Application of detection reagent in malignant differentiation detection of Carbosi sarcoma

Abstract

The invention discloses application of a detection reagent in malignant differentiation detection of Carbosi sarcoma, and belongs to the technical field of biological medicine. Aiming at the problem of insufficient specificity of a malignant differentiation marker of Carbosi sarcoma in the prior art, the invention provides application of a detection reagent in detection of the malignant differentiation of Carbosi sarcoma, and the detection reagent can simultaneously detect two biomarkers, namely CXCL12 and VWF, and is used for identifying endothelial cell malignant differentiation subgroups in tissue focuses of Carbosi sarcoma. By detecting the expression of CXCL12 and VWF in a tested sample, the endothelial cell malignant differentiation subgroup can be stably identified, and meanwhile, the expression level of CXCL12 can be used as an indicator of disease progression: the higher the expression level of CXCL12 is, the higher the lesion progression degree is, the higher the risk is, and the expression level is also positively correlated with the counting of peripheral blood lymphocytes. According to the invention, high-specificity recognition and standardized reading of the endothelial cell malignant differentiation subgroups are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of detection reagents in the detection of malignant differentiation of Kaposi's sarcoma. Background Technology

[0002] Kaposi's sarcoma (KS) is a rare vascular proliferative tumor that can affect the skin, oral mucosa, lymph nodes, and internal organs. Clinically, it often presents as purple, reddish-blue, or brownish-black spots, papules, and nodules, which are prone to bleeding and ulceration. Epidemiological data shows that in 2020, an estimated 34,000 cases were reported globally, all related to Kaposi's sarcoma-associated herpesvirus (KSHV / HHV-8) infection. The pathological stages of KS include patchy, plaque, and nodular stages, with the skin lesions progressively worsening as the disease progresses. Early lesions often present with a granulomatous reaction, accompanied by significant immune cell infiltration and strong angiogenesis, with proliferation of spindle-shaped (fusiform) cells derived from endothelial cells and macrophages. Typical KS is characterized by the proliferation of spindle-shaped endothelial cells in the skin and sometimes in mucosa and internal organs, forming slit-like vascular cavities, often accompanied by inflammatory cell infiltration and extravasation of erythrocytes. In advanced nodular lesions, tumor cells gradually become dominant, and the lesions exhibit a fibrosarcoma-like appearance, but angiogenesis remains active. In terms of immunophenotype, KS cells can express panendothelial and lymphoendothelial markers (such as ERG) and may exhibit a poorly differentiated spindle cell phenotype.

[0003] KS is a highly angiogenic tumor, characterized by abnormal blood vessel walls and irregular morphology, suggesting that angiogenesis and lymphangiogenesis-related factors are continuously upregulated in the context of KSHV infection. Unlike physiological angiogenesis, pathological angiogenesis is closely related to tumor growth, invasion, and metastasis. Although the specific mechanisms of angiogenesis in KS are not fully understood, current research generally suggests that KSHV-induced angiogenic factors and inflammatory factors are key drivers of tumorigenesis and maintenance. Reports indicate that KSHV infection can upregulate molecules such as IL-6, IL-8, basic fibroblast growth factor (b-FGF), ephrin B2, cyclooxygenase-2 (COX-2), heme oxygenase-1 (HO-1), Ang2, and various matrix metalloproteinases (MMPs); simultaneously, KSHV-encoded proteins (such as vIL-6, vCCL-1, vCCL-2, vGPCR, etc.) and factors such as vIRF1, v-FLIP, and v-Cyclin can synergistically promote angiogenesis and activation of inflammatory pathways. In addition, KSHV-encoded miRNAs can further promote the formation and maintenance of angiogenesis-related phenotypes by regulating multiple cell signaling pathways.

[0004] Currently, the diagnosis and classification of Kaposi's sarcoma (KS) are based on pathological morphology, supplemented by immunohistochemical markers such as ERG and VWF, and comprehensively interpreted in conjunction with clinical staging methods such as patches, plaques, and nodules. Although single-cell sequencing can reveal endothelial cell heterogeneity and copy number abnormalities, it lacks validation of the spatial localization of target cells within the lesion, and the evidence for its association with clinical outcomes such as staging and progression is unstable. It is difficult to translate research findings into reproducible in vitro detection readouts, and it is also insufficient to support reliable risk stratification and clinical decision-making.

[0005] In summary, the existing technology has at least the following prominent problems: (1) Lack of specific biomarkers makes it difficult to accurately identify malignant endothelial subpopulations: Current techniques mostly use pan-endothelial cells or inflammatory and angiogenesis indicators, such as ERG, VWF, and VEGF. These molecules can also be elevated in reactive or benign endothelial cells, making it difficult to effectively distinguish between neoplastic and non-neoplastic endothelial cells. The identification of endothelial subpopulations truly related to malignant differentiation is unstable, affecting the accuracy of diagnosis and the reliability of disease course assessment. The lack of specific biomarkers combining CXCL12 and VWF makes it impossible to clearly define key malignant differentiation subpopulations of endothelial cells.

[0006] (2) Lack of unified standards in analysis and validation procedures, resulting in poor reproducibility of multiplex immunofluorescence assays: While existing single-cell sequencing can reveal endothelial heterogeneity, it generally lacks a standardized procedure for distinguishing malignant and benign cells based on euploidy, which can easily mask a small number of malignant proliferation signals, making it difficult to accurately identify key subpopulations. Further spatial validation often uses multiplex immunofluorescence, but multiplex immunofluorescence assays vary significantly between different laboratories: screening methods, staining conditions, and interpretation criteria for co-localization analysis of specific markers are not unified, leading to strong subjectivity in result interpretation and difficulty in cross-laboratory reproducibility. These deficiencies result in a lack of stable and reproducible evidence chains in the process from the discovery of malignant differentiation subpopulations of endothelial cells by single-cell sequencing to histological validation, thus limiting the promotion and clinical translation of relevant markers.

[0007] (3) Weak clinical correlation and limited translational application: Existing studies mostly involve sporadic correlations between single molecules and staging or inflammatory markers, with limited sample sizes and insufficient statistical stability. There is a lack of biomarker systems validated with expanded samples and clear reporting standards, making it difficult to directly apply to risk stratification, disease progression assessment, or efficacy monitoring. The interpretation criteria for the tests are inconsistent, resulting in insufficient clinical reproducibility and operability. At the same time, there is a lack of systematic joint validation with commonly used clinical indicators (such as peripheral blood lymphocyte count), making it difficult to support clinical subtyping. Summary of the Invention

[0008] Based on literature review and research insights into the insufficient specificity of existing Kaposi's sarcoma biomarkers, this invention provides a set of biomarkers for malignant differentiation of Kaposi's sarcoma and their detection methods. By combining the detection of CXCL12 and VWF, high-specificity identification of malignant differentiation subpopulations of endothelial cells can be achieved.

[0009] This invention begins with single-cell sequencing, extracting endothelial cell data from Kaposi's sarcoma samples, and using CopyKAT to classify them into malignant and benign types based on aneuploidy. Differential analysis, functional enrichment, and secondary clustering are performed on malignant endothelial cells to identify candidate subpopulations and determine malignant differentiation subpopulations of endothelial cells marked by CXCL12 and SEMA3G. Subsequently, endothelial cells are confirmed using VWF, and CXCL12 is used as the primary detection indicator to construct a "CXCL12+VWF" combined biomarker. Multiplex immunofluorescence is used to detect the malignant differentiation subpopulations of endothelial cells and their enrichment characteristics near vascular-associated clefts. CXCL12 is detected on expanded samples, and correlation analysis is performed with skin lesion types such as patches, plaques, and nodules to establish reproducible thresholds and stratification rules. This achieves high-specificity identification of malignant endothelial subpopulations, supporting risk stratification and clinical translational applications.

[0010] In a first aspect, the present invention provides the application of reagents for simultaneously detecting CXCL12 and VWF in the preparation of products for the detection, monitoring, or progression assessment of Kaposi's sarcoma malignant differentiation.

[0011] Secondly, the detection product is a reagent for detecting the expression levels of CXCL12 and VWF proteins, and the reagent is used to perform a multiplex immunofluorescence detection method.

[0012] Thirdly, when the expression levels of both CXCL12 and VWF proteins are positive, the sample is determined to be positive for the malignant differentiation subgroup of endothelial cells, thus indicating that Kaposi's sarcoma is positive for malignant differentiation; otherwise, the sample is determined to be negative for the malignant differentiation subgroup of endothelial cells.

[0013] Fourthly, a method for detecting malignant differentiation subsets of endothelial cells for non-diagnostic purposes, characterized in that the method comprises: (1) Obtain test samples from subjects and perform multiplex immunofluorescence or immunohistochemical detection on the test samples; (2) Identify blood vessels or irregular slit-like structures in the sample to be tested based on HE staining, and make the following judgments in the vicinity of the structure; (3) When the expression level of VWF is significantly higher than that of nearby cells, identify and extract a subset of endothelial cells; (4) When the expression level of CXCL12 is significantly higher than that of the control group, the sample is determined to be positive for the malignant differentiation subset of endothelial cells.

[0014] Fifthly, a kit for detecting, monitoring, or assessing the malignant differentiation of Kaposi's sarcoma, the kit comprising: Detection reagent for simultaneously detecting VWF protein expression level and CXCL12 protein expression level; The instruction manual states that when the expression of VWF and CXCL12 in the detection area is higher than that in the control group, the sample is determined to be positive for the malignant differentiation subset of endothelial cells, indicating the presence of Kaposi's sarcoma-related signals.

[0015] Sixthly, the detection reagent is a multiplex immunofluorescence reagent.

[0016] Compared with the prior art, the present invention has the following advantages: 1. This invention proposes a combined biomarker of CXCL12 and VWF, clearly defining the malignant differentiation subset of endothelial cells as a population of VWF-positive endothelial cells that are positive for or highly express CXCL12. This combination offers higher differentiation between malignant and benign endothelial cells, significantly reduces reactive signal interference, and avoids dilution or missed detection of key subsets; it can stably identify target cells closely related to malignant differentiation, improving diagnostic accuracy and the reliability of disease progression assessment.

[0017] 2. This invention constructs a complete chain of evidence from single-cell discovery to spatial validation: single-cell sequencing combined with CopyKAT aneuploidy analysis distinguishes between malignant and benign endothelial cells; secondary clustering and functional enrichment within the malignant population identify malignant differentiation subpopulations of endothelial cells; subsequently, multiplex immunofluorescence confirms their spatial enrichment near blood vessels or fissures; and finally, immunohistochemistry is used for quantitative validation in expanded samples. This closed-loop chain of evidence ensures the reproducibility and cross-laboratory consistency of the results.

[0018] 3. This invention has clear clinical relevance and potential for widespread application: In tissue samples from 57 patients with Kaposi's sarcoma, this invention verified that high expression of CXCL12 was significantly associated with advanced skin lesions (nodular stage) and positively correlated with elevated peripheral blood lymphocyte count. Based on these results, this invention establishes quantifiable interpretation criteria and stratification rules, which can be directly applied to the stratification of malignant differentiation risk, assessment of disease progression, and monitoring of treatment efficacy in Kaposi's sarcoma, providing feasible detection products and methods for clinical translation. Attached Figure Description

[0019] Figure 1This is a landscape image of single-cell sequencing of Kaposi's sarcoma skin tissue, showing: A. Clustering yielded 23 clusters, with the healthy control group (HC) on the left and the Kaposi's sarcoma group (KS) on the right; B. Annotation revealed 11 cell types and 1 unknown type; C. Annotation of marker gene expression in each cell type; D. The percentage of each cell type in the Kaposi's sarcoma and healthy controls, with blue representing the healthy control group (HC) and yellow representing the Kaposi's sarcoma group (KS).

[0020] Figure 2 This study analyzed the malignant differentiation of endothelial cells in Kaposi's sarcoma skin tissue, including: A. Using CopyKAT, endothelial cells in Kaposi's sarcoma skin tumor tissue were divided into malignant (aneuploid) and benign (euploid) cells through aneuploidy analysis, with red representing malignant cells (aneuploid) and green representing benign cells (diploid); B. The percentage of benign and malignant cell types in each subpopulation of Kaposi's sarcoma, with red representing malignant cells (aneuploid) and green representing benign cells (diploid); C. The distribution of malignant cells in the endothelial cell subpopulations of Kaposi's sarcoma; D. The expression profile of key genes in the malignant cell subpopulations.

[0021] Figure 3 Identification of key endothelial cell subsets for Kaposi's sarcoma.

[0022] Figure 4 The expression of CXCL12 in Kaposi's sarcoma tissue. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described are merely illustrative of the specific applications of this invention and are not intended to limit the scope of the invention. These embodiments are intended to help understand the principles, operating methods, and advantages of this invention, but do not preclude appropriate adjustments and modifications during the implementation of this invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0025] Example 1: Screening for Kaposi's sarcoma malignancy differentiation markers CXCL12 and VWF Kaposi's sarcoma (KS) is a rare vascular proliferative tumor that can affect the skin, oral cavity, lymph nodes, and internal organs. It is characterized by purple, reddish-blue, or brownish-black spots, papules, and nodules that are prone to bleeding and ulceration. In 2020, an estimated 34,000 cases of KS were reported globally, all attributable to Kaposi's sarcoma-associated herpesvirus (KSHV). Kaposi's sarcoma can be classified into patchy, plaque, and nodular stages, with a gradual increase in skin lesions. Early progressive lesions (plaques or plaques) typically present with a granulomatous reaction, accompanied by immune cell infiltration, strong angiogenesis, and proliferation of "spindle-shaped" cells derived from endothelial cells and macrophages—the tumor cells of KS. Typical KS is characterized by the proliferation of spindle-shaped endothelial cells in the skin, and sometimes in mucous membranes and internal organs. Kaposi's cells form slit-like vascular cavities, often accompanied by extravasation of inflammatory cells and erythrocytes. In advanced nodular lesions of KS, tumor cells eventually become the dominant cell type, and the lesions present as fibrosarcomas, despite the presence of angiogenesis. KS cells express panendothelial and lymphoendothelial markers (such as ERG) and may also appear as poorly differentiated spindle cells expressing markers for multiple cell types.

[0026] Kaposi's sarcoma is a highly angiogenic tumor characterized by abnormal and irregularly shaped angiogenesis, suggesting a relatively high production of numerous angiogenic and lymphangiogenic factors during KSHV infection. Unlike the general role of the host, pathological angiogenesis is associated with tumor growth and metastasis. Although the mechanisms of angiogenesis in KS tumor development are not fully understood, KSHV-induced angiogenic factors and inflammatory cytokines appear to be essential drivers of KS tumor development. KSHV infection can induce angiogenic factors IL-6, IL-8, basic fibroblast growth factor (b-FGF), ephrin B2, cyclooxygenase-2 (Cox-2), heme oxygenase-1 (HO-1), Ang2, and MMPs. Furthermore, many KSHV-encoded proteins, such as vIL-6, vCCL-1, vCCL-2, and vGPCR, have been shown to stimulate angiogenesis along with vIRF1, v-FLIP, and v-Cyclin. In addition, KSHV-encoded miRNAs can induce angiogenesis by activating multiple cellular signaling pathways.

[0027] This study performed single-cell sequencing analysis on three pairs of Kaposi's sarcoma tissue specimens, targeting endothelial cells, which are important in the development and progression of Kaposi's sarcoma. Using CopyKAT, endothelial cells in Kaposi's sarcoma skin tumor tissues were divided into malignant (tumor) cells and benign (stromal) cells. Differential genes and potential functions in endothelial cell subsets were analyzed. A malignant differentiation subset of endothelial cells with CXCL12 and SEMA3G as markers was screened and found to be significantly enriched in tumorigenesis-related pathways such as angiogenesis. This subset may be an important cell subset for the occurrence and maintenance of Kaposi's sarcoma tumor.

[0028] The specific screening steps and strategies are as follows: A biopsy was performed on the skin lesions of the subjects, and the following procedures and tests were conducted.

[0029] (1) Tissue processing: Cut the target tissue into small pieces the size of soybeans (about 100mg), wash twice with sterile PBS (or sterile physiological saline), and immediately place it in pre-cooled tissue preservation solution.

[0030] (2) Tissue cleaning: Remove the skin tissue from the tissue preservation solution and place it together with the preservation solution in a 9cm sterile culture dish. Remove the non-target parts with ophthalmic forceps and place it in a new culture dish. Clean it repeatedly 3 times with sterile HBSS pre-cooled on ice.

[0031] (3) Tissue digestion: Use sterile ophthalmic scissors to cut the tissue into a paste-like consistency, transfer it to a 15mL centrifuge tube, add 2mL of skin tissue dissociation solution for every 100mg of tissue, ensuring that the dissociation solution can submerge the tissue. If the tissue volume is greater than 300mg, it can be digested in separate tubes. Place the centrifuge tube in a 37℃ constant temperature shaker and digest at 140rpm. Examine the tissue under a microscope every 30-40min to observe the degree of cell digestion and the remaining tissue. If the tissue is not completely digested after 70min, collect the supernatant and transfer it to a 50ml low-adsorption centrifuge tube using a Pasteur pipette. Add 3 times the volume of pre-cooled 10% FBS DMEM to terminate the digestion and store it on ice. Residue digestion: Continue digestion of the remaining tissue, add 3-4mL of dissociation solution, and digest in a 37℃ constant temperature shaker at 140rpm. Examine the cell state under a microscope every 30min. Digestion can be terminated when the tissue is basically completely digested or becomes extremely small particles.

[0032] (4) Cell suspension filtration: All digested suspensions were collected and filtered using a 70μm sterile filter. The volume was adjusted to 15-25mL using pre-cooled 10% FBS DMEM. The suspensions were centrifuged at 350g (rcf) for 5min, the supernatant was discarded, and 1ml of supernatant was retained. The cell pellet at the bottom of the tube was gently pipetted a few times to mix the cells (this step is referred to as cell resuspension below). The cells were examined under a microscope to observe the cell state and cell quantity.

[0033] (5) Red blood cell lysis treatment: Transfer the suspension to a 15ml low-absorption centrifuge tube, add 2ml of red blood cell lysis buffer, invert to mix, incubate on ice for 5-8min, centrifuge at 300g (rcf) for 5min, carefully remove the supernatant, and resuspend the cell pellet in pre-cooled PBS.

[0034] (6) Washing cells: Adjust the volume of the cell suspension to 10 mL with pre-cooled 10% FBS DMEM, invert and mix well, centrifuge at 300g (rcf) for 5 min, discard the supernatant, and resuspend the cell pellet in 0.15-0.2 mL of pre-cooled PBS.

[0035] (7) Suspension quality inspection: Cell suspension: trypan blue staining solution / fluorescent AO / PI staining solution = 1:1 volume ratio, and then the activity concentration is detected on a microscope / cell counter.

[0036] (8) Sequencing and quality control: The prepared cell suspension is labeled, lysed, nucleic acid captured and library constructed to obtain labeled transcriptome library, and the corresponding data is obtained by NGS sequencing. The corresponding data is processed by library construction quality control, double cell removal, batch effect removal and other treatments to obtain single cell sequencing standard data.

[0037] (9) Cell annotation and bioinformatics analysis: Cell annotation is performed based on data such as cell markers and cell transcriptome expression profiles to obtain cell types and distribution. Figure 1 C).

[0038] (10) Analysis of endothelial cell subsets and malignant cell characteristics: Endothelial cell subsets were extracted, and their expression levels were calculated to obtain further subdivisions and expression characteristics. Figure 1 D).

[0039] (11) Judgment criteria: The above endothelial cells were extracted and divided into malignant (aneuploid proliferative) cells and benign (euploid proliferative) cells by aneuploidy analysis using CopyKAT. The malignant (aneuploid proliferative) cell subpopulation with a higher proportion in the tumor tissue was obtained by comparison. Combined analysis, the cell subpopulation with the highest correlation with the occurrence of KS tumors was screened. Correlation analysis was performed ( Figure 2 D) Obtain the corresponding markers to distinguish each subgroup.

[0040] The results show: 1. Increased number of endothelial cells in Kaposi's sarcoma tumor tissue. Single-cell sequencing was performed on three pairs of Kaposi's sarcoma tissue specimens. Figure 1 The study revealed a significant overall proportion of endothelial cells in Kaposi's sarcoma tumor tissue, with a marked increase compared to healthy controls, potentially related to angiogenesis within the tumor. Expression profiling showed a significant increase in the VEF and SELE genes in endothelial cells.

[0041] 2. Malignant differentiation subsets of endothelial cells in Kaposi's sarcoma tumor tissue may be highly correlated with tumor occurrence and progression. Endothelial cell data were extracted from single-cell sequencing, and CopyKAT was used to classify endothelial cells in KS skin tumor tissue into malignant (aneuploid proliferative) cells and benign (euploid proliferative) cells through aneuploidy analysis. Figure 2 Based on the euploidy characteristics of cell proliferation, the subpopulations were analyzed, and some subpopulations showed significant aneuploidy, suggesting that malignant proliferation of endothelial cells mainly occurs in some cells. Secondary clustering of aneuploid proliferating cells yielded 6 subpopulations. Enrichment analysis indicated that the malignant differentiation subpopulation of endothelial cells with CXCL12 and SEMA3G as markers was significantly enriched in tumorigenesis-related pathways such as angiogenesis, and may be an important cell subpopulation for the occurrence and maintenance of Kaposi's sarcoma tumor.

[0042] Example 2: Identification of Malignant Differentiation Endothelial Cell Subpopulations in Kaposi's Sarcoma Skin Tissue Multiple immunofluorescence assays confirmed that the malignant differentiation subset of endothelial cells was significantly enriched near the blood vessels of Kaposi's sarcoma, and was significantly higher in the tumor area than in the control area, suggesting that this subset may be highly associated with the occurrence and progression of Kaposi's sarcoma.

[0043] The screening steps and strategies are as follows: Biopsies were performed on the skin lesions of the subjects. The corresponding skin tissue specimens were fixed in 4% formalin solution, dehydrated, embedded and prepared into paraffin samples. 4μm sections were then prepared for the following processing and testing.

[0044] (1) Dewaxing paraffin sections to water: The sections were placed in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, environmentally friendly dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then washed with distilled water.

[0045] (2) Antigen retrieval: Tissue sections were placed in a retrieval box filled with EDTA antigen retrieval buffer (pH 8.0) and microwaved for antigen retrieval. Microwave on medium heat for 8 minutes, turn off for 8 minutes, then microwave on medium-low for 7 minutes. During this process, excessive evaporation of the buffer should be prevented, and the slides should not be allowed to dry. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 minutes each time. (The retrieval solution and conditions are determined based on the tissue.) (3) Circle drawing and hydrogen peroxide blocking: After slightly drying the slides, draw circles around the tissue with a histochemical pen (to prevent antibody migration). Place the slides in a 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 min to block endogenous peroxidase. Place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker for 5 min each time. (4) Serum blocking: Shake off the PBS, add BSA, and block for 30 min. (For primary antibodies of goat origin, use 10% rabbit serum for blocking; for primary antibodies of other origin, use 3% BSA for blocking.) (5) Add the first primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in PBS at a certain ratio to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight. (Add a small amount of water to the humidified chamber to prevent antibody evaporation) (6) Add the corresponding HRP-labeled secondary antibody: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add the HRP-labeled secondary antibody corresponding to the species of the primary antibody to the tissue and incubate at room temperature for 50 minutes.

[0046] (7) Add iF488-TSA (or FITC-TSA): Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add TSA to the circle and incubate at room temperature in the dark for 10 minutes. After incubation, place the slide in TBST and wash three times on a decolorizing shaker for 5 minutes each time. (8) Microwave treatment: Place the tissue sections in a retrieval box filled with EDTA antigen retrieval buffer (pH 8.0) and heat in a microwave oven for 8 minutes on medium heat, turn off for 8 minutes, turn to medium-low heat for 7 minutes to remove the primary and secondary antibodies that have been bound to the tissue. During this process, prevent excessive evaporation of the buffer and do not dry the sections.

[0047] (9) Add the second primary antibody: Add the primary antibody prepared in PBS at a certain ratio to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight. (Add a small amount of water to the humidified chamber to prevent antibody evaporation).

[0048] (10) Add the corresponding HRP-labeled secondary antibody: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add the fluorescent secondary antibody corresponding to the species of the primary antibody to the circle to cover the tissue, and incubate at room temperature in the dark for 50 minutes.

[0049] (11) Adding iF555-TSA: Place the slide in PBS (pH 7.4) and wash three times on a destaining shaker, 5 min each time. After slightly drying the slide, add TSA to the circle and incubate at room temperature in the dark for 10 min. After incubation, place the slide in TBST and wash three times on a destaining shaker, 5 min each time. (12) Counterstaining cell nuclei with DAPI: After slightly drying the slices, add DAPI staining solution to the circle and incubate at room temperature in the dark for 10 min.

[0050] (13) Autofluorescence quenching: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add autofluorescence quenching agent to the circle for 5 minutes and rinse with running water for 10 minutes.

[0051] (14) Mounting: After slightly drying the sections, mount them with anti-fluorescence quenching mounting medium.

[0052] (15) Microscopic examination and photography: The slides were observed and images were acquired under a fluorescence microscope. (DAPI was excited by ultraviolet light at wavelengths of 330-380 nm and emitted blue light at wavelength of 420 nm; FITC was excited by wavelengths of 465-495 nm and emitted green light at wavelengths of 515-555 nm; CY3 was excited by wavelengths of 510-560 nm and emitted red light at wavelength of 590 nm.)

[0053] (16) Judgment criteria: Based on HE staining, identify blood vessels or irregular slit-like structures (dysplastic blood vessels) in tissue samples. In the vicinity of such structures, when the expression level of VWF is significantly higher than that of nearby cells, identify and extract a subset of endothelial cells; when the expression level of CXCL12 is significantly higher than that of the control group, determine that the sample is positive for the malignant differentiation subset of endothelial cells.

[0054] The results show: In Kaposi's sarcoma tumor tissue, malignant differentiation subsets of endothelial cells are significantly enriched near abnormally proliferating or dysplastic blood vessels.

[0055] Multiplex immunofluorescence assays were used to identify key malignant differentiation subsets of endothelial cells in Kaposi's sarcoma. Colocalization analysis showed that the malignant differentiation subsets of endothelial cells identified by VWF and CXCL12 were significantly enriched near blood vessels or related fissures in Kaposi's sarcoma, and may play an important role in tumor abnormalities (such as abnormal angiogenesis or dysplasia). Figure 3 This image shows the identification of key endothelial cell subsets in Kaposi's sarcoma. Multiplex immunofluorescence was used to identify the malignant differentiation subsets of key endothelial cells in Kaposi's sarcoma. DAPI was used to stain the cell nuclei, with VWF showing red fluorescence and CXCL12 showing green fluorescence.

[0056] Example 3: Correlation between CXCL12 expression in Kaposi's sarcoma tumor tissue and the severity of Kaposi's sarcoma. Immunohistochemical analysis in expanded samples revealed that the expression level of the marker gene CXCL12 in key endothelial cell malignant differentiation subsets was highly correlated with the type of Kaposi's sarcoma lesions. The positive rate of CXCL12 in nodular or plaque stage lesions was significantly higher than that in plaque stage lesions (P<0.001, χ2=33.715), suggesting that CXCL12 may be an indicator of the progression of Kaposi's sarcoma.

[0057] Step 1 Sample Preparation Biopsies were performed on the skin lesions of the subjects. The corresponding skin tissue specimens were fixed in 4% formalin solution, dehydrated, embedded and prepared into paraffin samples. 4μm sections were then prepared for the following processing and testing.

[0058] (1) Dewaxing paraffin sections to water: The sections were placed in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, environmentally friendly dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then washed with distilled water.

[0059] (2) Antigen retrieval: Tissue sections were placed in a retrieval box filled with EDTA antigen retrieval buffer (pH 8.0) and microwaved for antigen retrieval. Microwave on medium heat for 8 minutes, turn off for 8 minutes, then microwave on medium-low for 7 minutes. During this process, excessive evaporation of the buffer should be prevented, and the slides should not be allowed to dry. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker for 5 minutes each time. (The retrieval solution and conditions are determined based on the tissue.) (3) Circle drawing and hydrogen peroxide blocking: After the slide is slightly dried, circle the tissue with a histochemical pen (to prevent the antibody from flowing away). Place the slide in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 min to block endogenous peroxidase. Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 min each time.

[0060] (4) Serum blocking: Shake off the PBS, add BSA, and block for 30 min. (For primary antibodies of goat origin, use 10% rabbit serum for blocking; for primary antibodies of other origin, use 3% BSA for blocking.) (5) Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in PBS at a certain ratio to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight. (Add a small amount of water to the humidified chamber to prevent antibody evaporation) (6) Add the corresponding HRP-labeled secondary antibody: Place the slide in PBS (pH 7.4) and wash it three times on a decolorizing shaker for 5 minutes each time. After slightly drying the slide, add the HRP-labeled secondary antibody corresponding to the species of the primary antibody to the tissue and incubate at room temperature for 50 minutes.

[0061] Step 2: Combined detection of VWF and CXCL12 (1) DAB staining: After the section is slightly dried, add the mixed DAB staining solution (solution A and solution B) to the circle, incubate at room temperature in the dark for 10 min, and observe the staining under a microscope. If the staining is insufficient, it can be stained again.

[0062] (2) Counterstaining cell nuclei with hematoxylin: After slightly drying the slices, add hematoxylin staining solution to the circle, incubate at room temperature for 2-5 minutes, and then invert with cold water or invert blue reagent.

[0063] (3) Dehydration and mounting: Place the slide in a gradient of 75% alcohol, 95% alcohol and anhydrous ethanol and let it stand for 5 minutes in each liquid. After clearing with xylene, shake off the excess water and add epoxy resin or other mounting medium to mount the slide.

[0064] (4) Microscopic examination and photography: The slides are observed and images are acquired under an optical microscope. (Yellow or reddish-brown indicates staining with the corresponding antibody, and blue indicates staining with the cell nucleus).

[0065] Step 3 Judgment Criteria Based on HE staining, blood vessels or irregular slit-like structures (dysplastic blood vessels) in tissue samples are identified. When the expression level of CXCL12 in the vicinity of such structures is significantly higher than that in the control group, the sample is determined to be CXCL12 positive.

[0066] The results show: Immunohistochemical staining of CXCL12 expression levels in tissue specimens from 57 patients with Kaposi's sarcoma revealed that high CXCL12 expression was associated with highly progressive skin lesions (nodules / plaques) in Kaposi's sarcoma, suggesting that CXCL12 may play an important role in the progression of Kaposi's sarcoma. Blood routine analysis of Kaposi's sarcoma patients showed that the lymphocyte count was significantly higher in the high CXCL12 expression group compared to the low CXCL12 expression group (6.96 vs. 6.22).

[0067] Figure 4 This image shows the expression of CXCL12 in Kaposi's sarcoma tissue. CXCL12 was stained by immunohistochemistry in the tissue specimens. The left side shows a Kaposi's sarcoma tissue specimen from a patient in the patchy stage, and the right side shows a Kaposi's sarcoma tissue specimen from a patient in the nodular stage. The top row is at 40x magnification, and the bottom row is at 100x magnification.

[0068] Table 1. Correlation analysis between CXCL12 expression level and clinical characteristics of Kaposi's sarcoma ; The biomarkers used in this study were a combination of CXCL12 and VWF, which can identify a malignant differentiation subset of endothelial cells. In Kaposi's sarcoma tissue, this subset manifests as a malignantly proliferating cell subpopulation and plays a crucial role in the occurrence and progression of Kaposi's sarcoma tumors. Analysis of the correlation between CXCL12 expression levels and the clinical characteristics of Kaposi's sarcoma patients revealed that higher CXCL12 expression levels were more likely to present with highly progressive clinical manifestations such as nodular / plaque lesions. Correspondingly, the lymphocyte count was also significantly higher in the CXCL12 expression group compared to the low-CXCL12 expression group, indicating, to some extent, the level of inflammatory response in the patients.

[0069] Furthermore, this invention provides a kit for detecting, monitoring, or assessing the malignant differentiation of Kaposi's sarcoma. The kit includes detection reagents for detecting the expression levels of CXCL12 and VWF, along with accompanying instructions. When both CXCL12 and VWF show positive signals in the detection area, it indicates the presence of a malignant endothelial cell subpopulation in the sample, suggesting that the test sample is associated with malignant differentiation of Kaposi's sarcoma. This allows the technical solution of this invention to be commercially available as a kit, facilitating its widespread use in research and clinical translation.

[0070] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Application of reagents for simultaneous detection of CXCL12 and VWF in the preparation of products for the detection, monitoring or progression assessment of Kaposi's sarcoma malignant differentiation.

2. The application according to claim 1, characterized in that, The detection product is a reagent for detecting the expression levels of CXCL12 and VWF proteins, and the reagent is used to perform a multiplex immunofluorescence detection method.

3. The application according to claim 2, characterized in that, When both CXCL12 and VWF protein expression levels are positive, the sample is determined to be positive for the malignant differentiation subset of endothelial cells, thus indicating that Kaposi's sarcoma is malignantly differentiated; otherwise, the sample is determined to be negative for the malignant differentiation subset of endothelial cells.

4. A method for detecting malignant differentiation subsets of endothelial cells for non-diagnostic purposes, characterized in that, The method includes: (1) Obtain test samples from subjects and perform multiplex immunofluorescence or immunohistochemical detection on the test samples; (2) Identify blood vessels or irregular slit-like structures in the sample to be tested based on HE staining, and make the following judgments in the vicinity of the structure; (3) When the expression level of VWF is significantly higher than that of nearby cells, identify and extract a subset of endothelial cells; (4) When the expression level of CXCL12 is significantly higher than that of the control group, the sample is determined to be positive for the malignant differentiation subset of endothelial cells.

5. A kit for detecting, monitoring, or assessing the malignant differentiation of Kaposi's sarcoma, characterized in that, The kit includes: Detection reagent for simultaneously detecting VWF protein expression level and CXCL12 protein expression level; The instruction manual states that when the expression of VWF and CXCL12 in the detection area is higher than that in the control group, the sample is judged to be positive for endothelial cell malignant differentiation, and it indicates the presence of Kaposi's sarcoma malignant differentiation-related signals.

6. The reagent kit according to claim 5, characterized in that, The detection reagent is a multiplex immunofluorescence reagent.

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