Application of THY1 in diagnosis and prognosis of glioblastoma

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of THY1 in diagnosis and prognosis of glioblastoma. According to the invention, a THY1-positive cancer-related fibroblast (THY1 + CAF) subgroup is identified in glioblastoma for the first time, and it is found that the subgroup and tumor blood vessels are highly co-localized in spatial distribution, and the abundance of the subgroup and the blood vessel density are significantly positively correlated; tHY1 + CAF is a key cell component for promoting angiogenesis in a GBM tumor microenvironment; tHY1 can be used as a marker of GBM specific CAFs, and is used for auxiliary diagnosis, prognosis evaluation (high THY1 + CAF may indicate worse prognosis and stronger angiogenesis phenotype) and curative effect monitoring of GBM. Meanwhile, the target THY1 or THY1 + CAF provides a brand-new and potential action target for developing a GBM new therapy for resisting angiogenesis and even remodeling an immunosuppressive microenvironment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of THY1 in the diagnosis and prognosis of glioblastoma. Background Technology

[0002] THY1 is a glycosylphosphatidylinositol-anchored protein belonging to the immunoglobulin superfamily, with a molecular weight of approximately 25-37 kDa. Under normal physiological conditions, it participates in fibroblast proliferation, migration, and wound healing, T cell activation and apoptosis, as well as cell-cell interactions. In solid tumors, THY1 often serves as a marker for tumor stem cells. By binding to different ligands (such as integrins αvβ3, αvβ5, and Syndecan-4), it mediates interactions between tumor cells, between tumor cells and stromal cells, and between tumor cells and the extracellular matrix, thus influencing tumor cell adhesion and migration.

[0003] Glioblastoma (GBM) is the most malignant and poorly prognostic primary tumor of the central nervous system. Current treatments (surgery, radiotherapy, temozolomide chemotherapy) have limited effectiveness, resulting in a short median survival. The tumor microenvironment (TME) plays a crucial role in GBM progression and treatment resistance. Cancer-associated fibroblasts (CAFs) have been shown to drive tumor progression in various solid tumors by promoting angiogenesis, remodeling the extracellular matrix, and suppressing immunity, making them important therapeutic targets. However, the existence of functional CAFs in GBM has long been controversial due to the brain's traditionally perceived lack of fibroblasts. While recent studies have suggested the presence of CAF-like cells in GBM, their specific subtypes, molecular markers, spatial distribution characteristics, and their clearly defined functions and mechanisms in GBM, particularly in angiogenesis, remain unclear. This limits the development of precision diagnostic and therapeutic methods targeting CAFs in GBM.

[0004] There is an urgent need to identify key biomarkers of specific CAFs in GBM, elucidate their functions, and explore their potential as novel therapeutic targets. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned problems by providing an application of THY1 in the diagnosis and prognosis of glioblastoma.

[0006] To achieve its objective, the present invention employs the following technical solution:

[0007] A first aspect of the present invention provides a diagnostic marker for glioblastoma, said marker being a THY1-positive cancer-associated fibroblast subset, THY1. + CAF.

[0008] A second aspect of the present invention provides a prognostic biomarker for glioblastoma, said biomarker being a THY1-positive cancer-associated fibroblast subset, THY1. + CAF.

[0009] A third aspect of the present invention provides a detection reagent for the aforementioned markers.

[0010] The detection reagent includes an anti-THY1 antibody.

[0011] Preferably, the detection reagent further includes flow cytometry reagent.

[0012] A fourth aspect of the present invention provides a kit for diagnosing glioblastoma or predicting the prognosis of glioblastoma, the kit comprising the detection reagents described in any one of the preceding claims.

[0013] A fifth aspect of the invention provides the use of a biomarker in the preparation of products for diagnosing glioblastoma or predicting the prognosis of glioblastoma, said biomarker being a THY1-positive cancer-associated fibroblast subset, THY1. + CAF.

[0014] THY1 in tumor tissue of glioblastoma patients + CAF levels were significantly higher than in normal tissues; high THY1 + CAF predicts a worse prognosis for patients.

[0015] The sixth aspect of the present invention provides THY1 + CAF is used in any of the following:

[0016] (1) Application as a target in screening anti-angiogenic drugs in the GBM tumor microenvironment;

[0017] (2) Application as a target in screening drugs for the treatment of glioblastoma.

[0018] Among them, THY1 + CAF subgroups are highly colocalized with tumor blood vessels in spatial distribution, and their abundance is significantly positively correlated with blood vessel density.

[0019] The beneficial effects of this invention are:

[0020] Through single-cell transcriptome sequencing and bioinformatics analysis, combined with multiplex immunofluorescence verification, this invention, for the first time, identifies THY1-positive cancer-associated fibroblasts (THY1) in glioblastoma. + The CAF (Cardiac Acid Fiber) subgroup was identified, and it was found that this subgroup was highly co-localized with tumor vessels in terms of spatial distribution, with its abundance being significantly positively correlated with vessel density. This suggests that THY1 +CAFs are key pro-angiogenic cellular components in the GBM tumor microenvironment. Therefore, THY1 can serve as a marker of GBM-specific CAFs for the auxiliary diagnosis and prognostic assessment of GBM (high THY1 levels are associated with high prognosis). + CAF (which may predict a worse prognosis and a stronger angiogenic phenotype) and efficacy monitoring. Simultaneously, targeting THY1 or THY1... + CAF provides a novel and promising target for developing new GBM therapies that combat angiogenesis and even reshape the immunosuppressive microenvironment. Attached Figure Description

[0021] Figure 1 The following are shown: (A) UMAP diagram showing the cell populations present in the GBM microenvironment; (B) UMAP diagram showing the results of periderm cell re-clustering; (C) showing the specific genes highly expressed in the CAF subsets.

[0022] Figure 2 The following were displayed: (A) a bubble chart showing the specific genes highly expressed in each subpopulation; (B) GSEA analysis of THY1. + The signaling pathway enriched by CAF expression genes.

[0023] Figure 3 Multiple immunohistochemical staining was performed on paraffin sections of glioblastoma to determine the immunolocalization of PDGFRβ, CD31, and Thy1 in GBM tissue.

[0024] Figure 4 Analyzing THY1 for QuPath + Correlation between CAF and blood vessels, graphPad plotting results.

[0025] Figure 5 Flow cytometry plot for detecting CAF cells expressing THY1 in sorted GBM samples.

[0026] Figure 6 For THY1 + The results of co-culturing CAF and HUVEC were used to detect cell migration and invasion.

[0027] Figure 7 For THY1 + The results of cell proliferation detection by co-culturing CAF and HUVEC-GFP with EdU staining were obtained.

[0028] Figure 8 For THY1 + Cell proliferation was detected by co-culturing CAF with HUVEC-GFP.

[0029] Figure 9 For THY1 +Results of co-culture of CAF and HUVEC to detect changes in angiogenesis.

[0030] Figure 10 THY1 was displayed + CAF relative abundance (A) and Kaplan–Meier survival curve (B). Detailed Implementation

[0031] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0032] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0033] Example 1

[0034] I. Single-cell sequencing analysis

[0035] 1. Analyze using the C-SEURAT R package.

[0036] Samples from 10 patients with glioblastoma (GBM) were analyzed using BD Rhapsody. TM Single-cell transcriptome libraries were constructed using a single-cell capture platform, and high-throughput sequencing was performed by BGI Genomics. A paired-end 150 bp (PE150) sequencing strategy was used to ensure complete transcript coverage and quantitative accuracy. Raw sequencing data underwent quality control and filtering, including removal of low-quality reads, adapter sequences, and potential cell debris and double-cell contamination. Subsequently, the data were standardized, reduced in dimensionality, and subjected to cluster analysis using the C-SEURATR analysis pipeline.

[0037] 2. Systematic annotation of 149,130 ​​single cells based on marker gene expression profiles.

[0038] High expression of PTPRZ1, GFAP, and FABP4 was used to label tumor cells; PTPRC (CD45) was used to label immune cells; OLIG2 was used to label oligodendrocyte lineage; co-expression of RGS5 and CD146 (MCAM) was used to label CAF cells; and endothelial cells were labeled with PECAM1 (CD31) and CDH5 (VE-cadherin). Visualization was performed using UMAP dimensionality reduction. Figure 1 A), which is divided into different cell types.

[0039] 3. Louvain Modular Clustering

[0040] Further analysis of the heterogeneity of CAF cells within the glioblastoma microenvironment was conducted, and all RGS5 cells were extracted separately from the annotated cell population. + CD146 + CAF cell subsets (n = 8,423 in total) were subjected to Seurat clustering at high resolution (resolution = 0.8). Data standardization and hypervariable gene screening were performed using SCTransform. Principal component analysis (PCA) was performed to select the top 20 significant principal components, followed by Louvain modular clustering based on the shared nearest neighbor (SNN) graph. Figure 1 B).

[0041] In five pericyte subclusters, cluster 2 was found to significantly overexpress the MCAF (mesenchymal cell-associated fibroblast) marker THY1 (CD90). Figure 1 C).

[0042] 4. Pathway enrichment

[0043] Using Seurat::FindAllMarkers (min.pct = 0.25, logfc.threshold = 0.5, Wilcoxon test), cluster-by-cluster screening was performed for significantly overexpressed genes. Figure 2 A), take the top 5 values ​​of avg log2FC as the feature markers for each subgroup.

[0044] The top 50 genes highly expressed by MCAF were entered into the public database Metascape to obtain the signaling pathways involved in regulation. Figure 2 B), enrichment of extracellular matrix composition and highly angiogenesis-related pathways.

[0045] II. Flow Cytometry Detection and Sorting Validation of THY1 + CAF cell expression and its correlation with blood vessels

[0046] 1. Staining verification of THY1 + Correlation between CAF and vascular expression

[0047] Multiple immunohistochemical staining was performed on paraffin sections of glioblastoma from the patient to detect THY1. +The expression of CAF in tissue sections was studied. Clinical GBM patient tissue samples used in this study were provided by the Department of Neurosurgery, First Affiliated Hospital of Chongqing Medical University. Freshly collected tissue samples were immediately placed on ice after ex vivo. Blood clots and electrocautery-damaged areas were removed, and the samples were cut into small pieces. One-third of the pieces were embedded in paraffin, one-third were embedded using OCT, and the remaining pieces were collected in EP tubes for protein and RNA sampling. The research methods used in this experiment were approved by the Ethics Committee of Chongqing Medical University.

[0048] The antibodies used were: COL1A1 antibody: Cell Signal technology, 72026S; PDGFRβ antibody: CellSignal technology, 3169; THY1 antibody: ABclonal, A12623; CD31 antibody: Proteintech, 11265-1-AP.

[0049] Following the instructions of the Aifang Biotechnology (China) Three-Label Four-Color Multiplex Fluorescence Staining Kit (catalog number AFIHC024), COL1A1, PDGFRB, THY1, and CD31 were stained and labeled on paraffin sections of GBM tissue. After mounting, the entire slide was scanned using an Axioscan 7 fully automated digital slide scanner.

[0050] result( Figure 3 This indicates that CAF cells expressing THY1 exist in GBM tissue and are spatially close to blood vessels.

[0051] 2. QuPath analysis of THY1 + Correlation between CAF and blood vessels

[0052] The stained image was segmented, and THY1 was selected. + PDGFRβ + Detect CD31 in the region. + PDGFRB + The cell count was summarized and statistically analyzed, and then plotted using GraphPad, such as... Figure 4 As shown: THY1 + The spatial expression of CAF cells is highly positively correlated with that of vascular endothelial cells, indicating that THY1 + CAF cells may affect the expression and function of blood vessels.

[0053] 3. Flow cytometry detection of THY1 in sorted GBM samples + CAF

[0054] Fresh GBM tissue samples taken post-surgery were washed twice with PBS buffer in the tumor center area on a cell culture table to remove surface blood. Blood clots and electrocautery charred areas were removed using sterile forceps. The tissue was then vertically minced with a sterile blade. A tissue digestion solution was prepared: 9 ml DMEM high-glucose medium + 1 ml 10 mg / ml collagenase + 200 μL 200× DNase. The tissue fragments were resuspended in the digestion solution and digested on a shaker at 37°C for 1 hour. Digestion was terminated with 20 ml DMEM high-glucose medium. Undigested tissue was removed by filtration through a 70 μm cell sieve. The tissue was centrifuged at 280 g for 10 min at 4°C, and the supernatant was removed. A 30% percolate solution was prepared: 3 ml 100% percolate + 7 ml PBS buffer. The pellet was resuspended in 3 ml of the 30% percolate solution, and then 4 ml of PBS buffer was slowly added to allow for separation. The pellet was then centrifuged at 800 g for 20 min at 4°C on a low speed. Remove the lipids from the intermediate layer, then remove the supernatant. Resuspend the precipitate in 1 ml of 1× Lysine Resin solution. After 3 min, add 6 ml of PBS buffer to stop the staining. Centrifuge at 300 g for 5 min at 4°C and remove the supernatant. Resuspend the precipitate in 300 μl of PBS buffer. Add anti-CD45, CD31, THY1, CD140b, Ep-CAM, and 7AAD at a 1:100 ratio for 20 min of flow cytometry staining. Then add 3 ml of PBS buffer to stop the staining. Centrifuge at 300 g for 5 min at 4°C and remove the supernatant. Resuspend the precipitate in 3-5 ml of PBS buffer and perform flow cytometry to sort CD45. - CD31 - THY1 + PDGFRB + Cell populations were sorted and centrifuged at 300g for 5 min at 4℃ to collect the precipitate, which was then transferred to 24-well plates for culture. Flow cytometry antibody sources: CD140b: BD pharmingen, 564124; CD31: Invitrogen, 11-0319-42; THY1: Invitrogen, 2873293; Ep-CAM: BioLegend, 324204; CD45: BioLegend, 304006; 7AAD: BioLegend, 420404.

[0055] result( Figure 5 This indicates that the cells sorted by flow cytometry are CAF cells expressing THY1.

[0056] III. Cellular Experiment Validation of THY1 + Effects of CAF on vascular function

[0057] 1. THY1 + CAF and HUVEC co-culture assays for cell migration and invasion

[0058] Prepare CAF and HUVEC (human umbilical vein endothelial cells) in 6cm culture dishes. HUVECs were starved 12 hours prior to culture by changing the medium to serum-free medium. The medium was removed, and the surface residual medium was washed with 1ml PBS buffer. 500ul of trypsin was added for 1-2 minutes of digestion. Digestion was stopped by adding 1.5ml of high-glucose DMEM medium. The cells were centrifuged at 900rpm for 5 minutes, resuspended in 1ml DMEM, and 10ul of the suspension was added to 90ul PBS and mixed thoroughly. Cells were counted using a cell counting chamber. 15,000 CAF cells were seeded in 24-well plates, and 10,000 HUVEC cells were seeded in 8μm chambers. For the invasion group, 60ul of Matrigel (pre-diluted 1:8 with DMEM) was added to the upper chamber. 200μl of DMEM was added to the upper chamber, and 600μl of ECM to the lower chamber. The cells were incubated for 16 hours. After removing the fluid from the lower chamber, wash once with 1 ml of PBS buffer. At this point, HUVECs have penetrated from the upper chamber to the membrane of the lower chamber. Add 1 ml of 4% PFA (paraformaldehyde) for fixation at room temperature for 20 min, then wash once more with 1 ml of PBS buffer. Add 1 ml of crystal violet for staining at room temperature for 30 min, then gently wash the chamber with ddH2O, retaining only cell staining. Gently wipe the stain from the upper chamber with a cotton swab and allow to air dry. Images were taken using a Zeiss Axio Imager 2 research-grade upright microscope, and image counts were performed.

[0059] result( Figure 6 This indicates that THY1 + CAF co-culture treatment significantly promoted the migration and invasion of HUVECs.

[0060] 2. THY1 + CAF was co-cultured with HUVEC-GFP, and cell proliferation was detected by EdU staining.

[0061] Cell digestion and cell counting were performed as described above. Cell slides were placed in 24-well plates. 5000 HUVEC-GFP cells were placed in each well (500 μL ECM). The cell suspension was mixed and spread onto the slides, then cultured for 48 h. The supernatant was removed, and the cells were washed once with PBS buffer, fixed with 4% PFA at room temperature for 10 min, washed three times with PBS buffer for 5 min each time, blocked with 5% goat serum for 30 min, and punched with 0.3% Triton for 10 min. Staining was performed according to the EdU kit procedure. After DAPI staining for 10 min, the slides were mounted. Images were taken using a Zeiss Axio Imager 2 research-grade upright microscope. EdU was statistically analyzed using ImageJ. + Number of HUVEC-GFP cells.

[0062] result( Figure 7 This indicates that THY1 +Co-culturing CAF with HUVEC-GFP resulted in EdU... + There was no significant difference in the number of HUVECs.

[0063] 3. THY1 + CAF was co-cultured with HUVEC-GFP, and cell proliferation was detected by photographing.

[0064] Cell digestion and cell counting were performed as described above. 5000 HUVEC-GFP cells were mixed and seeded into 6-well plates, with 1.5 ml of ECM per well. Incucyte software was set to photograph the plates every 24 hours to record changes in cell count.

[0065] result( Figure 8 This indicates that, compared to THY1 + In CAF co-culture, the number of HUVEC cells was not significantly different from that of the control group.

[0066] 4. THY1 + Co-culturing CAF and HUVECs to detect angiogenesis changes

[0067] Cell digestion and cell counting were performed as described above. HUVEC cells were pre-treated with serum-free medium, starved for 4 hours, and then 50 μl of matrix gel was seeded into 96-well plates and incubated at 37°C for 30 minutes. 5000 HUVEC cells were prepared, mixed thoroughly, and seeded onto the matrix gel, with 100 μl of ECM per well, and incubated at 37°C for 5 hours. Images were taken using a Zeiss Axio Imager 2 research-grade upright microscope. ImageJ quantitative statistical analysis was performed.

[0068] result( Figure 9 This indicates that, compared to THY1 + Co-culture with CAF significantly promotes angiogenesis.

[0069] IV. Database Analysis THY1 + Expression and prognostic role of CAF in patient tissues

[0070] Download 170 bulk RNA-seq TPM matrices from TCGA-GMB, upload them to CIBERSORTx (LM22 signature, 100 permutations) for deconvolution, and extract THY1. + Relative abundance of CAF. The abundance of this subgroup increased significantly with increasing WHO classification, with an average increase of 1.9-fold between classification IV and classification II (p < 2.2 × 10⁻⁶). -16 (Mann–Whitney U test) Figure 10 A).

[0071] THY1 obtained from CIBERSORTx+ CAF abundance was divided into high / low groups based on median value, and the data were pooled with the overall survival data of patients corresponding to TCGA, and the Kaplan-Meier survival curves were analyzed using the log-rank test. THY1 + Patients with high CAF levels had significantly shorter survival (median OS 11.4 vs 18.7 months, p = 3.1 × 10⁻⁶). -5 () Figure 10 B), indicating that this subgroup is an independent adverse prognostic factor for GBM.

[0072] V. Analysis and Summary

[0073] This study, through a multi-dimensional and multi-platform systematic investigation, identified and functionally validated THY1 in the microenvironment of glioblastoma (GBM). + The discovery of CAF, a key pro-angiogenic cell subset, reveals its potential as a diagnostic biomarker and therapeutic target for GBM. Single-cell transcriptome analysis showed that THY1 was specifically and highly expressed in the GBM pericyte subset, and its marker genes were significantly enriched in the angiogenesis pathway, positively correlated with the expression of endothelial markers CD34 and PECAM1, as well as the pro-angiogenic factor TGFB1. Multiplex immunofluorescence confirmed the expression of THY1. + CAF and CD31 + Vascular spatial colocalization (r=0.77, P<0.0001), CD45 was successfully sorted by flow cytometry. - CD31 - THY1 + PDGFRβ + Cell populations were analyzed and their fibroblast characteristics were verified. Functional experiments showed that THY1... + Co-culture of CAF and HUVEC significantly promoted endothelial cell migration, invasion, and angiogenesis, but did not affect cell proliferation, suggesting that it regulates angiogenesis by enhancing endothelial motility rather than directly promoting proliferation. Clinical data analysis showed that THY1 + CAF abundance increased with increasing WHO classification (1.9-fold increase in classification IV vs. classification II, p < 2.2 × 10⁻⁶). -16 Patients with high abundance had significantly shorter overall survival (median OS 11.4 vs 18.7 months, p=3.1×10⁻⁶). -5 ), which is an independent adverse prognostic factor.

[0074] In summary, THY1 + CAF is a GBM-specific CAF subgroup that can serve as a diagnostic biomarker and prognostic indicator, while also providing potential new targets for anti-angiogenic therapy.

Claims

1. A diagnostic marker for glioblastoma, characterized in that: The biomarker is the THY1-positive cancer-associated fibroblast subset THY1. + CAF.

2. A prognostic marker for glioblastoma, characterized in that: The biomarker is the THY1-positive cancer-associated fibroblast subset THY1. + CAF.

3. The detection reagent for the marker as described in claim 1 or 2.

4. The detection reagent according to claim 3, characterized in that: The detection reagent includes an anti-THY1 antibody.

5. The detection reagent according to claim 4, characterized in that: The detection reagents also include flow cytometry reagents.

6. A kit for diagnosing glioblastoma or predicting the prognosis of glioblastoma, characterized in that: The kit includes the detection reagents as described in any one of claims 3 to 5.

7. The use of a biomarker in the preparation of products for diagnosing glioblastoma or predicting the prognosis of glioblastoma, wherein the biomarker is a THY1-positive cancer-associated fibroblast subset, THY1. + CAF.

8. The application according to claim 7, characterized in that: THY1 in tumor tissue of glioblastoma patients + CAF levels were significantly higher than in normal tissues; high THY1 + CAF predicts a worse prognosis for patients. 9.THY1 + CAF is used in any of the following: (1) Application as a target in screening anti-angiogenic drugs in the GBM tumor microenvironment; (2) Application as a target in screening drugs for the treatment of glioblastoma.

10. The application according to claim 9, characterized in that: THY1 + CAF subgroups are highly colocalized with tumor blood vessels in spatial distribution, and their abundance is significantly positively correlated with blood vessel density.

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