Application of POSTN + fibroblast subpopulation in screening and preparing medicine for improving immune checkpoint blocking treatment drug resistance of head and neck squamous cell carcinoma patient

Abstract

The invention discloses an application of a POSTN + fibroblast subgroup in screening and preparing a medicine for improving immune checkpoint blocking treatment drug resistance of a patient with head and neck squamous cell carcinoma. The action mechanism of small extracellular vesicles secreted by POSTN < + > fibroblasts in head and neck squamous cell carcinoma is studied, two fibroblast subtypes of POSTN <-> and POSTN < + > are identified through single-cell RNA sequencing, and the POSTN < + > subtype highly expresses FAP and other markers and is related to immune checkpoint blocking treatment drug resistance. Research finds that POSTN is enriched on the surface of sEVs secreted by POSTN + fibroblasts, and macrophages can be driven to be polarized to an M2 type by activating a BMP4 / BMPR2 / Smad signal channel. High expression of POSTN and FAP indicates that ICB treatment response of a patient is poor, and POSTN and FAP can be used as predictive biomarkers. The pathway can be blocked by down-regulating the POSTN, the ICB curative effect is improved, and a new target and a new strategy are provided for treatment of head and neck squamous cell carcinoma.

Description

Technical Field

[0001] This application belongs to the field of tumor microenvironment and immune regulation, and relates to POSTN. + Application of fibroblast subsets in screening and preparing drugs to improve resistance to immune checkpoint blockade therapy in patients with head and neck squamous cell carcinoma. Background Technology

[0002] Head and neck squamous cell carcinoma (HNSCC) is a common malignant tumor, accounting for more than 90% of all head and neck cancer cases. While early-stage HNSCC patients generally have a better prognosis, those with advanced-stage HNSCC often have a significantly worse prognosis. Although immunotherapy—especially immune checkpoint blockade (ICB) therapy targeting PD-1 / PD-L1—has improved survival in patients with recurrent / metastatic HNSCC, the molecular mechanisms of ICB resistance remain unclear. Increasing evidence suggests that the tumor immune microenvironment is a key factor determining treatment efficacy.

[0003] The tumor microenvironment (TME) plays a crucial role in tumor growth and progression by regulating the interactions between cancer cells and matrix components, including various cellular and extracellular molecules. As a core component of the TME, cancer-associated fibroblasts (CAFs) promote immune escape and treatment resistance by remodeling the extracellular matrix (ECM) and secreting growth factors / cytokines. CAFs not only affect the infiltration of immunosuppressive cells but also lead to CD8+ inflammatory responses. + T cell exhaustion, regulation of natural killer (NK) cell activation, and alteration of macrophage polarization. Furthermore, CAF plays a crucial role in constructing the immunosuppressive TME. Targeting STAT3 or IL-6 can block this signaling pathway, thereby reprogramming the characteristics of the immunosuppressive TME. Recent research suggests that CAF may be associated with reduced efficacy of immune checkpoint blockade (ICB) therapy.

[0004] Studies have confirmed that cancer-associated fibroblasts (CAFs) exhibit high heterogeneity across various cancers, with certain subtypes associated with resistance to immune checkpoint blockade (ICB). ICB-resistant CAF subtypes may differ across different solid tumor types. FAP + CAFs can be divided into eight subsets, among which the TGF-β signaling pathway and extracellular matrix-associated subsets mediate primary immunotherapy resistance through a PD-1 / CTLA4-dependent Treg feedback loop. In non-small cell lung cancer (NSCLC), the Meflin-expressing CAF subset is associated with enhanced ICB efficacy and prolonged survival because this pro-immunogenic subset promotes CD4+ expression. + T-cell infiltration and angiogenesis in the immunosuppressive tumor microenvironment (TME). Conversely, LRRC15 + CAF mediates CD8+ T-cell dysfunction and resistance to immune checkpoint inhibitors are associated with poorer immunotherapy outcomes. Multi-cancer meta-analysis identified six conserved pan-CAF subtypes with unique molecular signatures (CXCL12, CXCL14, IL6) and transcriptional regulators (MEF2C, TWIST1, NR1H3, RELB, FOXM1), revealing the specific roles of these subtypes in tumor progression, anti-PD-1 / PD-L1 resistance, and therapeutic potential.

[0005] Macrophages and fibroblasts are widely distributed in various tissues. Tissue-resident macrophages maintain homeostasis through phagocytosis, coordinate immune responses, and promote tissue repair. Based on their functional differentiation status, macrophages are mainly divided into M1 and M2 types. Pro-inflammatory factors (such as TNF-α and IL-6) or bacterial products (such as LPS) induce M1 polarization, while immunomodulatory cytokines (such as TGFβ and IL-10) promote M2 polarization. M2 macrophages secrete pro-angiogenic factors (VEGF) and tissue remodeling enzymes (MMPs), thereby accelerating tumor progression; while M1 macrophages release anti-angiogenic factors (such as IL-12 and CXCL10) and exhibit anti-tumor effects.

[0006] Postiostrin (POSTN) is a secreted stromal cell protein that plays a crucial role in tumor cell proliferation, angiogenesis, and metastasis in various cancers. Studies have found that upregulation of POSTN is associated with enhanced cell migration, chemotherapy resistance, and poor prognosis in various cancers. Previous research has shown that cancer-associated fibroblasts (CAFs) in healthy gingival tissue secrete significantly more POSTN than normal fibroblasts (NFs). POSTN secreted by glioma stem cells can recruit M2 tumor-associated macrophages (TAMs), and TAM density is positively correlated with POSTN expression. Similarly, high POSTN levels in ovarian cancer ascites are associated with CD163... + TAM infiltration is associated with decreased relapse-free survival. POSTN + CAFs are a key driver of immunotherapy resistance, acting through the IL-6 / STAT3 signaling pathway and SPP1. + Macrophages work synergistically to create an immunosuppressive microenvironment that inhibits T cell infiltration, thereby weakening the response to immune checkpoint blockade (ICB). Furthermore, POSTN + Myofibroblast CAFs (myCAFs), as a pro-tumor subset, are associated with SPP1. + Macrophage colocalization remodels the extracellular matrix, drives immunosuppression through T cell rejection / exhaustion mechanisms, and is associated with disease progression and poor prognosis.

[0007] Extracellular vesicles (EVs) are a class of nano- to micron-sized particles enclosed in a lipid bilayer, naturally present in all cell types. Their main types include: exosomes derived from endosomes (endosome-derived vesicles formed through the fusion of multiple vesicle bodies and released into the extracellular space), microvesicles (submicron-sized particles derived from the cell membrane), and apoptotic bodies. Functionally, EVs can transport substances to target sites, achieving long-distance action. Based on size, EVs can be classified as: <200 nm are called small EVs (sEVs), and 200-1000 nm are classified as large EVs. Exosomes secreted by human cardiac explant-derived progenitor cells (CPCs) express a short POSTN isoform. Lysyl oxidase (LOX) binds to POSTN and localizes to the surface of CAF-derived sEVs (CAFs: cardiac adipose cells). However, how POSTN-rich CAF sEVs regulate macrophage polarization remains unclear.

[0008] In summary, the CAF subtypes in HNSCC (especially POSTN) + CAFs (corporeal angiotensin-converting enzymes) construct immunosuppressive TMEs by regulating macrophage polarization, which is a significant cause of ICB resistance; however, targeted intervention strategies against this mechanism remain lacking. Therefore, elucidating POSTN... + The molecular mechanisms by which CAFs and their secreted EVs regulate macrophage polarization, and the development of interventions targeting this pathway, are of great clinical significance for improving the response of ICB treatment in HNSCC patients. Summary of the Invention

[0009] In view of the shortcomings of the prior art, the purpose of this application is to provide POSTN. + Application of fibroblast subsets in screening and preparing drugs to improve resistance to immune checkpoint blockade therapy in patients with head and neck squamous cell carcinoma.

[0010] This invention integrates single-cell RNA sequencing data from two groups of head and neck squamous cell carcinomas (HNSCC) and successfully identifies two different POSTNs. - Fibroblast subtypes and POSTN +Fibroblast subtypes. Further studies revealed significant co-localization of fibroblast activation protein (FAP) and POSTN in the HNSCC tissue matrix. Notably, the expression levels of FAP and POSTN were significantly positively correlated with the degree of macrophage infiltration. Mechanistically, POSTN-enriched substances secreted by cancer-associated fibroblasts (CAFs) can drive macrophage differentiation towards M2 polarization. When macrophages were treated with POSTN-silenced CAF sEVs, the expression level of bone morphogenetic protein 4 (BMP4) was significantly reduced, further confirming that BMP4 promotes M2 polarization through the BMPR2 / Smad signaling pathway. Finally, combining single-cell RNA sequencing data with the results of the Cancer Genome Atlas (TCGA)-HNSCC analysis, this invention elucidates the crucial role of POSTN in regulating macrophage M2 polarization.

[0011] This invention provides the following technical solutions: Technical Solution 1: Application of BMP4 protein and its encoding gene in screening and preparing drugs to improve resistance to immune checkpoint blockade therapy in patients with head and neck squamous cell carcinoma.

[0012] The BMP4 drives macrophages to M2 polarization by activating the BMPR2 / Smad signaling pathway, and the characteristic of the head and neck squamous cell carcinoma patients is that macrophages are abnormally polarized to M2.

[0013] The drug described above improves resistance to immune checkpoint blockade therapy in patients with head and neck squamous cell carcinoma, thereby enhancing sensitivity and efficacy of immune checkpoint blockade therapy.

[0014] Technical Solution 2, POSTN + Application of fibroblast subsets in screening and preparing drugs to improve resistance to immune checkpoint blockade therapy in patients with head and neck squamous cell carcinoma.

[0015] Furthermore, the POSTN + Fibroblasts secrete small extracellular vesicles, the surface of which is enriched with POSTN protein; these small extracellular vesicles regulate macrophage polarization by activating the BMP4 / BMPR2 / Smad signaling pathway, thereby promoting the upregulation of BMP4 expression in macrophages.

[0016] Furthermore, the POSTN +The fibroblast subpopulation originated from head and neck squamous cell carcinoma. This subpopulation highly expressed ECM-associated CAF-related proteins (eCAF) and antigen-presenting CAF-related proteins (apCAF). The eCAF-related proteins include FAP, FN1, SFRP2, THY1, COL1A1, FBLN1, and LUM, while the apCAF-related proteins include CD74, HLA-DRA, HLA-DRB5, HLA-DRB1, and HLA-DPA1. This subpopulation was identified from single-cell RNA sequencing data of head and neck squamous cell carcinoma using Seurat clustering and UMAP clustering analysis, and it also highly expressed POSTN.

[0017] Furthermore, with POSTN + POSTN associated with fibroblast subsets - The fibroblast subset highly expressed myofibroblast CAF-related proteins (myCAF) and inflammation CAF-related proteins (iCAF); the myCAF-related proteins include ACTA2, MYH11, TAGLN, PDGFA, MCAM, and MYLK, and the iCAF-related proteins include IL6, CXCL12, C3, and CFD; this cell subset was identified from single-cell RNA sequencing data of head and neck squamous cell carcinoma using Seurat clustering and UMAP clustering analysis, and it did not express POSTN.

[0018] Furthermore, the drug contains POSTN + Small extracellular vesicles secreted by fibroblasts, and pharmaceutically acceptable excipients.

[0019] Furthermore, the dosage form of the drug is selected from: injection or oral preparation; the pharmaceutically acceptable excipient is one or more of physiological saline, phosphate buffer, and dextran solution.

[0020] Furthermore, POSTN is provided. + Application of fibroblast subsets in the preparation of a diagnostic kit for resistance to immune checkpoint blockade therapy in patients with head and neck squamous cell carcinoma. The kit is used to detect a combination of biomarkers for head and neck squamous cell carcinoma, namely POSTN and FAP, wherein high expression of POSTN and high expression of FAP indicate poor response to ICB treatment.

[0021] The expression levels of POSTN and FAP were obtained by detecting fibroblasts in the intermediate region of head and neck squamous cell carcinoma tissue, and the two showed co-expression characteristics in this region.

[0022] Compared with the prior art, this application has at least the following improvements and beneficial effects: (1) This invention clarifies the role of a specific CAF subtype for the first time, and identifies POSTN through single-cell sequencing. + and POSTN - Fibroblast subtypes, clarify POSTN + Subtypes with high expression of biomarkers such as FAP are directly associated with ICB treatment resistance in patients with head and neck squamous cell carcinoma, providing a new explanation for the ICB resistance mechanism.

[0023] (2) This invention reveals the sEV-mediated cell communication mechanism and confirms POSTN + POSTN is enriched on the surface of sEVs secreted by fibroblasts, which drive macrophage M2 polarization through the BMP4 / BMPR2 / Smad pathway, elucidating a new pathway for the interaction between CAF and macrophages.

[0024] (3) Discover novel predictive biomarkers and determine that co-expression of POSTN and FAP can be used as a predictive indicator of ICB treatment response in patients with head and neck squamous cell carcinoma, providing a precise basis for the selection of clinical treatment plans.

[0025] (4) It provides potential therapeutic targets, confirms that downregulating POSTN can block the immunosuppressive pathway and enhance the efficacy of ICB, laying the foundation for developing targeted therapy strategies for head and neck squamous cell carcinoma, and has important clinical translational value. Attached Figure Description

[0026] Figure 1 Single-cell sequencing reveals POSTN in head and neck squamous cell carcinoma + With POSTN - Fibroblast subsets; among which Figure 1 A is an integrated head and neck squamous cell carcinoma single-cell sequencing dataset (GSE103322 and GSE139324). Based on known marker genes, Seurat clustering UMAP dimensionality reduction analysis identified five cell types, including B cells, epithelial cells, fibroblasts, myeloid cells, and T cells. Figure 1 B is a violin plot showing the expression of POSTN in five cell types; Figure 1 C represents the re-clustering of Fibroblasts using UMAP dimensionality reduction analysis, which identified nine cell types, including clusters 0-8. Figure 1 D is a violin plot showing the expression of POSTN in 9 cell types; Figure 1 E is used to determine POSTN using the POSTN score threshold method. - fibroblasts and POSTN + fibroblasts subgroup; Figure 1F is a heatmap showing known apCAF, iCAF, and mCAF subgroup markers in POSTN. - fibroblasts and POSTN + Expression in fibroblasts; Figure 2 POSTN and FAP expression were positively correlated with the efficacy of ICB treatment in patients with head and neck squamous cell carcinoma; among them Figure 2 Diagram A shows POSTN, FAP, THY1, LUM, and SFPR2 in POSTN. - fibroblasts and POSTN + Expression levels in fibroblasts subsets; Figure 2 B indicates that POSTN expression is positively correlated with FAP expression in the TCGA-HNSCC database. Figure 2 C indicates that POSTN and FAP expressions are correlated with TIDE scores; Figure 2 D represents H&E staining and the expression levels of POSTN and FAP in oral squamous cell carcinoma and normal mucosal tissue; Figure 3 The expression levels of POSTN and FAP were positively correlated with the degree of macrophage infiltration; among them Figure 3 A study found in the TCGA-HNSCC database that POSTN and FAP were correlated with macrophage infiltration; Figure 3 B indicates that POSTN and FAP are positively correlated with CD163 expression in the TCGA-HNSCC database; Figure 3 C indicates that POSTN and CD163 are co-localized and expressed in the stroma of oral squamous cell carcinoma. Figure 4 POSTN is located on the surface of the CAF-secreted sEVs membrane; among which Figure 4 A is a Western blot analysis of the expression level of POSTN in CAFs, NFs and their sEVs; Figure 4 B represents the detection of POSTN expression levels in CAF-secreted sEVs using sucrose density gradient centrifugation. Figure 4 C indicates that POSTN was located on the surface of the CAF secreted sEVs membrane by immunogold labeling. Figure 4 D and 4E show that ELISA detection revealed an increase in the number of sEVs, and a corresponding increase in the POSTN content on the sEVs membrane surface (4D); after antibody blocking, the POSTN content on the sEVs membrane surface decreased significantly. Figure 5 To ensure that downregulating POSTN does not affect macrophage uptake of CAF and secretion of sEVs; among which Figure 5A is the Western blot analysis of POSTN expression levels in CAFs and sEVs transfected with shNC and shPOSTN; Figure 5 B represents the concentration and particle size distribution of secreted sEVs from CAFs transfected with shNC / shPOSTN, as determined by nanoflow cytometry. Figure 5 C shows the typical morphology of THP-1-derived macrophages induced by PMA; Figure 5 D represents the mRNA expression level of CD68 detected by qRT-PCR; Figure 5 E represents the flow cytometry analysis of macrophage uptake of CAFs (transfected with shNC / shPOSTN). Secretion of sEVs (PKH26-labeled) via endocytosis; Figure 6 To downregulate POSTN and inhibit CAF secretion, sEVs induce macrophage polarization; among which Figure 6 A represents the detection of mRNA expression levels of CD80, CD86, CD163, and CD206 in CAF (transfected with shNC / shPOSTN) sEVs-induced macrophages by qRT-PCR. Figure 6 B shows the expression levels of FITC-CD163 and CD206 in macrophages as detected by flow cytometry. Figure 6 C represents the Western blot analysis of the expression levels of CD80, CD86, CD163, and CD206 proteins in macrophages; Figure 7 Transcriptomic sequencing revealed the signaling pathway by which POSTN-downregulated CAF secretion of sEVs induces macrophage polarization; among which Figure 7 A shows the distribution of macrophage samples induced by CAF-S6 secreted sEVs after transfection with shNC / shPOSTN via PCA. Figure 7 B is a Venn diagram showing the number of differentially enriched genes in macrophages (shNC sEVs vs CTRL; shPOSTN sEVs vs shNC sEVs); Figure 7 C is a heatmap showing the top differentially expressed genes in each treatment group; Figure 7 D, the volcano diagram, shows that shPOSTN sEVs induce significantly lower BMP4 expression in macrophages than shNC sEVs; Figure 7 E indicates GSEA enrichment, showing enrichment of the hallmark pathway of shPOSTN / shNC sEVs-induced macrophages; Figure 7 F,7G enrichment of GO and KEGG reveals key DEG-related pathways and significant KEGG pathways. Figure 8 The BMP4 / BMPR2 / Smad pathway is involved in regulating macrophage polarization induced by CAF secretion of sEVs downregulated by POSTN; among which Figure 8 A represents the qRT-PCR detection of BMP4 mRNA expression levels in macrophages induced by sEVs secreted by CAFs transfected with shNC / shPOSTN; Figure 8 B represents the mRNA expression levels of TNF-α, IL-6, TGF-β, and IL-10 in macrophages induced by different concentrations of BMP4 factor; Figure 8 C represents the expression levels of CD163 and CD206 in macrophages induced by 100 ng / mL BMP4, as detected by flow cytometry. Figure 8 D represents Western blot analysis of the expression levels of MPR2, pSmad1 / 5 / 9, and Smad5 / 9 proteins in macrophages induced by 100 ng / mL BMP4. Figure 8 E represents the Western blot analysis of the expression levels of pSmad1 / 5 / 9, Smad5 / 9, CD163, and CD206 proteins in macrophages in both the LDN193189 inhibitor and control groups. Detailed Implementation

[0027] Experimental methods involved in the embodiments: (1) Data acquisition, quality control and processing Single-cell RNA sequencing (scRNA-seq) data were obtained from two datasets: GSE103322 (Cell, 2017, 171:1611-1624.e24.) and GSE139324 (Nat Commun, 2021, 12:3349.; Immunity, 2020, 52:183-199.e9.). Both datasets were derived from the Gene Expression Integrated Database (GEO, https: / / www.ncbi.nlm.nih.gov / geo / ). The GSE103322 dataset contains samples from 18 patients with head and neck squamous cell carcinoma (HNSCC) (5902 single cells in total). The GSE139324 dataset contains 26 samples, all of which are paired samples of peripheral blood mononuclear cells and tumor-infiltrating immune cells from HNSCC patients (18 HPV- and 8 HPV-). +Six cases involved peripheral blood mononuclear cells from healthy donors, and five cases involved resident immune cells from healthy donor tonsil tissue (a total of 131,224 single cells). By merging the two datasets, a total of 136,206 single cells were obtained. Data analysis was performed using the R language package, specifically using Seurat's analysis tool (Cell, 2021, 184:3573-3587.e29.). To ensure data integrity, standard quality control measures were implemented, specifically: nFeature-RNA <300 and <7000, %mt <10%, and the top 2000 genes with the largest variations were selected. Nonlinear dimensionality reduction was performed using the Uniform Manifold Approximation and Projection (UMAP) method, with 15 principal components and a resolution of 0.5. Transcriptome FPKM (Fragments Per Million Mapped Fragments per Thousand Bases) data from TCGA-HNSCC patients were downloaded from the database (https: / / www.cbioportal.org / ). To eliminate batch effects within each subgroup, Harmony software (version 0.1.0) was used in this invention. Subsequent re-analysis of cells in each group followed the standard Seurat protocol. Immune infiltration levels were quantified using single-sample gene set enrichment analysis (ssGSEA), and the relevant data were downloaded from the database (http: / / cis.hku.hk / TISIDB / download.php).

[0028] (2) Cell clustering and annotation This invention uses Seurat software for cell cluster analysis. To reduce the dimensionality of the single-cell RNA sequencing dataset, principal component analysis (PCA) was performed on the top 2000 highly variable genes with the largest variance. The FindClusters function in Seurat software was used to identify major cell subpopulations at the default resolution setting (res=0.7). All cell markers were annotated according to previously reported conventional markers, specifically including: epithelial markers (EpCAM, SFN, KRT14, KRT5); fibroblast markers (FN1, DCN, COL1A1, COL1A2, COL3A1, COL6A1); T cell markers (CD2, CD3D, CD3E, CD3G); B cell markers (CD19, CD79A, MS4A1, JCHAIN, MZB1); and myeloid markers (CD68, LYZ, CD14, IL3RA, LAMP3, CLEC4C, TPSAB1). Subsequently, the fibroblasts containing 1761 single cells were re-clustered into POSTN. + Fibroblasts and POSTN - Two subpopulations of fibroblasts were identified for further analysis.

[0029] (3) Bioinformatics analysis of the TCGA database Gene expression data from TCGA-HNSCC patients (https: / / portal.gdc.cancer.gov / projects / TCGA-HNSC) were downloaded via the TCGA biolink R package. Transcription count per million reads (TPM) was converted to a Log2 value (TPM). + 1) For subsequent analysis. High-expression and low-expression groups were divided based on median values ​​of POSTN and FAP. The Tumor Immune Dysfunction and Rejection (TIDE) algorithm was used to predict the potential response to immune checkpoint blockade (ICB). Furthermore, ssGSEA was used to quantify immune infiltration levels. To further evaluate the impact of POSTN and FAP expression levels and immune infiltration levels, ssGSEA was also used for quantitative analysis.

[0030] (4) Immunohistochemistry We analyzed 4-micron thick paraffin-embedded tumor tissue sections from 40 patients with oral squamous cell carcinoma (OSCC) who were unsuitable for surgical resection. The clinical and histopathological characteristics of these head and neck squamous cell carcinoma cases are recorded in Table S1b. Diagnosis was made using hematoxylin-eosin (H&E) staining, with 14 normal oral mucosa tissues serving as a control group. Immunohistochemical detection was performed using the SPlink assay kit (SP-9000, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.). After dewaxing with xylene and hydration with ethanol, endogenous peroxidase activity was blocked with methanol containing 3% hydrogen peroxide, and non-specific binding sites were blocked with 10% goat serum. Anti-FAP antibody (catalog number ab314075, 1:200; Abogen Biosciences, Cambridge, UK) and anti-POSTN antibody (catalog number ab14041, 1:100, Abogen Biosciences) were incubated overnight at 4°C. The immunoreaction was performed by staining with 3,3'-diaminobenzidine, and cell nuclei were counterstained with hematoxylin. Negative control experiments were performed using PBS instead of the primary antibody. Air-dried sections were observed using a Leica Thunder DM6B optical microscope, and the integrated optical density and area of ​​the target region were measured using Image Pro® Plus 6.0 software. After calculating the average density for each field of view, the average of at least 10 fields of view was taken as the average density of the case.

[0031] (5) Immunofluorescence Four sections of head and neck squamous cell carcinoma (HNSCC) were dewaxed and hydrated. The heat antigen retrieval method was used, with sections incubated at 95°C for 5 minutes in pH 6.0 citrate buffer (catalog number C1010, Beijing Solarbio Science & Technology Co., Ltd.). Non-specific binding sites were blocked using 10% QuickBlock blocking buffer (Shanghai Beyotime Biotechnology Co., Ltd.). The sections were then incubated overnight at 4°C with anti-POSTN antibody (catalog number ab14041, 1:100 dilution, Abogen Biosciences) and CD163 antibody (catalog number sc-33715, 1:1000 dilution, Santa Cruz Biotechnology Co., Ltd.). The following day, the cells were washed with 1×TBST solution for 30 minutes, and then incubated for 60 minutes with either goat anti-rabbit IgG H&L-AF488 secondary antibody (catalog number ab150077, 1:200 dilution, Abogen) or goat anti-mouse IgG H&L-AF594 secondary antibody (catalog number ab150116, 1:200 dilution, Abogen). The nuclei were counterstained with DAPI (catalog number 1:3000, Thermo Fisher Scientific). Finally, the stained sections were observed using an optical microscope (Leica Thunder DM6B), and images were recorded for analysis.

[0032] (6) Cell culture Primary CAF cells (CAF-S5 and CAF-S6) derived from OSCC and normal fibroblasts (NFs) derived from healthy gingiva were cultured in DMEM / F12 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, ScienCell, Carlsbad, CA, USA). THP-1 cells were purchased from iCell Bioscience and cultured in RIPA 1640 medium (Gibco) supplemented with 10% FBS (ScienCell). All cell cultures were supplemented with 100 U / mL penicillin and 100 U / mL streptomycin (Gibco) and maintained at a constant temperature of 37°C and 5% CO2 humidity.

[0033] (7) Stable transfection of POSTN POSTN was silenced using CAF cells containing concentrated lentiviral particles (control shRNA or shPOSTN). 8 mg / mL Polybrene (catalog number TR-1003; Sigma-Aldrich, St. Louis, Missouri, USA) was added to the transfection medium, and the cells were cultured overnight. Forty-eight hours after transfection, stable clones were selected using medium containing 2 mg / mL puromycin (Sigma-Aldrich) for two weeks.

[0034] (8) PMA induces THP-1 to enter macrophages THP-1 cells (2 × 10^6 cells) were seeded in T25 culture flasks and cultured for 48 hours in RPMI 1640 medium containing 50 ng / mL phorbol 12-myristate 13-acetate (PMA, catalog number P8139, Sigma-Aldrich). The cells were then transferred to fresh RPMI medium and cultured for another 24 hours to stabilize their growth.

[0035] (9) Cell therapy To verify the mechanism by which BMP4 promotes macrophage polarization towards the M2 phenotype, researchers seeded THP-1 cells (2 × 10^6 cells) in 6-well plates and induced differentiation by treating with 50 ng / mL PMA for 48 hours. After cell stabilization, control and experimental groups were set up: the control group received no stimulation, while the experimental group was stimulated with 50 ng / mL or 100 ng / mL recombinant human BMP4 (HY-P7007, Medicilon Shanghai) for another 48 hours. The expression levels of the M2 phenotype markers CD163 and CD206 were then detected by flow cytometry. To further verify the role of the Smad signaling pathway, 10 µL of the DN193189 inhibitor (HY-12071, Medicilon) was added to each well during BMP4 stimulation for 48 hours. Finally, cellular proteins were collected for Western blot analysis to assess the expression of markers related to Smad and M2 phenotype polarization.

[0036] (10) Isolation, characterization and labeling of sEVs The isolation method for small cell exosomes (sEVs) was as described in previous literature (Int. J. Oral. Sci. 2023, 15, 32.). First, fetal bovine serum (FBS) de-EV was obtained by centrifugation at 100,000 × g for 3 hours. When the confluence of CAF and NF cells reached 80%-90%, the cells were washed three times with PBS and cultured for 72 hours in DMEM / F12 medium containing 5% de-EV FBS. The resulting medium was collected as conditioned medium (CM). The CM was then subjected to a gradient centrifugation: 500 × g for 10 min, 2500 × g for 20 min, and 12,000 × g for 30 min. After filtration through a 0.22 μm filter, the precipitate was collected by ultracentrifugation at 100,000 × g for 70 min. The precipitate was diluted with 20 mL of PBS and then ultracentrifuged again at 100,000 × g for 70 min. The concentration of sEVs was determined using a nanoflow cytometer (N30E, NanoFCM, Xiamen). The morphology and structure of sEVs were observed using a transmission electron microscope (JEM-2000EX*, NEC Corporation). Western blotting was used to verify the expression of positive and negative markers for sEVs. The particle size and concentration of sEVs were determined using a nanoflow cytometer (N30E, NanoFCM). PKH26 labeling reagent (PKH26PCL, Sigma-Aldrich, Louisville, Missouri, USA) was used for labeling according to the manufacturer's instructions. The labeled sEVs were then separated by ultracentrifugation at 100,000 × g for 70 minutes.

[0037] (11) OptiPrep density gradient purification Small extracellular vesicles (sEVs) were purified using the OptiPrep density gradient method, with specific procedures described in a previous reference (Nat Commun, 2015, 6:7164.). In short, 60% w / v OptiPrep (catalog number D1556, Sigma-Aldrich) was mixed with 0.25 M sucrose / 10 mM Tris buffer (pH 7.5) and aliquoted into ultra-clear centrifuge tubes (catalog number 344059, Beckman Coulter, California, USA) to prepare 60%, 50%, 40%, 30%, 25%, 15%, 10%, and 5% (w / v) iodixanol solutions. The ultracentrifuged sEVs were placed on top of the gradient solution and centrifuged at 100,000 × g for 18 hours using an Optima XPN-100 ultracentrifuge (Beckman Coulter) with a Ti41 rotor to create a continuous gradient, and eight fractions were collected. After each component was diluted with PBS, it was centrifuged again at 100,000×g for 70 minutes in an ultra-high-definition centrifuge tube to collect the precipitate, which was then washed with PBS and resuspended.

[0038] (12) sEV colloidal gold labeling Purified extracellular vesicles (sEVs) were incubated on a formvar carbon mesh in PBS for 20 minutes, followed by washing three times with PBS and adding 50 mmol / L glycine solution to remove free aldehyde groups. After washing the mesh nine times with PBS, it was blocked with PBS solution containing 5% bovine serum albumin (BSA) for 1 hour, and then incubated overnight at 4°C with primary antibodies against CD9 (10 μg / mL, Abcam) and POSTN (10 μg / mL, Abcam). The samples were then co-incubated with a secondary antibody against goat anti-rabbit IgG labeled with 5 nm colloidal gold particles (Thermo Fisher Scientific, Waltham, Massachusetts, USA). After washing with PBS containing 0.1% BSA, the mesh was fixed with 2.5% glutaraldehyde for 15 minutes and finally rinsed thoroughly with deionized water. After counterstaining with phosphotungstic acid for 5 minutes, the samples were imaged using a transmission electron microscope (JEM-2000EX*, Japan).

[0039] (13) Macrophage uptake of PKH26-labeled sEVs Purified sEVs were placed in 50 µL of PBS buffer and fluorescently labeled with 0.4 µL of PKH26 dye (Sigma-Aldrich) for 20 minutes. The labeled sEVs were then centrifuged at 100,000 × g for 70 minutes. After discarding the supernatant, equal volumes of sEVs were added to macrophages, and incubation was continued for 2 hours and 6 hours, respectively. Macrophages were then fixed with 4% paraformaldehyde for 10 minutes. After centrifugation at 1000 × g for 5 minutes, the macrophages were resuspended in 1 mL of PBS buffer, and the percentage fluorescence intensity was detected using a flow cytometer (Novoset 2040R, ACEA Biosciences, Hangzhou, China).

[0040] (14) CAF sEV-induced macrophage and flow cytometry analysis To verify whether CAF-S5 / -S6 derived from shNC and shPOSTN sEVs could induce macrophage polarization towards M1 and M2 types, researchers added 5 × 10^9 sEV particles to 2 × 10^6 macrophages cultured in 6-well plates and incubated for 48 hours. The staining procedure was as follows: 100 μL of a mixture containing 1 × 10^6 macrophages was incubated with 5 μL of FITC-labeled anti-human CD80 antibody (catalog number 375405, Biolegend, USA), FITC-labeled anti-human CD86 antibody (catalog number 374203), FITC-labeled anti-human CD163 antibody (catalog number 333617), and FITC-labeled anti-human CD206 antibody (catalog number 321103) on ice for 15 minutes, followed by washing twice with PBS buffer. The stained cells were resuspended in 1 mL of PBS and analyzed using a NovoCyte 2040R flow cytometer (ACEABiosciences, USA).

[0041] (15) RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific). RNA was precipitated with isopropanol, washed with 100% ethanol, and resuspended in 30 μL DEPC buffer. RNA concentration was determined using a Nanodrop One instrument (Thermo Fisher Scientific). Reverse transcription was performed using the Primer Script RT kit (Dalian Takara Bio Inc.). Quantitative real-time PCR was performed using the SYBR Premix Ex Taq kit (Takara Bio Inc.) on a LightCycler® 480II instrument (Roche, Switzerland). GAPDH was used as an internal control gene. Data from each sample were semi-quantitatively analyzed using the 2−ΔΔCT method. Primers for CD68, CD80, CD86, CD163, CD206, and GAPDH were purchased from Sangon Biotech (Shanghai).The primer sequences are as follows: CD68 (forward): 5'-AGCCACAAAACCACCACTCA-3', (reverse): 5'-CTAGTGGTGGCAGGACTGTG-3'; CD80 (forward): 5'-AACTGTGTGGATCTCTGGGG-3', (reverse): 5'-ACTTCTGAGCTTGCTTTGCC-3'; CD86 (forward): 5'-TCAGGAGGGCTTTTAGTGGG-3', (reverse): 5'-AAA TGTCTGCTTGCCTTGGG-3'; CD163 (forward): 5'-CTTGACCAGTGTGCAATGCT-3', (reverse): 5'-GTGCCCTTGGGATTTGTCAG-3'; CD206 (forward): 5'-GGGACAGAGCACAGGTTACT-3'; (reverse): 5'-CATTTCCTGCAGCTCGAGTC-3'; BMP4 (forward): 5'-GGAGGAGGAGGAAGAGCAG A-3' (reverse): 5'-TTCTTCGTGGTGGAAGCTCC-3'; TNF-α (positive): 5'-CTTCCAGCTGGAGAAGGGTG-3' (reverse): 5'-CCCAAAGTAGACCTGCCCAG-3'; IL-6 (positive): 5'-AGTGAGGAACAAGCCAGAGC-3' (reverse): 5'-GGTCAGGGGTGGTTATTGCA-3'; TGF-β (positive): 5'-CTA CGCCGCCTACTACTGTG-3' (reverse): 5'-CGGGTTGATGAAGTGGACCA-3'; IL-10 (forward): 5'-AAGACCCAGACATCAAGGCG-3' (reverse): 5'-AGGCATTCTTCACCTGCTCC-3'; GAPDH (forward): 5'-CACATCGCTCAGACACCATG-3' (reverse): 5'-CATACGACTGCAAAGACCCG-3'. All experiments were repeated at least three times.

[0042] (16) Western blot Cell lysis buffer was prepared using RIPA lysis buffer (Catalog No. R0010, Beijing Solarbio Science & Technology Co., Ltd.), supplemented with a mixture of protease inhibitors (Catalog No. HY-K0010, Shanghai Medicilon Biotech Co., Ltd.) and a mixture of phosphatase inhibitors I (Catalog No. HY-K0021, Shanghai Medicilon Biotech Co., Ltd.). Lysis was performed at 4°C for 30 minutes. Protein concentration was determined using a BCA protein quantification kit (Catalog No. P0009, Shanghai Beyotime Biotechnology Co., Ltd.). Equal volumes of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Merck Millipore, Inc.). The membranes were blocked with 5% skim milk for 1 hour to prevent nonspecific binding, and then incubated with primary antibody overnight at 4°C. Primary antibodies include: POSTN antibody (catalog number ab14041, 1:500 dilution, Abogen Biosciences), CD9 (catalog number ab236630, 1:1000 dilution, Abogen Biosciences), CD81 (catalog number ab79559, 1:1000 dilution, Abogen Biosciences), calconazole (catalog number 10427-2-AP, Protectives), CD80 (catalog number ab1341201, 1:1000 dilution, Abogen Biosciences), CD86 (catalog number abs115477, 1:1000 dilution, Abogen Biosciences), CD163 (catalog number sc-33715, 1:1000 dilution, Santa Cruz Biotechnology), and CD206 (catalog number ab646). The following antibodies were used: 93 (1:1000 dilution, Abogen Biosciences), BMPR2 (catalog number abs147034, 1:1000 dilution, Abogen Biosciences, Shanghai), pSmad1 (Ser463 / 465) / Smad5 (Ser463 / 465) / Smad9 (Ser465 / 467) (catalog number 13820, 1:1000 dilution, Cell Signaling Technologies), Smad5 (catalog number 12534, 1:1000 dilution, Cell Signaling Technologies), Smad9 (catalog number abs131190, 1:1000 dilution, Abogen Biosciences), and GAPDH antibody (catalog number 10494-1-AP, 1:5000 dilution, Protec). After washing the membrane three times with 1×TBST buffer, goat anti-rabbit IgG secondary antibody (catalog number 35401, 1:5000 dilution, Cell Signaling Technologies) was added and incubated for 2 hours. The membrane was washed three times with 1×TBST, and then the protein bands were detected using an Amersham ImageQuant 800 (Cytiva, Shanghai, China) enhanced chemiluminescence (ECL) detection system. The intensity of the bands was quantified using ImageJ software.

[0043] (17) Enzyme-linked immunosorbent assay (ELISA) To confirm the presence of POSTN on the surface of CAF sEVs, a POSTN ELISA kit (catalog number H2452c, Wuhan Abogen Biosciences Co., Ltd.) was purchased. CAF sEVs isolated from cell culture medium were serially diluted and added to each well (100 μL per well). After 90 minutes, biotinylated POSTN antibody was added to each well, and the mixture was incubated at 37°C for 1 hour. Subsequently, horseradish peroxidase-labeled streptavidin (100 μL) was added, and the mixture was incubated at 37°C for 30 minutes, followed by reaction with 3,3',5,5'-tetramethylbenzidine substrate at 37°C for 15 minutes. After adding 50 μL of stop solution, the absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific). To block POSTN on the surface of CAF sEVs, 10 μg / mL of POSTN blocking antibody (Abogen) was dissolved in 100 μL of PBS and incubated with CAF sEVs overnight at 4°C. After washing with PBS (20 mL), unbound antibody was removed by ultracentrifugation (100,000 × g, 70 min). All experiments were repeated at least three times.

[0044] (18) RNA sequencing analysis Total RNA was extracted from macrophages treated with CAF sEV-derived shNC and shPOSTN for 48 hours using TRIzol® reagents (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA quality was then assessed using a 5300 Bioanalyser (Agilent Technologies), and quantification was performed using an ND-2000 spectrophotometer (NanoDrop Technologies). Sequencing libraries were constructed from high-quality RNA samples. RNA purification, reverse transcription, library construction, and sequencing were all performed at Mabpharm Biopharmaceuticals Co., Ltd. (Shanghai, China) according to the Illumina (San Diego, USA) manufacturer's instructions. In short, RNA sequencing was performed using the Illumina NovaSeq / HiSeq Xten platform with NovaSeq kits. Raw paired-end reads were trimmed and quality-controlled using Fastp default parameters, and then aligned to the reference genome using HISAT2 software in directional mode. Aligned reads from each sample were assembled using StringTie based on the reference genome. To identify differentially expressed genes (DEGs) among different samples, the expression level of each transcript was calculated using the TPM (transcripts per million) read method, and gene abundance was quantified using RSEM. Finally, differential expression analysis was performed using DEGseq (Bioinformatics, 2019, 26:136-138.). This invention identifies differentially expressed genes (DEGs) through functional enrichment analysis (including GO and KEGG). These genes, when the Bonferroni-corrected p-value is ≤0.05, show significant enrichment in GO functional annotation and metabolic pathways compared to the whole transcriptome background. GO functional enrichment analysis was performed using Goatools software, while KEGG pathway analysis was performed using KOBAS (Nucleic Acids Res, 2011, 39, W316–W322.).

[0045] (19) Statistical analysis Statistical analysis was performed using R software (version 4.1.1). Histograms were created using the `histfunction` function in R to visualize the data distribution. For comparisons of categorical variables, chi-square test, Wilcoxon rank-sum test, or t-test were selected based on the data distribution. Relationships between variables were assessed using the Spearman correlation coefficient. Ancillary statistical analyses were performed using GraphPad Prism 7.0 and ImageJ software. Statistical significance was defined as p-value < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001). Unpaired t-tests were used for all data comparisons. Experiments were repeated at least three times, and data are presented as mean ± standard error.

[0046] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0047] Unless otherwise specified, all raw materials used in this invention are not subject to any particular restriction on their source; they may be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0048] Example 1: POSTN + FAP + Fibroblasts are associated with poor ICB response in HNSCC This invention utilizes sequencing datasets ( Figure 1 A) A comprehensive analysis of single-cell profiling data for head and neck squamous cell carcinoma (HNSCC) was performed, and five cell clusters were identified based on specific markers using the Seurat clustering method. Existing research indicates that POSTN expression is significantly increased in tumor-derived fibroblasts compared to normal tissues (Aging. Dis. 2023,14, 2177-2192.). vlnplot analysis showed that POSTN is mainly expressed in fibroblast clusters (…). Figure 1 B). Fibroblasts were further re-clustered into nine subgroups ( Figure 1 C), of which the fifth subgroup mainly expresses POSTN ( Figure 1 D). Based on UMAP cluster analysis, the fibroblast subset was classified as POSTN positive (POSTN... + ) and POSTN negative (POSTN) - Two major clusters ( Figure 1 E). Heatmap analysis showed that myCAF-related proteins (ACTA2, MYH11, TAGLN, PDGFA, MCAM, and MYLK) and inflammation-related CAF (iCAF) proteins (IL6, CXCL12, C3, and CFD) were mainly expressed in POSTN fibroblasts, while ECM-related CAF (eCAF) proteins (FAP, FN1, SFRP2, THY1, COL1A1, FBLN1, and LUM) and antigen-presenting CAF (apCAF) proteins (CD74, HLA-DRA, HLA-DRB5, HLA-DRB1, and HLA-DPA1) were mainly expressed in POSTN fibroblasts. + Fibroblasts ( Figure 1 F).

[0049] vlnplot further demonstrates the effects of POSTN, FAP, THY1, LUM, and SFRP2 on POSTN fibroblasts and POSTN + Differences in expression levels in fibroblasts ( Figure 2 A). Spearman correlation analysis showed a significant correlation between the expression levels of POSTN and FAP (A). Figure 2 B). Notably, patients with head and neck squamous cell carcinoma exhibiting high POSTN and FAP expression showed a poorer response to immune checkpoint blockade (ICB) therapy. Figure 2 C). H&E staining and immunohistochemical staining revealed higher expression levels of FAP and POSTN in the stroma of OSCC cases compared to the normal control group. Figure 2 D). These results confirm that POSTN + FAP + Fibroblasts are located in the mesenchymal region and are associated with adverse reactions to ICB treatment in HNSCC patients.

[0050] Example 2: POSTN + FAP + Fibroblasts may be associated with macrophage infiltration and the presence of POSTN on the surface of CAF sEVs. Studies have shown that POSTN + FAP + Evidence exists regarding intercellular communication between fibroblasts and macrophages (Aging. Dis. 2023, 14, 2177-2192.). Furthermore, macrophage infiltration rate is a key factor in immune checkpoint blockade (ICB) responses. Single-sample gene set enrichment analysis (ssGSEA) algorithm ( Figure 3 A) showed that in TCGA head and neck squamous cell carcinoma (HNSCC) cases, the expression levels of POSTN and FAP were significantly associated with macrophage immune infiltration. The expression levels of POSTN (R=0.42, p<2.2e-16) and FAP (R=0.38, p<2.2e-16) were strongly correlated with CD163 expression. Figure 3 B). Immunofluorescence staining showed co-expression of POSTN and CD163 in the interstitial region of HNSCC cases. Figure 3 C).

[0051] To further verify the expression level of POSTN in CAF and its secreted exosomes, this invention successfully extracted and verified the expression levels of POSTN in CAF-S5 / -S6 cell lines derived from primary oral squamous cell carcinoma (OSCC) and NFs cell lines derived from healthy human gingiva, following previous literature (Int. J. Oral. Sci. 2023, 15, 32.). The expression level of POSTN in CAF-S5 / -S6 and its secreted extracellular vesicles (sEVs) was significantly higher than that in normal fibroblasts (NFs) and their sEVs ( Figure 4 A). Density gradient fractionation analysis showed that POSTN protein was present on the surface of CAF-derived sEVs (A). Figure 4 B). Immunogold labeling assays further confirmed the presence of POSTN protein on the surface of CAF-S5 / -S6 sEVs. Figure 4 C). Enzyme-linked immunosorbent assay (ELISA) results showed that the concentration of sEV-POSTN in CAF-S5 / -S6 cells exhibited an increasing trend related to sEV concentration. Figure 4 D). Furthermore, after treatment with the POSTN blocking antibody, the detectable concentration of sEV-POSTN was significantly reduced ( Figure 4 E). These findings suggest that POSTN is located on the surface of CAF-S5 / -S6 sEVs.

[0052] Example 3: CAF-derived sEV-POSTN induces macrophage M2 polarization To investigate whether CAF-derived sEV-POSTN induces macrophage polarization, this invention constructed CAF-S5 / -S6 cell lines stably transfected with control shRNA (shNC) or POSTN-targeting shRNA (shPOSTN), and sEVs were isolated using differential ultracentrifugation. Compared with the shNC group, shPOSTN significantly reduced the expression level of POSTN in CAF-S5 / -S6 cells and their sEVs. Figure 5 A). In the CAF-S5 / -S6 cell lines transfected with shNC / shPOSTN, the average particle sizes of sEVs were 95 nm and 80 nm, respectively, and their particle concentrations reached approximately 1.9 × 10¹ per mL of culture medium. 0 and 2.7×10¹ 0 indivual( Figure 5 B). Human THP-1 cells were induced to differentiate into M0 macrophages after treatment with PMA (50 sng / mL) for 48 hours. These cells exhibited a typical adherent morphology. Figure 5 C), and the expression of macrophage markers such as CD68 was significantly increased (*** p<0.001, Figure 5D). Flow cytometry showed that, compared with the shNC transfection group, there was no significant difference in the uptake of PKH26-labeled sEVs by CAF-S5 / -S6 macrophages in the shPOSTN transfection group. Figure 5 E).

[0053] qRT-PCR assays showed that when macrophages were co-incubated with shPOSTN-transfected CAF-S5 / -S6 sEVs, the downregulation of M2 macrophage markers (CD163 and CD206) was significantly higher than that of M1 macrophage markers (CD80 and CD86) (**p<0.01, ). Figure 6 A). Flow cytometry analysis confirmed that knockdown of the POSTN gene significantly reduced the expression levels of M2 macrophage markers (CD163 and CD206), while the expression levels of M1 macrophage markers (CD80 and CD86) remained stable. Figure 6 B). Compared with sEVs derived from shNC, the expression levels of M2 markers (CD163 and CD206) were significantly downregulated in macrophages treated with sEVs derived from the CAF-S5 / -S6 cell line transfected with shPOSTN. Figure 6 C). However, in sEVs derived from CAF-S5 / -S6 cell lines transfected with shNC / shPOSTN, no significant difference was observed in the expression levels of M1 markers (CD80 and CD86). Figure 6 C). The results showed that POSTN knockdown of sEVs secreted by CAF-S5 / -S6 could regulate M2 macrophage polarization.

[0054] Example 4: BMP4 drives macrophage M2 polarization by activating the BMP R2 / Smad signaling pathway To further investigate the mechanism by which POSTN regulates macrophage M2 polarization, this invention performed RNA sequencing on macrophages stimulated with autologous exosomes (sEVs) transfected with shPOSTN and shNC small interfering RNA (shRNA) from the CAF S6 cell line. Principal component analysis (PCA) showed significant differences in gene expression between the shPOSTN sEVs treatment group and the control group (CTRL) or the shNC sEVs treatment group. Figure 7 A). Venn diagram analysis revealed 15 common genes and 242 unique genes in both groups of samples ( Figure 7 B). The heatmap further shows that, compared with the shNC sEVs treatment group, gene expression in the shPOSTN sEVs treatment group showed a significant up- and down-regulated trend (B). Figure 7 C). Volcano plot analysis identified 443 differentially expressed genes (DEGs), of which 262 upregulated genes and 181 downregulated genes were significantly present in the shPOSTN sEVs treatment group ( Figure 7 D). Among these differentially expressed genes, BMP4 was identified as a key growth factor that promotes tumor growth by inducing macrophage polarization toward an M2-like phenotype and by creating a pro-tumor immune microenvironment.

[0055] Gene set enrichment analysis (GSEA) showed that, compared with the shNC sEVs group, the inflammatory response in macrophages treated with shPOSTN sEVs was significantly upregulated. Figure 7 E). Gene ontology (GO) analysis revealed significant alterations in 20 biological processes in macrophages treated with shPOSTN sEVs, with particularly pronounced changes in cellular anatomy, binding, and cellular processes. Figure 7 F). Kyoto Encyclopedia of Genetics and Genomes (KEGG) pathway analysis showed significant enrichment of pathways such as cytokine-receptor interactions and viral protein-cytokine-receptor interactions, which are closely related to the inflammatory response during macrophage M2 polarization. Figure 7 G).

[0056] qRT-PCR analysis showed that, compared with the sEVs group transfected with CAF-S5 / -S6 cells by shPOSTN, the BMP4 level in macrophages treated with sEVs transfected with CAF-S5 / -S6 cells by shNC was significantly increased. Figure 8 A). Furthermore, in macrophages treated with 50 ng / mL and 100 ng / mL BMP4, the mRNA expression of pro-inflammatory factors (TNF-α and IL-6) was significantly downregulated, while the mRNA expression of anti-inflammatory factors (TGF-β and IL-10) was significantly upregulated. Figure 8 B). Flow cytometry showed that BMP4 induced upregulation of the expression of M2 macrophage markers CD163 and CD206. Figure 8 C). Notably, in macrophages treated with 100 ng / mL BMP4 for 48 hours, the expression levels of BMPR2 and pSmad1 / 5 / 9 were also significantly increased (C). Figure 8 D). To verify the mechanism by which BMP4 induces macrophage polarization towards M2 via the Smad signaling pathway, this invention uses the type I BMP receptor inhibitor LDN193189 in combination with BMP4. The results showed that the expression levels of pSmad1 / 5 / 9, CD163, and CD206 in macrophages were significantly reduced. Figure 8 E). These findings suggest that extracellular vesicles secreted by POSTN-deficient CAFs inhibit BMP4 secretion—which is supposed to activate the BMPR2 / Smad signaling pathway to drive macrophages toward M2 polarization. This discovery provides a potential therapeutic strategy for targeting tumor-associated macrophages (TAMs).

[0057] In recent years, researchers have increasingly favored single-cell RNA sequencing (scRNA-seq) technology to explore the heterogeneous characteristics of cancer cell fibroblasts (CAFs) in solid tumors. The results of scRNA-seq analysis vary significantly depending on the specific experimental method, each with its own unique advantages and technical limitations. These limitations include spatial information loss and underrepresentation of certain cell types, stemming from the technical challenges of isolating these cells from tissues. Despite these technical limitations, scRNA-seq has successfully identified CAF subsets with unique gene expression profiles and functional characteristics, such as myCAFs, iCAFs, and apCAFs. These subsets influence tumor progression and immune responses in different ways through various mechanisms, including the secretion of specific cytokines, chemokines, and ECM components. While immune checkpoint blockade (ICB) targeting PD-1 / PD-L1 has improved survival in patients with recurrent / metastatic head and neck squamous cell carcinoma (HNSCC), its mechanism of action remains not fully elucidated. Several researchers have pointed out that the efficacy of ICB associated with CAF subsets varies among different solid tumors. Researchers have also found that POSTN… + Fibroblasts highly express α-smooth muscle actin (αSMA), and these cells play a crucial role in regulating the endocrine system (ECM) while maintaining tissue structural integrity. Previous studies have shown that POSTN... + Fibroblasts are characterized by myCAFs because they express characteristic genes associated with ECM (POSTN, MMP14, and MMP11).

[0058] This invention, by integrating single-cell RNA sequencing data from two groups of head and neck squamous cell carcinomas (HNSCC), reveals the heterogeneous characteristics of fibroblasts and identifies POSTN. - fibroblasts and POSTN + Two distinct subgroups of fibroblasts. Studies have found that myCAF and apopaF characteristics are present in POSTN. + iCAF is significantly highly expressed in fibroblasts, while its characteristic feature is present in POSTN. - It is enriched in fibroblasts. Functionally, apCAF plays a crucial role in promoting the tumor immunosuppressive microenvironment by regulating T cell activity; while POSTN... + Fibroblasts inhibit CD8 + T-cell infiltration weakens the anti-tumor immune response and creates an immunosuppressive microenvironment, thereby affecting the efficacy of immunotherapy. Therefore, POSTN + The potential interaction between fibroblasts and apopafenone (apCAF) has become an important direction to be explored in the study of the tumor microenvironment in HNSCC.

[0059] Functional enrichment analysis showed that POSTN + Fibroblasts are primarily enriched in genes regulating collagen metabolism and extracellular matrix assembly. However, POSTN + The specific mechanisms by which fibroblasts lead to resistance to immune checkpoint blockade (ICB) drugs remain to be elucidated. POSTN + Fibroblasts can enhance CD4 + CD25 + T lymphocytes express PD-1 and CTLA4 proteins on their surface and are involved in immunotherapy resistance. FAP + αSMA + Fibroblasts function by producing collagen, forming multilayered tumor structures, and blocking contact between T cells and tumor cells. LRRC15 + High expression levels of fibroblasts are associated with poor prognosis after immunotherapy. Furthermore, this invention found that patients with head and neck squamous cell carcinoma (HNSCC) exhibiting high expression of POSTN and FAP had significantly poorer responses to ICB treatment. Co-expression of FAP and POSTN in HNSCC was significantly associated with poor prognosis, suggesting their potential as prognostic biomarkers. The study found a positive correlation between POSTN and FAP expression levels and macrophage infiltration in TCGA-HNSCC. Further comprehensive analysis of TCGA-HNSCC data indicated that HNSCC tumors with high POSTN / high FAP expression exhibit characteristic immune escape features and lower ICB response rates, potentially suggesting an immune resistance phenotype.

[0060] cadherin can recruit and induce monocytes to differentiate into M2-like tumor-associated macrophages (TAMs) through cytokines (CXCL12, CCL5, IL-6, IL-33) and the glycoprotein chitinase 3-like-1. (POSTN) + Fibroblasts attract and induce SPP1 via the IL-6 / STAT3 signaling pathway. + Macrophages in TAM can hinder effective T cell infiltration and reduce the efficacy of immunotherapy. In an immunotherapy cohort study, co-expression of POSTN... + Fibroblasts and SPP1 +Patients with macrophage-related diseases often have poor treatment outcomes. Exosomes (EVs) can also serve as an adjunct to existing immunotherapies. Despite the significant success of immune checkpoint blockade (ICB), a considerable proportion of patients still develop resistance to these drugs. PD-L1-positive exosomes have been used as potential targets; they enhance macrophage clearance of these antibodies by binding to and targeting them, thereby weakening the efficacy of anti-PD-L1 therapy. It is anticipated that blocking PD-L1 expression or release in exosomes may enhance the efficacy of ICB and overcome patient resistance. Human CPC-derived exosomes carry short POSTN isoforms (amino acids 22-669) that promote cardiomyocyte proliferation by activating FAK phosphorylation, actin polymerization, and nuclear translocation in the Yes-related protein (YAP) signaling pathway. The results of this invention indicate that the concentration of sEV-POSTN in CAF-S5 / -S6 increases in a sEV concentration-dependent manner, and treatment with POSTN-blocking antibodies significantly reduces detectable sEV-POSTN concentrations.

[0061] The findings of this invention demonstrate that CAF-enriched sEV-POSTN promotes M2 macrophage polarization by activating the BMP4-induced BMPR2 / Smad signaling pathway. These findings highlight the potentially crucial role of sEVs in CAF-macrophage interactions. CAFs, by regulating key signaling pathways, have become a potential target for anti-tumor therapy. To enhance the efficacy of chemotherapy and immunotherapy, researchers are exploring FAPs in preclinical and clinical trials. + CAF-guided therapies, including antibody-drug conjugates and CAR-T cells, have been explored. However, the clinical benefit of these approaches for patients with head and neck squamous cell carcinoma (HNSCC) remains unclear. This study reveals the benefits of POSTN... + The role of fibroblasts in predicting response to immunotherapy for HNSCC was investigated, and potential targets for enhancing treatment efficacy were identified. However, this study has certain limitations. Future work should include retrospective analyses of HNSCC patients in the immunotherapy group and large-scale prospective trials to validate these findings. Furthermore, further research is needed to evaluate the targeting of POSTN. + The clinical feasibility of fibroblast-macrophage interaction as a therapeutic strategy.

[0062] In summary, this invention successfully identified POSTN. - Fibroblast subsets and POSTN +POSTN is a characteristic molecular marker of fibroblast subsets. Subsequent studies found that POSTN accumulates on the membranes of small cell exosomes (sEVs) derived from CAF and is activated via the BMP4-mediated BMPR2 / Smad signaling pathway, promoting M2 macrophage polarization and thus enhancing immune checkpoint (ICB) resistance. Targeted downregulation of POSTN can block this immunosuppressive pathway and significantly improve the therapeutic effect of ICB, indicating that this protein has important application value in the treatment of head and neck squamous cell carcinoma (HNSCC).

[0063] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of BMP4 protein and its encoding gene in screening and preparing drugs to improve resistance to immune checkpoint blockade therapy in patients with head and neck squamous cell carcinoma, characterized in that, BMP4 drives macrophages to M2 polarization by activating the BMPR2 / Smad signaling pathway, and the head and neck squamous cell carcinoma patients are characterized by abnormally enhanced macrophage polarization to M2. 2.POSTN + The application of fibroblast subsets in screening and preparing drugs to improve resistance to immune checkpoint blockade therapy in patients with head and neck squamous cell carcinoma is characterized by, The POSTN + Fibroblasts secrete small extracellular vesicles, the surface of which is enriched with POSTN protein; these small extracellular vesicles regulate macrophage polarization by activating the BMP4 / BMPR2 / Smad signaling pathway, thereby promoting the upregulation of BMP4 expression in macrophages.

3. The application according to claim 2, characterized in that, The POSTN + The fibroblast subset originates from head and neck squamous cell carcinoma, and this cell subset highly expresses ECM-associated CAF-associated protein eCAF and antigen-presenting CAF-associated protein apCAF.

4. The application according to claim 3, characterized in that, The eCAF-related proteins include FAP, FN1, SFRP2, THY1, COL1A1, FBLN1, and LUM, while the apCAF-related proteins include CD74, HLA-DRA, HLA-DRB5, HLA-DRB1, and HLA-DPA1. This cell subpopulation was identified from single-cell RNA sequencing data of head and neck squamous cell carcinoma using Seurat clustering and UMAP clustering analysis, and it highly expressed POSTN.

5. The application according to claim 2, characterized in that, With POSTN + POSTN associated with fibroblast subsets - Fibroblast subsets highly express myofibroblast CAF-related protein myCAF and inflammation CAF-related protein iCAF.

6. The application according to claim 5, characterized in that, The myCAF-related proteins include ACTA2, MYH11, TAGLN, PDGFA, MCAM, and MYLK, and the iCAF-related proteins include IL6, CXCL12, C3, and CFD. This cell subpopulation was identified from single-cell RNA sequencing data of head and neck squamous cell carcinoma using Seurat clustering and UMAP clustering analysis, and it does not express POSTN.

7. The application according to claim 2, characterized in that, The drug contains POSTN + Small extracellular vesicles secreted by fibroblasts, and pharmaceutically acceptable excipients.

8. The application according to claim 7, characterized in that, The dosage form of the drug is selected from: injection or oral preparation.

9. The application according to claim 7, characterized in that, The pharmaceutically acceptable excipients are one or more of physiological saline, phosphate buffer, and dextran solution. 10.POSTN + The application of fibroblast subsets in the preparation of a diagnostic kit for resistance to immune checkpoint blockade therapy in patients with head and neck squamous cell carcinoma is characterized by, The kit is used to detect a combination of biomarkers for head and neck squamous cell carcinoma, the combination of biomarkers being POSTN and FAP, wherein high expression of POSTN and high expression of FAP indicate poor response to immune checkpoint blockade therapy; the expression levels of POSTN and FAP are obtained by detecting fibroblasts in the intermediate region of head and neck squamous cell carcinoma tissue.

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