature for 15 to 20 hours. In this application, "room temperature" means 20°C to 30°C.

[0048] In this application, the reaction conditions for the synthesis step (3) of the compound shown in Formula I are not particularly limited, as long as they can achieve the purpose of this application. For example, the reaction can be carried out at room temperature for 1 to 3 hours.

[0049] In this application, the reaction conditions for step (4) of the synthesis of the compound shown in Formula I are not particularly limited, as long as they achieve the purpose of this application, for example, overnight at room temperature. In this application, "overnight" means 12 to 20 hours.

[0050] In this application, the reaction conditions for the synthesis step (5) of the compound shown in Formula I are not particularly limited, as long as they can achieve the purpose of this application. For example, the reaction can be carried out at 35°C to 40°C for 4 to 8 hours.

[0051] In one embodiment of this application, the wavelength of the deep red light irradiation is 640–680 nm. Preferably, the wavelength of the deep red light irradiation is 650–670 nm. For example, the wavelength of the deep red light irradiation can be 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, or a range consisting of any two of these values. The deep red light irradiation wavelength is within the above range, exhibiting high and rapid labeling efficiency, good biocompatibility, and tissue penetration.

[0052] In one embodiment of this application, the concentration of the photocatalyst is 50–200 nM. For example, the concentration of the photocatalyst can be 50 nM, 100 nM, 150 nM, 200 nM, or a range consisting of any two of these values. When the concentration of the photocatalyst is within the above range, CAT-Tissue labeling exhibits good labeling effect and low labeling background.

[0053] In one embodiment of this application, the concentration of the probe is 100–200 μM. For example, the concentration of the probe can be 100 μM, 130 μM, 150 μM, 180 μM, 200 μM, or a range consisting of any two of these values. When the probe concentration is within the above range, CAT-Tissue labeling exhibits good labeling effect and low labeling background.

[0054] In one embodiment of this application, the incubation temperature for adding the photocatalyst is 2–8°C, and the incubation time is 100–140 min. For example, the incubation temperature for adding the photocatalyst can be 2°C, 4°C, 6°C, 8°C, or any combination of two of these values, and the incubation time can be 100 min, 110 min, 120 min, 130 min, 140 min, or any combination of two of these values. Incubation temperatures and times within the above ranges help maintain the activity and spatial relationships of cells within the tissue and facilitate the full binding of the Ce6 nanobody conjugate.

[0055] In one embodiment of this application, the incubation temperature for adding the probe is 2–8°C, and the incubation time is 25–40 min. For example, the incubation temperature for adding the probe can be 2°C, 4°C, 6°C, 8°C, or a range of any two of these values, and the incubation time can be 25 min, 30 min, 35 min, 40 min, or a range of any two of these values. Incubation temperatures and times within the above ranges help maintain the viability and spatial relationships of cells within the tissue and facilitate sufficient infiltration and uniform distribution of the probe.

[0056] In one embodiment of this application, the method further includes: performing flow cytometry and transcriptome analysis to confirm the effect of neighboring cell labeling. In one embodiment of this application, a flowchart of the method is shown below. Figure 1 As shown.

[0057] This application does not impose specific limitations on the methods of flow cytometry and transcriptome analysis; any method that achieves the purpose of this application may be used, and conventional methods in the art can be employed. For example, for flow cytometry, 1 million cells to be analyzed constitutes 1 test, with a chromosome volume of 100 μL. The single-cell suspension is washed two to three times with FACS Buffer (DPBS + 2 vol% FBS), and incubated at room temperature for 10 minutes with 0.2 μL / test of anti-mouse CD16 / 32 (BioLegend, 101320) and Zombie fixable viability dye (BioLegend, 423101, 423111, or 423113). Then, staining is performed with FACS Buffer containing 0.2 μL / test (unless otherwise specified) of flow cytometry antibody, and the solution is incubated at 4°C in the dark for 30 minutes. The cells are washed three times with FACS Buffer, resuspended in FACS Buffer, transferred to flow cytometry tubes, and analyzed or sorted using a flow cytometer. For intracellular protein detection, after staining and washing with cell surface markers in the previous step, resuspend the cell pellet in 150 μL FIX / Perm Buffer (BD, 562574) mixture, fix at 4°C for 45 minutes, add 120 μL Perm / Wash Buffer (BD, 562574), centrifuge and discard the supernatant. Add 200 μL Perm / Wash Buffer to each well, centrifuge and discard the supernatant. Add 100 μL Perm / Wash Buffer containing flow cytometry antibody to each well and incubate at 4°C in the dark for 45 minutes. Centrifuge and discard the supernatant, wash twice with 200 μL Perm / Wash Buffer per well. Resuspend in 150 μL FACS Buffer for analysis.

[0058] For transcriptome analysis, after tissue sectioning and CAT-Tissue labeling, a single-cell suspension of tumor tissue is prepared from the labeled sections. After washing once with FACS buffer, the suspension is then processed using EasySep. TMThe MouseTIL(CD45) Positive Selection Kit (StemCell, 100-0350) was used to enrich TILs. After cell counting, Zombie Aqua (BioLegend, 42310) and flow cytometry antibody mixture [Streptavidin-APC (BioLegend, 405243), anti-mCD45-Pacific Blue (BioLegend, 103125), anti-mCD8α-PE (BioLegend, 100708), anti-mCD4-FITC (BioLegend, 100406), and anti-mNK1.1-BV605 (BioLegend, 108739, 0.2 μL / test each] were added sequentially for staining. After washing, sorting was performed using flow cytometry. For CD8... +T cells were sorted based on their biotin signal levels, with the top 30% (Biotin Top 30%) and bottom 30% of cells receiving the strongest signal (Biotin Bottom 30%). After sorting, 50–100 μL of TRIzon (Kangwei Century, CW0580S) was added to each cell, and the mixture was thoroughly lysed by pipetting or vortexing. 20 μL of chloroform was added to each 100 μL of the extract, and the mixture was thoroughly mixed. The cells were then centrifuged at 12,000 rpm for 15 min at 4°C, and the clear, colorless aqueous phase was transferred to a new RNase-free EP tube. Total RNA was then extracted using RNA Clean & Concentrator Kits (Zymo, R1015). Next, mRNA was enriched from the total RNA using Oligo dT magnetic beads. After fragmentation, first-strand cDNA was synthesized using random hexamer primers, followed by second-strand cDNA synthesis. After end repair, A-tailing, adapter ligation, fragment selection, amplification, and purification, the library is ready. After passing library inspection, the quantified library is mixed and sequenced on the Illumina platform according to the effective library concentration and data volume. Image data of the sequencing fragments obtained by the high-throughput sequencer are converted into sequence data (reads) through base identification in FASTQ format, mainly containing the sequence information of the sequencing fragments and their corresponding sequencing quality information. First, the adapter sequences in the sequencing data are cut using `trim_galore`, and the cut sequences are aligned to the reference genome using STAR. Then, the aligned sequences are sorted using `picard`, and `featureCounts` is used to count the sorted sequence information to obtain the gene expression matrix. In RStudio, the DESeq2 package is used to perform principal component analysis (PCA) and differential gene expression analysis (DGE) on the gene expression matrix. The thresholds for significant up / downregulation of genes are set as follows: Benjamin-Hochberg FDR(p.adjust) < 0.05 and |log2(foldchange)| ≥ 1. For genes that were significantly upregulated or downregulated, GO pathway enrichment analysis was performed using the clusterProfiler and enrichplot packages. GSEA analysis was performed using the fgsea package on the top 30% of biotin and the bottom 30% of biotin.

[0059] The following examples illustrate the implementation of this application in more detail. Various tests and evaluations were conducted according to the methods described below. Furthermore, unless otherwise specified, "parts" and "%" are quality standards.

[0060] Probe screening and characterization of protein labeling reactions:

[0061] Ce6 was used as a photocatalyst, and various nucleophilic probes were screened. The specific steps are as follows:

[0062] Photocatalysts (0, 1 μM, 10 μM), probes (100 μM), and BSA (1 mg / mL) were mixed thoroughly in PBS (pH 7.4). 100 μL of the mixture was spread into a 96-well plate and incubated under a 660 nm red LED lamp (30 mW / cm²). 2 Irradiate at room temperature (0, 5s, 10s, 30s, 60s, 120s, 300s). Take 10μL of sample, add 30μL of water and 10μL of 5X SDS-PAGE non-reducing loading buffer (CWBIO) sequentially, mix well, heat at 95℃ for 10 minutes, and take 5μL of sample for loading. Perform electrophoresis using 4-15% gradient SDS-PAGE gel (Beyotime, P0466S), and transfer to a 0.22μm polyvinylidene fluoride (PVDF) membrane (Merck Milipore, ISEQ00010). Then, the sample was blocked with BSA, incubated with primary antibody using Mouse anti-biotin mAb (Santa Cruz, sc-101339, 1:1000 dilution), washed with PBST (0.01M PBS pH 7.4, with 0.1 vol% Tween 20), and incubated with secondary antibody using HRP-linked anti-mouse IgG (Cell Signaling Technology, 7076S, 1:5000 dilution). After washing with PBST, chemiluminescence imaging was performed using an Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0500) and a ChemiDoc (Bio-Rad) scanner.

[0063] The results showed that Biotin-An exhibited the best labeling efficiency, demonstrating a significant biotin labeling signal even at a low photocatalyst concentration (1 μM). Figures 2a to 2c Photocatalytic proximity labeling kinetics experiments showed that Ce6 rapidly completed the labeling reaction under 660 nm deep red light irradiation, reaching a plateau phase in about half a minute of illumination. Figure 2d Therefore, Ce6 exhibits excellent proximity labeling ability and a superior labeling reaction rate under deep red light irradiation, making it an ideal deep red light photocatalyst, while Biotin-An is the best nucleophilic probe.

[0064] Construction of nanobody dihydroporphyrin E6 conjugate:

[0065] (I) Synthesis steps of Ce6-DBCO

[0066] To achieve spatial targeting of the photocatalyst through coupling with nanobodies, we need to modify and derivatize Ce6 to incorporate bioorthogonal reactive groups. The synthetic steps are as follows:

[0067]

[0068] (1) Ce6 (0.20 g, 0.335 mmol, 1.0 equiv.) was completely dissolved in methanol (16 mL, 0.02 M), and sulfuric acid (0.9 mL, 16.8 mmol, 50 equiv.) was added dropwise. The mixture was stirred at room temperature for about 5 hours, and the reaction progress was monitored by TLC. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was added to quench the reaction, and the reaction solution was extracted with ethyl acetate. The resulting extract was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain a black solid product, dm-Ce6. The crude product was used for the next step of the reaction.

[0069] (2) The crude dm-Ce6 product (0.335 mmol, 1.0 equiv.) was completely dissolved in dichloromethane (16 mL, 0.02 M), followed by the addition of DIPEA (0.293 mL, 1.68 mmol, 5.0 equiv.) and HATU (0.1940 g, 0.51 mmol, 1.5 equiv.). The mixture was stirred at room temperature for approximately 1 hour, and the reaction progress was monitored by TLC. After the starting materials had completely reacted, mono-Boc protected ethylenediamine (0.108 mL, 0.67 mmol, 2.0 equiv.) was added, and the mixture was stirred at room temperature for approximately 16 hours. After the reaction was complete, the reaction was quenched with saturated sodium bicarbonate aqueous solution, and the reaction mixture was extracted with dichloromethane. The obtained extract was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography (silica gel, dichloromethane / methanol, v / v ratio 50:1) to give a black solid compound dm-Ce6-NH-Boc (0.2318 g, 0.302 mmol). The two-step yield was 90.1%.

[0070] 1H NMR(400MHz,Chloroform-d)δ9.62(s,1H),9.55(s,1H),8.80(s,1H),7.98(dd,J=17.8,11.5Hz,1H),6.85(br.s,1H),6.26(dd,J=17.8,1.4Hz,1H),6.05(dd,J=11.5,1.4Hz,1H),5.48(d,J=18.9Hz,1H),5.33(br.s,1H),5.22(d,J=18.9Hz,1H),4.47(q,J=7.1Hz,1H),4.38(d,J=9.9Hz,1H),3.82-3.65(m,6H),3.60(s,3H),3.50-3.36(m,8H),3.21(s,3H),2.76(s,3H),2.54(dt,J=15.7,7.6Hz,1H),2.29-2.09(m,2H),1.88-1.76(m,1H),1.74-1.64(m,6H),1.40(s,9H),-1.64(br.s,1H),-1.84(br.s,1H).

[0071] 13 C NMR(101MHz,Chloroform-d)δ173.90,173.54,170.05,168.96,166.80,156.55,144.67,138.98,136.03,135.01,134.88,134.83,134.59,130.22,129.84,129.38,128.00,121.66,102.28,101.36,98.79,93.76,79.55,77.25,53.10,52.23,51.64,49.25,40.86,40.70,38.60,37.88,31.12,29.71,28.39,23.08,19.66,17.71,12.15,11.90,11.28.

[0072] FT-HRMS(ESI):m / z calcd.for C 43 H 55 N6O7([M+H] + )767.4127,found 767.4127.

[0073] (3) dm-Ce6-NH-Boc (0.2318 g, 0.302 mmol, 1.0 equiv.) was completely dissolved in dichloromethane (6 mL, 0.05 M), and trifluoroacetic acid (0.60 mL) was added dropwise. The mixture was stirred at room temperature for about 2 hours, and the reaction progress was monitored by TLC. After the reaction was complete, the solvent was removed by rotary evaporation, and the crude product was purified by column chromatography (silica gel, dichloromethane / methanol, volume ratio 30:1) to obtain a black solid compound dm-Ce6-NH2 (0.1308 g, 0.196 mmol). The yield was 65%.

[0074] 1 H NMR (400MHz, DMSO-d6) δ9.79(s,1H),9.73(s,1H),9.39(br.t,J=5.6Hz,1H),9.12(s,1H),8.30(dd,J=17.8,11.6Hz,1H),6.44( dd,J=17.8,1.4Hz,1H),6.17(dd,J=11.6,1.4Hz,1H),5.48(d,J=19.0Hz,1H),5.32(d,J=19.0Hz,1H),4.63(q,J=7.1Hz,1H),4.4 2(d,J=10.4Hz,1H),3.97-3.75(m,4H),3.70(s,3H),3.58(s,3H),3.53(s,3H),3.51(s,3H),3.32-3.22(m,6H),2.71(dt,J=16. 0,8.0Hz,1H),2.35(ddd,J=16.3,7.2,5.1Hz,1H),2.14(dt,J=15.5,8.3Hz,1H),1.71-1.57(m,6H),-1.77(s,1H),-2.03(s,1H).

[0075] (4) dm-Ce6-NH2 (0.1308 g, 0.196 mmol, 1.0 equiv.) and NHS-DBCO (0.1183 g, 0.294 mmol, 1.5 equiv.) were thoroughly dissolved in dichloromethane (19.6 mL, 0.01 M), and DIPEA (1.0 mL, 5.88 mmol, 30 equiv.) was added. The mixture was stirred overnight at room temperature, and the reaction progress was monitored by TLC. After the reaction was complete, water was added to quench the reaction, and the reaction solution was extracted with ethyl acetate. The resulting extract was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography (silica gel, dichloromethane / methanol, v / v ratio 50:1) to obtain the black solid compound dm-Ce6-DBCO (0.0505 g, 0.053 mmol). The yield was 27%.

[0076] 1 H NMR(400MHz,Chloroform-d)δ9.60(s,1H),9.50(s,1H),8.88-8.76(m,1H),7.94(dd,J=17.8,11.5Hz,1H),7.29-7.00(m,5H),6.90(t,J=7.8Hz,1H),6.75-6.55(m,2H),6.54-6.41(m,1H),6.22(dd,J=17.8,1.4Hz,1H),6.11(t,J=7.6Hz,1H),6.01(dd,J=11.5,1.4Hz,1H),5.64-5.39(m,1H),5.35-5.16(m,1H),4.60-4.41(m,2H),4.40-4.21(m,2H),3.71-3.49(m,9H),3.46-3.34(m,7H),3.23-3.08(m,4H),2.73-2.61(m,1H),2.60-2.45(m,1H),2.33-2.06(m,3H),2.04-1.91(m,1H),1.89-1.56(m,9H),-1.53--1.71(m,1H),-1.73--1.91(m,1H).

[0077] 13 C NMR(101MHz,Chloroform-d)δ173.89,173.56,173.22,172.25,169.77,168.87,166.98,154.22,150.95,149.05,147.63,144.67,138.84,136.11,135.12,134.94,134.76,134.49,131.57,130.14,129.80,129.38,129.17,128.43,128.27,128.01,127.61,127.21,126.84,125.00,122.60,122.19,121.61,114.54,107.51,102.46,101.35,98.78,93.68,54.98,53.15,52.06,51.68,49.20,39.98,39.62,37.86,31.40,31.17,30.58,29.83,23.22,19.65,17.77,12.15,11.98,11.26.

[0078] FT-HRMS(ESI):m / z calcd.for C 57 H60 N7O7([M+H)) + )954.4549,found 954.4558.

[0079] (5) dm-Ce6-DBCO (0.0505 g, 0.053 mmol, 1.0 equiv.) was fully dissolved in tetrahydrofuran (5 mL), and 1 mL of an aqueous solution containing LiOH·H2O (0.022 g, 0.53 mmol, 10 equiv.) was added. The mixture was stirred at 37 °C for about 6 hours, and the reaction progress was monitored by TLC. After the reaction was completed, a saturated ammonium chloride aqueous solution was added to quench the reaction, and the reaction solution was extracted with dichloromethane. The resulting extract was dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography (silica gel, dichloromethane / methanol / formic acid, volume ratio 20:1:0.1) to obtain a black solid compound Ce6-DBCO (0.0162 g, 0.017 mmol). The yield was 32%.

[0080] 1 H NMR (400MHz, DMSO-d6) δ12.53(br.s,2H),9.84(s,1H),9.74(s,1H),9.19(s,1H),9.10-8.99(m,1H),8.23(dd,J=17.8,11.6Hz,1H),8.01(t,J=5.5Hz, 1H),7.69(t,J=7.8Hz,1H),7.52-7.37(m,4H),7.32-7.19(m,3H),6.37(d,J =17.9Hz,1H),6.11(d,J=11.6Hz,1H),5.52(d,J=19.0Hz,1H),5.31(d,J=19 .0Hz,1H),4.88(d,J=14.0Hz,1H),4.75(d,J=14.0Hz,1H),4.67(q,J=7.2H z,1H),4.49(d,J=11.8Hz,1H),3.81(q,J=7.6Hz,2H),3.69-3.44(m,10H),3 .27(s,3H),2.77-2.60(m,2H),2.42-2.25(m,2H),2.24-2.05(m,2H),1.85( dq,J=16.4,6.6Hz,1H),1.76-1.58(m,7H),-2.03(br.s,1H),-2.19(s,1H).

[0081] 13C NMR(101MHz,DMSO-d6)δ174.85,174.37,171.98,171.63,171.61,168.35,163.54,152.02,151.98,148.78,148.73 ,144.33,136.28,135.82,134.35,134.12,132.79,130.78,130.64,130.29,130.06,129.72,129.38,128.58,128.4 0,128.06,127.22,125.55,122.92,122.88,122.54,121.85,114.66,108.53,104.56,100.82,98.71,95.14,55.42,55.25,55.16,53.27,48.61,38.80,37.58,31.32,31.08,30.26,30.04,23.55,19.39,18.17,12.54,12.21,11.45.

[0082] FT-HRMS(ESI): m / z calcd.for C 55 H 56 N7O7([M+H)) + )926.4236,found 926.4250.

[0083] (II) Construction and characterization of Ce6 nanobody conjugates

[0084] We expressed Nb-LPETG (a nanobody with an LPETG-His-tag C-terminus) in E. coli, then used mgSrtA (a sortase transpeptidase) to link the GGGK(N3)GGK(N3)K(N3) peptide, and finally reacted it with Ce6-DBCO via a click reaction to obtain the photocatalyst nanobody conjugate Nb-Ce6. The synthetic steps of the Ce6 nanobody conjugate are as follows: Figure 3a As shown, Figure 3b The chemical structure of Ce6-DBCO is shown. The specific steps are as follows:

[0085] Protein expression and purification: Based on previous research and literature reports of nanobody sequences (ZHER nanobody sequence as shown in SEQ ID NO:1, P43 nanobody sequence as shown in SEQ ID NO:2, dP43 nanobody sequence as shown in SEQ ID NO:3), we added 10 ng of plasmid (ZHER plasmid sequence as shown in SEQ ID NO:4, P43 plasmid sequence as shown in SEQ ID NO:5, dP43 plasmid sequence as shown in SEQ ID NO:6) to 100 μL of DH10B E. coli competent cells, mixed well, and incubated on ice for 15 minutes. We then heat-shocked the cells in a 42℃ water bath for 1 minute and incubated on ice for 3 minutes. After adding antibiotic-free Luria-Bertani (LB) medium and culturing on a shaker at 37℃ for 30–60 minutes, the bacterial culture was spread onto bacterial plates for further culture. Bacterial clones were selected and spotted into LB medium containing ampicillin or kanamycin (LB medium composition: 10g trypsinone, 5g yeast extract, and 10g sodium chloride dissolved in 1L deionized water) for protein expression. The bacterial culture was centrifuged at 4500g for 15 minutes, the supernatant was discarded, and 40mL of PBS was added. The mixture was then vortexed and sonicated on ice to release the protein into the solution. The bacterial pellet was collected by centrifugation at 14000rpm at 4℃ for 30 minutes. The supernatant was purified using an AKTA protein purifier with a nickel column in a gradient of NTA concentration. His-tagged nanobodies were purified, and their molecular weight was confirmed by mass spectrometry. If the sample contained endotoxin protein, the nanobodies could be further purified using a size exclusion column in rapid protein liquid chromatography with PBS as the mobile phase, and their molecular weight was confirmed by mass spectrometry.

[0086] Preparation of catalyst nanobody conjugates: 5 equivalents of peptide GGGK(N3)GGK(N3)K(N3) (provided by Propeptide Bio) were mixed with purified nanobody, and 0.1 equivalents of mg SrtA enzyme (self-expressed according to literature (J. Am. Chem. Soc. 2019, 141, 1833-1837)) were added. The mixture was incubated at room temperature in the dark for 40 minutes, and the reaction progress was monitored by mass spectrometry to avoid the formation of side hydrolysis products. After confirming the reaction was complete, 2 mM MTSET was added to quench the reaction, and unreacted raw material nanobody and Sortase enzyme with His tag were removed by passing through 1.2 times nickel-NTA agarose beads. Then, unreacted peptide and MTSET were removed by desalting to obtain purified Nb-N3. Then, 3 equivalents of Ce6-DBCO were added, and a click reaction was performed at room temperature for 1 hour to obtain Nb-Ce6. The reaction progress was monitored by mass spectrometry. After the reaction was complete, the mixture was transferred to a -30°C freezer for subsequent labeling experiments.

[0087] UPLC-MS characterization: Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) analysis was performed on an ACQUITY UPLC I-Class SQD 2 (Waters) system equipped with ESI and a BEH C18 Acquity column (1.7 μm, 2.1 × 50 mm).

[0088] Figures 3c to 3e Mass spectrometry characterization of Ce6 conjugate nanobodies ZHER-Ce6 (targeting HER2), P43-Ce6 (targeting hPD-L1), and dP43-Ce6 (mutation control) are shown. The black line represents the mass spectrum of the deconvoluted Nb-N3 nanobodies after the peptide linker, and the red line represents the deconvoluted Nb-Ce6 nanobodies after the click reaction. The molecular weight of the Ce6 catalyst nanobodies was confirmed by mass spectrometry, showing that three Ce6 photocatalyst molecules were successfully conjugated to each nanobodies.

[0089] Validation of cell markers:

[0090] First, we conducted experimental studies on targeted cell self-labeling. Figure 4a The specific steps are as follows:

[0091] MDA-HER2-GFP cells (the human breast cancer cell line MDA-HER2-GFP was provided by Chen Peng's group at the College of Chemistry and Molecular Engineering, Peking University) were digested with 0.25% trypsin (gibco, 25200056), quenched with DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody), and counted. 3 × 10⁶ cells were placed in each group. 5 Cells were resuspended in 100 μL of DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody). Photocatalyst nanobody conjugate (ZHER-Ce6) was added to a final concentration of 50 nM, mixed, and incubated at room temperature for 20 min. After incubation, cells were washed three times with DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody), and then resuspended in 200 μL of DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody). Biotin-An stock solution was added to a final concentration of 100 μM, and the cells were seeded into 96-well plates. The 96-well plates were incubated at 660 nm for 30 seconds. Cells were then transferred to V-plates and washed three times with FACS buffer in each well. Streptavidin-PE was added for staining. After washing, flow cytometry was used for analysis.

[0092] The results showed that using aniline biotin as a nucleophilic probe, a significant biotin labeling signal was observed in the target cells MDA-HER2-GFP after 30 seconds of irradiation with 660 nm deep red light. However, under the same conditions, no significant biotin labeling signal was observed in non-target cells MDA-WT that did not undergo light irradiation or did not express the nanobody-recognized target HER2. Figure 4b Under fluorescence confocal microscopy, it was observed that the photocatalytically labeled biotin was concentrated extracellularly and exhibited good co-localization with HER2-GFP, demonstrating the specificity and spatial selectivity of this labeling method. Figure 4c ).

[0093] Similarly, we also verified that P43-Ce6 has a specific self-labeling ability for MC38 cells expressing hPD-L1. Figure 4d The specific steps are as follows:

[0094] MC38-hPD-L1 cells (the mouse colon adenocarcinoma cell line MC38-hPD-L1 was provided by Chen Peng's group at the College of Chemistry and Molecular Engineering, Peking University) were digested with 0.25% trypsin, quenched with DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody), and counted. 3 × 10n cells were placed in each group. 5 Cells were resuspended in 100 μL of DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody). Photocatalyst nanobody conjugates (P43-Ce6, dP43-Ce6, or ZHER-Ce6) were added to a final concentration of 50 nM, mixed, and incubated at room temperature for 20 min. After incubation, cells were washed three times with DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody), and then resuspended in 200 μL of DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody). Biotin-An stock solution was added to a final concentration of 100 μM, and the cells were seeded into 96-well plates. The 96-well plates were incubated at 660 nm for 30 seconds. Cells were then transferred to V-plates and washed three times with FACS Buffer in each well. Streptavidin-APC staining was performed. After washing, the cells were analyzed by flow cytometry.

[0095] We used P43-Ce6 as the experimental group to evaluate the photocatalytic labeling efficiency of the photocatalyst nanobody conjugates, and ZHER-Ce6 (isotype control) and dP43-Ce6 (inactivated mutant) as control groups to evaluate the labeling background caused by non-specific adsorption of the photocatalyst nanobody conjugates. Flow cytometry analysis showed that only the P43-Ce6 experimental group exhibited a significant biotin labeling signal, while the control group showed a low labeling background due to non-specific adsorption. Figures 4e to 4fThese experimental results demonstrate the feasibility of a strategy that uses photocatalyst-based nanobody conjugates to target cells expressing specific antigens, thereby achieving selective cell labeling. Furthermore, this method exhibits good labeling efficiency and low background signal, laying the foundation for further labeling of cell interactions.

[0096] Example 1: Capture of interacting cells in a co-culture system

[0097] We set out to investigate the ability of p43-Ce6 to capture cell-cell interactions. To evaluate the effectiveness of p43-Ce6 cell tagging, we constructed a CD40-CD40L-mediated HEK 293T cell interaction system (…). Figure 5a The specific steps are as follows:

[0098] HEK 293T cells were passaged into 12-well plates at a ratio of 1:3 and cultured for 16 hours. In each well, plasmids (CD40L-PD-L1 sequence as shown in SEQ ID NO:7, CD40L sequence as shown in SEQ ID NO:8, CD40 sequence as shown in SEQ ID NO:9, and CD45 sequence as shown in SEQ ID NO:10) were transfected with 1.25 μg / 1 mL DMEM medium and 1.5 μL / μg PEI plasmid transfection reagent. Transfection was performed in DMEM (containing 1 vol% FBS). After 8 hours, the medium was changed to DMEM (containing 10 vol% FBS and 1 vol% antibiotics). After the medium change, the cells were cultured for 24 hours. Transfection efficiency was observed using a fluorescence microscope. Cells transfected with CD40L-PD-L1 and CD40L (obtained by transfecting HEK 293T cells with the above plasmids) were digested with 0.25% trypsin, resuspended in DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody) for quenching, and centrifuged. 50 nM P43-Ce6 was added, and the cells were incubated at room temperature for 20 minutes, followed by cell counting. After incubation, the cells were washed three times with DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody), and resuspended in DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody) after each wash. Simultaneously, cells transfected with CD40-GFP and CD45-GFP were digested with 0.25% trypsin, resuspended in DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody) for quenching, centrifuged, resuspended in DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody), and cell counted. Biotin-An probe was added to a final concentration of 100 μM. Cells transfected with CD40L-PD-L1 or CD40L were used as bait cells, with 3 × 10⁶ cells per group. 5 Cells transfected with CD40-GFP or CD40L were used as prey cells, with 1.5 × 10⁶ cells per group. 5The two cell lines were mixed and incubated together at 37°C for approximately 1 hour. The 96-well plate was then illuminated at 660 nm for 30 seconds. Each well was washed twice with 180 μL of DMEM (containing 10 vol% FBS and 1 vol% penicillin antibody) and FACS Buffer. Zombie Violet and Streptavidin-APC were added sequentially for staining. After washing, the cells were analyzed by flow cytometry.

[0099] For bait cells, plasmids of either tdTomato-CD40L-PD-L1 or tdTomato-CD40L were transfected. For prey cells, plasmids of either CD40-EGFP or CD45-EGFP were transfected. When CD40L-CD40 interaction was present and target PD-L1 was expressed (CD40L-PD-L1 and CD40), significant self-labeling and mutual-labeling signals were observed. When only target PD-L1 was expressed but no cell interaction was present (CD40L-PD-L1 and CD45), only self-labeling signals from bait cells were observed, while almost no prey cells were labeled by the biotin probe. This demonstrates the specificity for capturing neighboring interacting cells of the target cell. When only CD40L-CD40 interaction was present but target PD-L1 was not expressed (CD40L and CD40), neither self-labeling nor mutual-labeling signals were significantly detected, indicating that P43-Ce6 has good targeting ability. Figures 5b to 5c These experimental results demonstrate that our photocatalytic proximity labeling system exhibits good specificity and selectivity, making it suitable for capturing cell interactions.

[0100] To more realistically reflect the interaction patterns between tumor cells and T cells, we constructed an interaction model mediated by TCR-pMHC to evaluate the universality of our deep-red light photocatalytic proximity labeling strategy. Figure 5d The specific steps are as follows:

[0101] K562-HLA-A*02:01-hPD-L1-GFP (human chronic myeloid leukemia cells K562-HLA-A*02:01 were provided by Chen Peng's group at the College of Chemistry and Molecular Engineering, Peking University. Based on this cell line, a stable transfection line K562-HLA-A*02:01-hPD-L1-GFP was constructed using lentivirus, hereinafter referred to as K562) was centrifuged, resuspended, and counted. The cells were diluted to 1×10⁻⁶ with RPMI 1640 (containing 10 vol% FBS and 1 vol% penicillin antibody). 6Cells / mL. Add 1 μg / mL of antigenic peptide and incubate at 37°C for 2 hours. Centrifuge and resuspend. Add P43-Ce6 to a final concentration of 50 nM and incubate at room temperature for 20 min. After incubation, wash the cells three times with RPMI 1640 (containing 10 vol% FBS and 1 vol% penicillin antibody), and resuspend them to 6 Million / mL with RPMI 1640 (containing 10 vol% FBS and 1 vol% penicillin antibody). Simultaneously, centrifuge and resuspend JC5-1G4-RFP (human leukemia T lymphocytes JC5-1G4-RFP provided by Chen Peng's group at the College of Chemistry and Molecular Engineering, Peking University, hereinafter referred to as JC5), and count them. Dilute to 2 Million / mL with RPMI 1640 (containing 10 vol% FBS and 1 vol% penicillin antibody). Add Biotin-An to a final concentration of 200 μM. 50 μL of each of the K562 and JC5 groups were added to 96-well plates, mixed thoroughly by pipetting, and incubated at 37°C for 2 hours. The 96-well plates were then irradiated at 660 nm for 60 seconds. Each well was washed twice with 180 μL of RPMI 1640 (containing 10 vol% FBS and 1 vol% penicillin antibody) and FACS Buffer. Staining was performed sequentially with Zombie Aqua (BioLegend, 423101) and flow cytometry antibody mixture [0.2 μL / test Streptavidin-APC (BioLegend, 405243) and 0.2 μL / test anti-hCD69-Pacific Blue (BioLegend, 310919)]. After washing, the samples were analyzed by flow cytometry.

[0102] For K562 cells, we constructed HLA-A2:02*01 and hPD-L1 using lentiviral infection. For JC5 cells, we constructed HLA-A2:02*01-restricted and NYESO-restricted nucleotides. 157-165 Antigen peptide-specific 1G4TCR. We first used NYESO-3A (sequence shown in SEQ ID NO:11, K...) D of TCR-pMHC=6.6μM), NYESO-7H (sequence shown in SEQ ID NO:12, K D The antigenic peptide (of TCR-pMHC = 100 μM) or negative control (NC, sequence as shown in SEQ ID NO: 13) was incubated with K562 cells, then P43-Ce6 was added to bind to it, and finally co-cultured with JC5 cells and photocatalytic proximity labeling was performed. Flow cytometry results showed that Biotin + The proportion of JC5 cells was positively correlated with the affinity of the 1G4 TCR-pMHC variant. Figures 5e to 5fThese findings demonstrate that CAT-Tissue can detect and quantify the strength of TCR-pMHC-mediated cell interactions with high sensitivity and specificity.

[0103] Example 2: Validation and application of in situ proximity labeling strategy on mouse tumor tissue sections

[0104] To highlight the advantages of non-genetically encoded and deep red light's tissue penetration, we further extended the photocatalytic proximity labeling strategy to in situ labeling of primary tissue samples. The mouse colon cancer MC38 xenograft model is widely used in preclinical studies of tumor chemotherapy and immunotherapy drugs; therefore, using this model to validate proximity labeling strategies has good relevance to clinical drug development. Revealing the composition, state, and function of tumor-adjacent immune cells helps in understanding tumorigenesis, development, and immune escape phenomena. Specifically, we transplanted 1×10-1 cells into the legs of CD57BL / 6N mice (ordered from Vital Rivers). 6 MC38-hPD-L1 cells were collected. After tumor formation at 12-14 days, the dissected tumor tissue was embedded in low-melting-point agarose and then sectioned into approximately 100 μm thick biopsy sections using a vibratory microtome. The sections were then incubated with Ce6 nanobody conjugates at 4°C, washed, and incubated with the Biotin-An probe. After irradiation with 660 nm light, the tissues were digested, and the intensity of Biotin signaling on different immune cells was analyzed using flow cytometry (FACS). Figure 6a The specific steps are as follows:

[0105] After dissecting tumor-bearing mice, the surface moisture of MC38-hPD-L1 tumor tissue was wiped dry and embedded in 2 wt% low-gel-temperature agarose (Sigma-Aldrich, A9414-25G). After solidification, the tissue-containing portion was cut into small pieces for vibratory sectioning. The tissue-containing pieces were attached to the stage using instant glue and immersed in ice-cold HBSS (containing 2 vol% serum and 1 vol% penicillin antibody). Using a Leica vibratome, 100 μm biopsies were obtained at a speed of 0.18 mm / s and an amplitude of 1.8. The biopsies were then transferred to well plates, kept in solution, and maintained on ice. The gel was carefully removed from the tissue sections, and the sections were cleaned. The tissue sections were then transferred to RPMI 1640 (containing 10 vol% FBS and 1 vol% penicillin antibody) containing 50 nM P43-Ce6 or dP43-Ce6. The sections were incubated on a shaker at 4°C for 2 hours. After incubation, the solution was aspirated and RPMI 1640 (containing 10 vol% FBS and 1 vol% penicillin antibody) was added, and the tissue sections were eluted on a shaker at 4°C for 5–10 minutes. This washing step was repeated 5 times. Then, the tissue sections were transferred to RPMI 1640 (containing 10 vol% FBS and 1 vol% penicillin antibody) containing 100 μM Biotin-An. The sections were incubated on a shaker at 4°C for half an hour. The tissue sections were then labeled by irradiation at 660 nm for 60 seconds. The sections were then washed twice each with RPMI 1640 (containing 10 vol% FBS and 1 vol% penicillin antibody) and HBSS on a shaker at 4°C for 5–10 minutes each time to remove free probe molecules. Labeled tissue sections were added to RPMI 1640 digestion solution containing mouse tumor tissue mild enzymatic digestion reagent (Reward, DHTE-5001), and minced with small scissors. The sections were then placed in a single-cell suspension preparer (Reward) or a shaker at 37°C for 40 minutes for digestion. After digestion, FBS was added to a final concentration of 10% to quench the digestive enzyme activity. The cell suspension was then filtered through a 70μm cell filter to obtain a single-cell suspension of tumor tissue for flow cytometry staining or sorting.

[0106] Flow cytometry results showed that, compared with the labeling results of the inactivated mutant dP43-Ce6 control group, CD8+ after P43-Ce6 labeling was significantly higher. + T cells, NK cells, and CD4 + T cells showed significant biotin + Cell population ( Figure 6b and Figure 6c These experimental results preliminarily validate our idea that photocatalytic proximity labeling technology can be used to achieve in-situ labeling of primary tissue section samples.

[0107] To further confirm the target specificity and spatial selectivity of this system on primary tissue sections, we used immunofluorescence (IF-Fr) of frozen sections to observe the co-localization of biotin signals with hPD-L1, and the effect of this system on immune cells adjacent to tumor cells (mCD45). + Specific markers of ) Figure 6d The specific steps are as follows:

[0108] Take the labeled sections and add 4% paraformaldehyde fixative (Beyotime, P0099-100ml) overnight at 4°C. Add 10wt%, 20wt%, and 30wt% sucrose aqueous solutions sequentially for dehydration, incubating at 4°C for 6–8 hours each time. Embed the sections with OCT frozen section embedding medium (Sakura) and place them in a -80°C freezer overnight. Perform cryosectioning using a CM-1950 cryostat (Leica). Then, bake the sections in a 60°C oven for 10 minutes. Wash the sections with PBST (PBS + 0.1 vol% Tween-20) for 5 minutes. Immerse the sections in sodium citrate antigen retrieval solution (Beyotime, P0081) and heat at 95–100°C for approximately 20 minutes. Wash the sections with PBST twice at room temperature for 5 minutes each time. After wiping the area around the sample dry, draw circles around the sample with a hydrophobic pen and allow to dry at room temperature for one minute. Block with 100 μL of PBST solution containing 10 vol% goat serum and 2 wt% BSA, incubate at room temperature for 4 hours. Wash sections with PBST incubate at room temperature for 5–10 minutes, repeat twice. Add 60–100 μL of PD-L1 solution. Rabbit mAb (1:400, CST, 13684T) in PBST solution, incubated overnight at 4°C. Wash sections with PBST at room temperature for 5–10 minutes, repeated five times. Add 60–100 μL of PBST solution containing AF647-streptavidin (1:400, Invitrogen, S21374), AF555-goat anti-rabbit IgG (1:400, Invitrogen, A27039), AF488-mCD45 (1:400, BioLegend, 103121), and DAPI (5 μg / mL, C1002), incubated at room temperature for 2 hours. Wash sections with PBST at room temperature for 5–10 minutes, repeated five times. Mount sections with Fluoromount-G (SouthernBiotech, 0100-01) and image using a confocal laser scanning microscope (Zeiss).

[0109] In the confocal fluorescence microscopy images, we found that the Biotin signal and the hPD-L1 signal have a certain degree of spatial overlap and correlation. In areas where tumor cells accumulate (hPD-L1...),... +Biotin signals are more pronounced in areas where immune cells accumulate (mCD45); while in areas where immune cells accumulate (mCD45)... + Biotin signal was weak. Stronger biotin labeling signals were observed only on immune cells that had infiltrated areas of tumor cell aggregation. Through imaging techniques, we obtained direct evidence of the specificity of biotin labeling on cells adjacent to tumors within the tissue.

[0110] After validating the feasibility and specificity of our technology for in situ labeling of tumor tissue sections, we utilized bulk RNA-seq to reveal differences in the transcriptomes of tumor-adjacent and non-adjacent immune cells, thereby establishing a correlation between spatial location and cellular function within the tissue and providing new insights for tumor immune surveillance and immunotherapy. Specifically, after photocatalytic proximity labeling of tissue sections, two cell populations, the Top30 and Bottom30 in terms of biotin signal intensity, were sorted. mRNA was extracted, reverse transcribed, library constructed, and sequenced. Differences in the transcriptomes of the Top30 and Bottom30 cells were then compared. Figure 7a The specific steps are as follows:

[0111] After tissue sectioning and CAT-Tissue labeling, a single-cell suspension of tumor tissue was prepared from the labeled sections. After washing once with FACS buffer, the suspension was processed using EasySep. TM The Mouse TIL (CD45) Positive Selection Kit (StemCell, 100-0350) was used to enrich TILs. After cell counting, Zombie Aqua (BioLegend, 423101) and flow cytometry antibody mixture [Streptavidin-APC (BioLegend, 405243), anti-mCD45-Pacific Blue (BioLegend, 103125), anti-mCD8α-PE (BioLegend, 100708), anti-mCD4-FITC (BioLegend, 100406), and anti-mNK1.1-BV605 (BioLegend, 108739), each at a concentration of 0.2 μL / test] were added sequentially for staining. After washing, the cells were sorted by flow cytometry. For CD8... +T cells were sorted into the top 30% and bottom 30% based on biotin signal levels. After sorting, 50–100 μL of TRIzon (Kangwei Century, CW0613S) was added to each cell, and the cells were thoroughly mixed by pipetting or vortexing to lyse them. 20 μL of chloroform was added to each 100 μL of the extract, and the cells were thoroughly mixed and extracted. The cells were then centrifuged at 12,000 rpm at 4°C for 15 min, and the clear, colorless aqueous phase was transferred to a new RNase-free EP tube. Total RNA was then extracted using RNA Clean & Concentrator Kits (Zymo, R1015). Next, mRNA was enriched from the total RNA using Oligo dT magnetic beads. After fragmentation, first-strand cDNA was synthesized using random hexamer primers, followed by second-strand cDNA synthesis. After end repair, A-tailing, adapter ligation, fragment selection, amplification, and purification, the library was ready. After library verification, the quantified libraries were mixed and sequenced on the Illumina platform according to the effective library concentration and data volume. Image data of the sequencing fragments obtained by the high-throughput sequencer were converted into sequence data (reads) through base identification in FASTQ format, mainly containing sequence information of the sequencing fragments and their corresponding sequencing quality information. First, the adapter sequences in the sequencing data were cut using `trim_galore`, and the cut sequences were aligned to the reference genome using STAR. Then, the aligned sequences were sorted using `picard`, and `featureCounts` was used to count the sorted sequence information to obtain the gene expression matrix. In RStudio, the DESeq2 package was used to perform principal component analysis (PCA) and differential gene expression analysis (DGE) on the gene expression matrix. The threshold for significant up / downregulation of genes was set as follows: Benjamin-Hochberg FDR(p.adjust) < 0.05 and |log2(foldchange)| ≥ 1. For significantly upregulated or downregulated genes, GO pathway enrichment analysis was performed using the `clusterProfiler` and `enrichplot` packages. GSEA analysis was performed on the top 30% and bottom 30% of biotin using the fgsea package.

[0112] For CD8 + Principal component analysis of T cell transcriptomes showed that Biotin + and Biotin - The two cell groups showed significant differences, and the parallelism between biological replicates was good. Figure 7b As shown in the volcano plot of differentially expressed genes analysis, compared to Biotin... - CD8+ T cell transcriptome, Biotin + CD8 + 201 genes were significantly upregulated in T cells, while 1242 genes were significantly downregulated. Figure 7c Furthermore, these significantly differentially expressed genes showed good parallelism across three biological replicates. GO-BP pathway enrichment analysis revealed that CD8+ genes near the tumor were... + Genes significantly upregulated by T cells (Top 30) were significantly enriched in pathways such as cytotoxicity, immune cell proliferation, and T cell activation. Figure 7d This aligns with traditional understanding of tumor immunity, specifically the presence of CD8 receptors near the tumor. + After T cells recognize the pMHC complex on the tumor surface via the TCR, they enter an activation process upon stimulation by tumor antigens, increasing the expression of Granzyme family proteins, thereby contributing to their cytotoxic function. Under continuous stimulation by tumor antigens, some tumor-adjacent antigen-specific CD8... + T cells then initiate an exhaustion process, exhibiting a dysfunctional phenotype. To further elucidate the role of biotin... + and Biotin - CD8 + Differences in T cell gene expression and function were observed using CD8. + GSEA analysis was performed on gene sets containing T cell activation / dysfunction gene signatures and tumor-specific gene signatures. Figure 7e In B16F10 melanoma, a gene set associated with T cell activation / dysfunction was significantly enriched in biotin. + CD8 + In T cells, and based on the tumor-specific T cell gene set reported by Schiettinger et al., we found that the tumor-specific upregulated and downregulated gene sets on Day 30 were enriched in biotin. + CD8 + T cells and biotin - CD8 + T cells. These conclusions are consistent with current understanding of tumor immunity, specifically the tumor antigen-specific CD8. + T cells interact with tumor cells through TCR-pMHC, thereby gradually initiating the activation and depletion process.

[0113] Next, to verify the reliability of the transcriptome findings, we performed flow cytometry staining analysis on markers associated with T cell activation, exhaustion, and dysfunction. The specific steps are as follows:

[0114] After tissue sectioning and CAT-Tissue labeling, a single-cell suspension of tumor tissue was prepared from the labeled sections. Cells were washed twice with FACS buffer and then counted. Then, Zombie Aqua (BioLegend, 423101) and flow cytometry antibody mixture [Streptavidin-APC (BioLegend, 405243), anti-mouse CD8α-Pacific Blue (BioLegend, 100725), anti-mouse CD4-FITC (BioLegend, 100406), anti-mouse PD-1-BV605 (BioLegend, 135220), anti-mouse TIM-3-PerCP / Cy5.5 (BioLegend, 119717), anti-mouse CD39-PE / Cy7 (BioLegend, 143805), anti-mouse CD137-PE (BioLegend, 106105), each at a concentration of 0.2 μL / test] were added sequentially for staining. After washing, the samples were analyzed by flow cytometry.

[0115] For granzyme B, intracellular protein detection is required. The procedure is as follows: After tissue sectioning and CAT-Tissue labeling, a single-cell suspension of tumor tissue is prepared from the labeled sections. After washing twice with FACSbuffer, cell counting is performed. Then, Zombie Aqua (BioLegend, 423101), flow cytometry antibody mixture [Streptavidin-APC (BioLegend, 405243), anti-mouse CD45-Pacific Blue (BioLegend, 103125), anti-mouse CD8α-PE (BioLegend, 100708), anti-mouse CD4-FITC (BioLegend, 100406), and anti-mouse NK1.1-BV605 (BioLegend, 108739), each at a ratio of 0.2 μL / test] are added sequentially for staining. Resuspend the cell pellet in 150 μL Fix / Perm Buffer (BD) mixture, fix at 4°C for 45 min, add 120 μL Perm / Wash Buffer (BD), centrifuge and discard the supernatant. Add 200 μL Perm / Wash Buffer to each well, centrifuge and discard the supernatant. Add 100 μL Perm / Wash Buffer containing anti-mouse Granzyme B-PE / Cy7 (BioLegend, 372213) flow cytometry antibody to each well and incubate at 4°C in the dark for 45 min. Centrifuge and discard the supernatant, wash twice with 200 μL Perm / Wash Buffer per well. Resuspend in 150 μL FACS Buffer and analyze by flow cytometry.

[0116] For perforin, intracellular protein detection is required. The procedure is as follows: After tissue sectioning and CAT-Tissue labeling, a single-cell suspension of tumor tissue is prepared from the labeled sections. After washing twice with FACSbuffer, cell counting is performed. Then, Zombie Aqua (BioLegend, 423101), flow cytometry antibody mixture [Streptavidin-APC (BioLegend, 405243), anti-mouse CD45-Pacific Blue (BioLegend, 103125), anti-mouse CD8α-PerCP / Cy5.5 (BioLegend, 100733), anti-mouse CD4-FITC (BioLegend, 100406), and anti-mouse NK1.1-BV605 (BioLegend, 108739), each at a ratio of 0.2 μL / test] are added sequentially for staining. Resuspend the cell pellet in 150 μL Fix / Perm Buffer (BD) mixture, fix at 4°C for 45 min, add 120 μL Perm / Wash Buffer (BD), centrifuge and discard the supernatant. Add 200 μL Perm / Wash Buffer to each well, centrifuge and discard the supernatant. Add 100 μL Perm / Wash Buffer containing anti-mouse Perforin-PE (BioLegend, 154305) flow cytometry antibody to each well and incubate at 4°C in the dark for 45 min. Centrifuge and discard the supernatant, wash twice with 200 μL Perm / Wash Buffer per well. Resuspend in 150 μL FACS Buffer and analyze by flow cytometry.

[0117] Compared with transcriptome conclusions ( Figure 7f This matches, Biotin + CD8 + The expression levels of PD-1, TIM-3, CD137, and CD39 on T cells were significantly higher than those on biotin. - CD8 + T cells ( Figure 7g Furthermore, analysis of endotoxin molecules indicated that Biotin... + CD8 + T cells showed higher expression of Granzyme B and Perforin, which is largely consistent with transcriptome findings. Figure 7h and Figure 7i This indicates that under the stimulation of tumor antigens, the tumor adjacent to CD8... +T cells are activated and express secretory cytotoxic molecules to exert their cytotoxic immunological functions. These results preliminarily validate the quantitative results of the transcriptome at the protein level and further reveal the presence of CD8 cells near the tumor. + T cells (Biotin) + Compared to tumor-negative CD8+, + T cells (Biotin) - In contrast, it exhibits phenotypic characteristics of activation, depletion, and inactivation, which is consistent with existing knowledge in the field of tumor immunology.

[0118] In summary, the neighboring cell labeling method of this application is highly efficient, sensitive, and non-invasive, and can effectively analyze the dynamic interaction between tumor and immune cells, providing an important experimental tool for tumor immune surveillance and / or immunotherapy.

[0119] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for neighboring cell labeling, comprising: A photocatalyst and a probe are added sequentially to the cells to be labeled, and neighboring cells are labeled under deep red light irradiation; wherein, the photocatalyst contains dihydroporphyrin E6 or its derivatives, and the probe contains biotin aniline or its derivatives.

2. The method according to claim 1, wherein, The photocatalyst is a nanobody dihydroporphyrin E6 conjugate; the nanobody dihydroporphyrin E6 conjugate includes His-tag nanobody, ZHER-dihydroporphyrin E6 complex targeting HER2, P43-dihydroporphyrin E6 complex targeting human PD-L1, and dP43-dihydroporphyrin E6 complex, an inactivating mutant of P43-dihydroporphyrin E6 complex.

3. The method according to claim 2, wherein, The synthesis steps of the nanobody dihydroporphyrin E6 conjugate include: Nanobodies expressing the C-terminal sequence LPETG-His-tag were linked to the GGGK(N3)GGK(N3)K(N3) peptide using the transpeptidase mgSrtA, and then subjected to an azide-alkyne cycloaddition reaction with the compound shown in Formula I to obtain nanobodies dihydroporphyrin E6 conjugates.

4. The method according to claim 3, wherein, The synthesis steps of the compound represented by Formula I include: (1) Protect the two carboxyl groups of dihydroporphyrin E6 by methyl esterification; (2) Carboxyl activation was performed using benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, followed by the addition of monotert-butoxycarbonyl-protected ethylenediamine, and condensation reaction was carried out using N,N-diisopropylethylamine or triethylamine as a base. (3) Use trifluoroacetic acid or 1,4-dioxane hydrogen chloride to carry out the deprotection reaction of tert-butyloxycarbonyl group; (4) Using N,N-diisopropylethylamine or triethylamine as a base, the condensation reaction of amino and N-hydroxysuccinimide active esters is realized; (5) The methyl ester protecting group was removed by hydrolysis with lithium hydroxide to obtain the compound shown in Formula I with a bioorthogonal reactive group.

5. The method according to claim 1, wherein, The wavelength of the deep red light irradiation is 640–680 nm.

6. The method according to claim 1, wherein, The concentration of the photocatalyst is 50–200 nM.

7. The method according to claim 1, wherein, The concentration of the probe is 100–200 μM.

8. The method according to claim 1, wherein, The incubation temperature for adding the photocatalyst is 2–8℃, and the incubation time is 100–140 min.

9. The method according to claim 1, wherein, The incubation temperature for adding the probe is 2–8℃, and the incubation time is 25–40 min.

10. The method according to claim 1, wherein, The method further includes performing flow cytometry and transcriptome analysis to confirm the effect of neighboring cell labeling.

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