Tumor Endothelial ITGA5 Expression Induces T cell Dysfunction in Cervical Cancer

Tumor Endothelial ITGA5 Expression Induces T cell Dysfunction in Cervical Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tumor Endothelial ITGA5 Expression Induces T cell Dysfunction in Cervical Cancer Lu Shen, XiaoQiu Dai, Yue Liu, Yayun Zhang, Xiaoxue Xi, Xiao Wu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9241604/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Tumor vascular endothelial cells (TVECs), which originate from normal vascular endothelial cells surrounding the tumor, play a critical role in shaping the immunosuppressive tumor microenvironment and contributing to resistance to immunotherapy in cervical cancer. However, the molecular mechanisms underlying TVEC-mediated regulation of anti-tumor immunity remain elusive. Here, we performed a comprehensive assessment of integrin family proteins in cervical cancer-derived TVECs and identified ITGA5 as a key regulator of T cell activity within the tumor microenvironment. Loss of ITGA5 in TVECs markedly enhanced antigen presentation by upregulating both MHC class I and class II pathways, thereby promoting T cell activation and cytotoxic function. Importantly, therapeutic blockade of ITGA5 significantly enhanced the efficacy of anti–PD-1 treatment, and the combination of ITGA5 antibody and PD-1 blockade synergistically inhibited tumor growth in orthotopic murine models. Collectively, these findings identify ITGA5 as a previously unrecognized immune regulator in tumor vasculature and suggest that targeting endothelial ITGA5 may represent a promising strategy to improve immunotherapy efficacy in cervical cancer. cervical cancer tumor vascular endothelial cells integrin ITGA5 immunosuppressive tumor microenvironment T cell dysfunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Cervical cancer remains the most prevalent gynecological malignancy, with a mortality rate of 5.57 per 100,000 women 1 . Advanced-stage disease carries a poor prognosis, with recurrence rates of 28%–64% in patients with stage IIB–IV disease 2 . Current therapeutic options for cervical cancer, including conventional chemotherapy, radiotherapy, anti-VEGFA-targeted therapy, and immune checkpoint blockade targeting PD-1/PD-L1, provide limited survival benefit and rarely achieve durable remission 3 . A fundamental contributor to cervical cancer lethality is the presence of tumor vascular endothelial cells (TVECs) which originated from normal vascular endothelial cells surrounding the tumor 4 , 5 . TVECs drive cervical cancer progression by promoting angiogenesis, metastasis and conferring resistance to chemotherapy and anti-angiogenic therapy 6 , 7 . Furthermore, TVECs play a pivotal role in orchestrating cervical cancer immunosuppressive microenvironment and promoting immunotherapy resistance 8 , 9 . Studies have shown that vascular endothelial cells can modulate T cell responses independently of professional antigen-presenting cell activation. This immunomodulatory function is achieved through the expression of co-stimulatory receptors, co-inhibitory receptors, adhesion molecules, and secretion of cytokines 10 – 14 . Integrins are a family of heterodimeric cell-surface receptors that interact with diverse ligands and regulate a wide range of physiological processes, including cell migration, invasion, and proliferation 15 , 16 . Accumulating evidence indicates that integrin signaling also plays important roles in tumor progression. For instance, our previous study showed that cervical cancer cells maintained a pro-angiogenic ITGA5/AKT/VEGFA signaling axis 17 . Beyond their roles in tumor cells, integrins are also critical regulators of immune responses, including the maintenance of T-cell function and the modulation of inflammatory signaling 18 , 19 . Consistently, targeting integrin α5 in fibroblasts has been shown to enhance anti-tumor immunity and improve the efficacy of immune checkpoint blockade in colorectal cancer 20 . However, a functional assessment of integrins in TVECs is still lacking. To address this gap, we performed a comprehensive assessment of integrin expression and function in cervical cancer-derived TVECs. We identify ITGA5 as a key regulator of T-cell activity within the cervical cancer microenvironment, providing a potential target for enhancing immunotherapy responsiveness. METHODS Cell lines and cell culture We purchased the human umbilical vein cell line, EA.hy926 from Procell Inc. (Wuhan, China). The endothelial cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Cat#C11995500BT) supplemented with 10% FBS (Gibco, Cat#A5256701) and 1% penicillin/streptomycin (Beyotime, Cat#C0222). Primary T cells were cultured in (RPMI) 1640 medium (Gibco, Cat#C11875500BT) supplemented with 10% FBS, 50 IU/ml interleukin-2 (IL-2) (Novoprotein, Cat#C013), 5 ng/mL interleukin-7 (IL-7) (Novoprotein, Cat#C086) and 5 ng/mL interleukin-15 (IL-15) (Novoprotein, Cat#C016). All cells were cultured in a humidified incubator at a constant temperature of 37°C with 5% CO 2 . All cell lines were authenticated using Short Tandem Repeat (STR) profiling and tested annually for mycoplasma contamination via PCR-based detection of cellular supernatants. Cells were used within 20 passages. Isolation and expansion of human T cells and ethics Peripheral blood was obtained from healthy volunteers who provided informed consent. All procedures involving human participants were conducted in accordance with the Declaration of Helsinki and were approved by the Ethics Committee of Suzhou Hospital Affiliated to Nanjing Medical University (KL901461). All blood samples were handled following the required ethical and safety procedures. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood by Ficoll density gradient centrifugation (Meilunbio, Cat#PWL100-1). T cells were isolated from PBMCs using CD3 magnetic beads (T&L Biotechnology, Cat#TL-622) and then stimulated with anti-CD3(T&L Biotechnology, Cat#GMP-TL101-0500) and anti-CD28 antibody (T&L Biotechnology, Cat#GMP-TL102-0500) for 48 h. We employed human CD4 or CD8 positive selection magnetic beads to isolate pure populations of CD4 + and CD8 + T cells for further functional assays. Co-culture experiment One day in advance, EA.hy926 cells were plated in 48-well plates at a density of about 50%. The next day, the supernatant was changed by adding cervical cancer cell supernatant at a 1:1 ratio and incubated for 48 h. For direct co-culture, we co-incubated EA.hy926 cells with CD4 + and CD8 + T cell at a ratio of 2:1 for 48 h. For indirect co-culture, CD4 + and CD8 + T cells were incubated for 48 h with a 1:1 mixture of supernatant collected from EA.hy926 cells. To delineate the functional and phenotypic changes induced by co-culture, we performed transcriptome sequencing on the purified CD4 + and CD8 + T cell populations. To avoid the confounding effects of exogenous cytokines, the culture medium for co-culture experiments was devoid of IL-2, IL-7, and IL-15. Multiplex Immunofluorescence Staining FFPE sections were deparaffinized in xylene and subsequently rehydrated through a series of graded ethanol solutions. Antigen retrieval was conducted in EDTA buffer (pH 8.0) with microwave heating (medium power 8 min, standing 8 min, medium-low power 7 min). After cooling and PBS washing, sections were blocked with 3% BSA for 30 min at room temperature. Primary antibodies diluted in PBS were applied to the tissue sections and incubated overnight at 4°C: ITGA5 (Abcam, Cat#Ab150361, 1:400), CD31 (Huilan Biotech, Cat#ABB00001, 1 :100), CD4 (Huilan Biotech, Cat#HL18254, 1:250), CD8 (Huilan Biotech, Cat#HL06846, 1:100), PD-1 (Huilan Biotech, Cat#HL20641, 1:75), PD-L1 (Huilan Biotech, Cat#HL21176, 1:250). Co-staining of CD4, CD8 and PD-1 as well as PD-L1, ITGA5 and PECAM was performed using a four-color mIHC fluorescence kit (Huilanbio Biological Technology, Shanghai, China) based on tyramide signal amplification (TSA) technology according to the manufacture’s instructions. After washing with PBS, the sections were incubated with the appropriate fluorophore-conjugated secondary antibodies for 50 min at room temperature in the dark. Following PBS washes, nuclei were stained with DAPI for 10 min. Slides were mounted with anti-fade medium and imaged under a Nikon fluorescence microscope. The excitation wavelength of DAPI was 330–380 nm, the emission wavelength was 440 nm; the excitation wavelength of CD8 and PD-L1 was 488 nm, the emission wavelength was 520 nm; the excitation wavelength of CD4 and ITGA5 was 647 nm, the emission wavelength was 690 nm; the excitation wavelength of PD-1 and PECAM was 555 nm, the emission wavelength was 570 nm. Flow Cytometry All flow cytometry experiments were performed using standard sample processing and staining protocols. Assays were performed on Navios EX (Beckman Coulter, USA) or BD LSRFortessa (BD Biosciences, USA). Live cells were identified as staining negative for DAPI. Raw data were analyzed with the FlowJo software v.10.8.1. The following antibodies were used for flow cytometry: ITGA5 (also known as CD49e, APC, Biolegend, Cat#328012), HLA-ABC (FITC, eBioscience, Cat#11−9983−42), HLA-DRB1 (FITC, eBioscience, Cat#E-AB-F1111C), CD3 (BV421, RY610, BD Biosciences, Cat#566697, 571134), LAG3 (BV480, PE, BD Biosciences, Biolegend, Cat#746609, 369306), PD−1 (BB515, BD Biosciences, Cat#565936), CD4 (BB700, BD Biosciences, Cat#568370), TIM3 (PE/APC, BD Biosciences, Biolegend, Cat#565570, 345012), CD62L (PE-CF594, BD Biosciences, Cat#562330), CCR7 (APC, BD Biosciences, Cat#566762), CD69 (PE/Cy7, BD Biosciences, Cat#557745), CD8 (APC/Cy7, BD Biosciences, Cat#557834), CTLA4 (PE/Cy7, Biolegend, Cat#349914), IL−2 (BV421, Biolegend, Cat#500327), Perforin (BV711, Biolegend, Cat#308130), IFN-γ (PE/Cy7, BD Biosciences, Cat#560741), Granzyme B (Alexa Fluor 700, BD Biosciences, Cat#560213), Zombie Aqua Fixable Viability Kit (Biolegend, Cat#423101). Real-time quantitative polymerase chain reaction Total RNA was extracted using TRlzol reagent (Thermofisher, Cat#15596018) and reverse transcribed into cDNA with the HiScript III qRT SuperMix kit (Vazyme, R323-01). qPCR was performed with Novostart SYBR qPCR Supermix Plus (Novoprotein, E096-01A) in a LightCycler 480 (ROCHE) instrument. The sequences of the primers were as follows: Human ITGA5 (Forward primer, 5’-TTACGGGACTCAACTGCACC-3’, Reverse primer, 5’-AGCCTGAAACACTCAGCCTC-3’), Human PD-L1 (Forward primer, 5’-GGACAAGCAGTGACCATCAAG-3’, Reverse primer, 5’-CCCAGAATTACCAAGTG AGTCCT-3’), Human HLA-A (Forward primer, 5’-AGATACACCTGCCATGTGCAGC-3’, Reverse primer, 5’-GATCACAGCTCCAAGGAGAACC-3’), Human HLA-B (Forward primer, 5’-CTGCTGTGATGTGTAGGAGGAAG-3’, Reverse primer, 5’-GCTGTGAGAG ACACATCAGAGC-3’), Human HLA-DRB1 (Forward primer, 5’-GAGCAAGATGCTGAGTGGAGTC-3’, Reverse primer, 5’-CTGTTGGCTGAAGTC CAGAGTG-3’), Human HLA-DRA (Forward primer, 5’-AGCTGTGGACAAAGCCAACCTG-3’, Reverse primer, 5’-CTCTCAGTTCCACAGG GCTGTT-3’) Plasmid construction and virus transfection Restriction enzymes used in this study were purchased from New England Biolabs (NEB). Plasmids were constructed using NEBuilder HiFi DNA Assembly Master Mix with polymerase chain reaction (PCR) products and backbone restriction digests. For guide plasmid cloning, protospacer oligos were annealed and then inserted using BsmBI golden gate assembly into lentiCRISPR v2-U6-sgRNA plasmids (Addgene, Cat#52961) as previously reported. SgRNAs used in this study were: sgNT 5’-CTCTGCTGCGGAAGGATTCG-3’, sgITGA5#1 5’-GGGCTTCAACTTAGACGCGG-3’, sgITGA5#2 5’-GGGGCAACAGTTCGAGCCCA-3’. For the generation of stable ITGA5-knockout cells, EA.hy926 cells were seeded in 96-well plates (3,000–4,000 cells/well) and transduced at 20–30% confluence with lentivirus carrying either ITGA5-targeting or control sgRNA at an MOI of 10, using HitransG/P as an enhancer. After 24 h, the medium was replaced with fresh complete medium for 48 h, followed by selection with puromycin (1:5,000) for 2 weeks. Knockout efficiency was confirmed by Western blot before downstream assays were performed at 7–10 days post-transduction. Public database data analysis Gene expression data from the cervical cancer-related datasets GSE171894, GSE197461, GSE236738, and GSE208653 were obtained from the GEO database, comprising 4 normal cervical tissue samples, 2 precancerous lesion samples, and 33 cervical cancer tissue samples. Using R language, we integrated the cells from all datasets and performed cell clustering. The mRNA expression levels of integrin alpha 5 (ITGA5) were analyzed, and samples with expression levels below the average were defined as the ITGA5-low group, while those above the average were designated as the ITGA5-high group. Using the GEPIA2 database, we examined the correlation between ITGA5 and vascular endothelial markers, as well as the overall survival rates of individuals with different endothelial ITGA5 expression levels. The TIMER database was employed to analyze the correlation between ITGA5 and the infiltration degree of various immune cell populations. Animal studies Six-week-old female C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were randomly assigned to four groups (n = 4 per group) and subcutaneously injected with 3 × 10⁶ TC-1 murine cervical cancer cells to establish syngeneic tumors. Beginning on day 5 post-implantation, mice were treated with anti-ITGA5 antibody, anti-PD-1 antibody, a combination of both antibodies, or PBS as control until day 17. Tumor dimensions were measured every two days with calipers, and tumor volumes were calculated using the formula: volume = (length × width²) / 2. The antibodies used for animal studies are listed as follows: ATN−161 (integrin α5β1 Antagonist, Selleck, Cat#S8454), In vivo MAb anti-mouse PD−1 (CD279) antibody (Bioxcell, Cat#BE0273). Statistical analysis GraphPad Prism 10 (GraphPad Software), R version 4.5.1 and GSEA version 4.4.0 were taken for statistical analyses. For comparisons between two groups, we used the two-tailed Student’s t test. Signifcant differences were determined as: *P < 0.05, **P < 0.01, ***P < 0.001. RESULTS High expression of ITGA5 in TVEC correlates with poor prognosis in cervical cancer In our previous study, immunohistochemistry analysis of cervical cancer tissues revealed that ITGA5 was not only expressed in tumor cells but also in stromal compartments, particularly in vascular endothelial cells 21 . This observation suggests a potential role for endothelial integrins in shaping the tumor microenvironment (Supplementary Fig. 1A), consistent with previous reports 22 , 23 . To further characterize integrin expression in endothelial cells, we analyzed integrin profiles in cervical cancer using publicly available single-cell RNA sequencing datasets. Specifically, we integrated four datasets from the GEO database (GSE236738, GSE208653, GSE197461, and GSE171894), comprising 4 normal cervical tissue samples, 2 precancerous lesion samples, and 33 cervical cancer tissue samples. Unbiased clustering analysis of all cells revealed 10 distinct populations, annotated by their unique marker gene expression profiles (Fig. 1 A,B). Among the integrin family members, ITGA5, ITGA6, and ITGB1 were the most highly expressed in endothelial cells (Supplementary Fig. 1B). Notably, ITGA5 was preferentially enriched in endothelial cells, characterized by the canonical markers PECAM1, VWF, and IGFBP7 (Fig. 1 B and Fig. 1 C). Importantly, analysis of the GEPIA database revealed that ITGA5 was the only integrin significantly associated with poor prognosis in cervical cancer patients (Supplementary Fig. 1C). Based on these findings, we focused on elucidating the role of endothelial ITGA5 in cervical cancer progression. Further analysis revealed that ITGA5 expression was positively correlated with endothelial markers CDH5 and PECAM1. Notably, ITGA5 expression was also positively correlated with VEGFR2 and CD34, markers enriched in tumor vascular endothelial cells (TVECs) relative to normal endothelial cells (NECs), supporting preferential upregulation of ITGA5 in TECs (Fig. 1 D). To validate these findings in clinical specimens, multi-color immunohistochemistry (mIHC) with CD31 and ITGA5 antibody confirmed colocalization of ITGA5 with CD31, with increased ITGA5 expression observed in endothelial cells within cervical cancer tissues (Fig. 1 E). To further assess the clinical significance of TVEC ITGA5, we analyzed TCGA and GTEx datasets and found that high ITGA5 normalized by PECAM1 expression reflecting endothelial cells with elevated ITGA5 levels, was significantly associated with reduced overall survival in patients with cervical squamous cell carcinoma (Fig. 1 F). Moreover, elevated ITGA5 normalized by PECAM1 expression correlated with poor prognosis across multiple cancer types (Fig. 1 F). Collectively, these findings demonstrate that ITGA5 is highly expressed in tumor-associated endothelial cells and is associated with poor clinical outcomes, highlighting a potential role for endothelial ITGA5 in cervical cancer progression. ITGA5-expressing endothelial cells impede T cell infiltration and induce T cell dysfunction. Integrins have been shown to promote tumor progression by generating immune tolerance via the inhibition of immune cell responses 21 . We therefore investigated whether ITGA5 modulates the cervical cancer microenvironment. Utilizing the TIMER2 database, we analyzed the correlation between ITGA5 expression and immune cell infiltration. TIMER2 analysis revealed an inverse correlation between ITGA5 mRNA expression and T cells infiltration, particularly CD8 + T cells (Fig. 2 A). Cervical cancer samples were further stratified into ITGA5-low and ITGA5-high groups based on the median expression level in the scRNA-seq dataset. In the ITGA5 high group, the infiltration levels of CD4 + and CD8 + T cells were significantly reduced, whereas no significant difference were observed in the infiltration of B cells, macrophages, dendritic cells, and other immune cell populations (Fig. 2 B). To further explore the functional states of T cells, we performed pseudotime analysis to reconstruct the differentiation trajectories and functional state evolution of CD4 + and CD8 + T cells based on ITGA5 expression level (Fig. 2 C,D). Compared to ITGA5 low group, both CD4 + and CD8 + T cells in ITGA5-high group exhibited a more rapid and pronounced trajectory toward functional exhaustion. To validate these findings at the tissue level, we performed multiplex immunofluorescence staining of cervical cancer tissue microarrays. Tumor tissue exhibited enrichment of PD-1 + CD8 + T cells and ITGA5 + CD31 + populations compared to normal cervical tissues, along with an increase proportion of PD-1 + CD4 + T cells (Fig. 2 E). Collectively, these results indicate that elevated ITGA5 expression is associated with an immunosuppressive microenvironment in cervical cancer, characterized by impaired T cell infiltration and increased T cell dysfunction. ITGA5 knockout in endothelial cells enhance MHC expression To elucidate the molecular mechanisms underlying endothelial ITGA5 regulated T cell infiltration, we reanalyzed publicly available scRNA-seq datasets. Compared with ITGA5 high endothelial cells, genes involved in antigen processing and presentation pathway ( HLA-DRB5 , HLA-DQA1 and HLA-DQB1., et al ) were enriched in ITGA5 low endothelial cells (Fig. 3 A). Consistently, gene set enrichment analysis (GSEA) revealed that antigen processing and presentation via both MHC class I and MHC class II pathways were indeed enriched in ITGA5 low endothelial cells (Fig. 3 B). Supporting this observation, previous studies have demonstrated that human renal vascular endothelial cells express the major histocompatibility complex class II (HLA-DR) molecule and are capable of presenting antigens to CD4 + T cells 24 . To experimentally validate whether ITGA5 regulates antigen processing and presentation pathway, we generated a CRISPR-mediated ITGA5 knockout in a human umbilical vein endothelial cell line (EA.hy926) (Fig. 3 C,D). Compared with control cells, ITGA5-deficient endothelial cells exhibited increased mRNA expression of HLA-A , HLA-B , HLA-DRA and HLA-DRB1 (Fig. 3 E). Consistent with this transcriptional changes, flow cytometry analysis revealed a corresponding increase in the surface expression of both MHC-II (HLA-DRB) and MHC-I (HLA-ABC) proteins (Fig. 3 F). Collectively, these results demonstrate that ITGA5 suppresses antigen presentation in endothelial cells by downregulating MHC class I and class II expression. ITGA5 knockout in endothelial cells enhances T cell function in a contact-dependent manner. To determine whether tumor-associated endothelial cells regulate T cell function, we performed a direct in vitro co-culture assay. Endothelial cells were first stimulated with tumor cell conditioned medium for 48 h and then co-cultured with Healthy donor-derived T cells for an additional 48 h. Following co-culture, T cells were isolated using magnetic-activated cell sorting (MACS) and separated into CD4⁺ and CD8⁺ subsets for subsequent flow cytometric analysis and transcriptomic sequencing (Fig. 4 A). GSEA revealed that CD8⁺ T cells co-cultured with ITGA5 deficient endothelial cells exhibited significant enrichment of genes involved in T cell mediated cytotoxicity, compared with CD8⁺ T cells co-cultured with control endothelial cells (Fig. 4 B). Consistent with these transcriptomic changes, flow cytometry analysis showed that both CD4⁺ and CD8⁺ T cells co-cultured with ITGA5 deficient endothelial cells displayed reduced surface expression of exhaustion markers (LAG3 and TIM3) and increased production of effector cytokines (IL-2 and IFN-γ), compared with the control group (Fig. 4 C,D). Together, these results demonstrate that ITGA5 deficiency in tumor-associated endothelial cells enhances T cell effector function and alleviates T cell exhaustion. Secreted factors from endothelial cells are insufficient to modulate T cell phenotype To evaluate the effects of secreted factors, we established an indirect co-culture system by treating healthy donor-derived T cells with conditioned medium collected from endothelial cells expressing different levels of ITGA5 and cervical cancer cells. Flow cytometry analysis revealed no differences in the CD62L + CCR7 + central memory T cell (TCM) population following stimulation with endothelial conditioned medium (Fig. 5 A). In addition, the expression of early activation marker CD69 and classical exhaustion markers (PD-1, TIM-3, and LAG-3) remained unchanged in T cells regardless of ITGA5 expression in endothelial cells (Fig. 5 B). These results indicate that modulation of T cell function by endothelial cells requires direct cell–cell contact, rather than being mediated solely by secreted factors. ITGA5 blockade enhances the anti-tumor efficacy of PD-1 immunotherapy in vivo To evaluate the therapeutic potential of ITGA5 blockade in vivo, we established a syngeneic cervical cancer model by subcutaneously injecting 3.5 million TC-1 murine cervical cancer cells into C57BL/6J mice. Following 5days after transplantation, mice were randomly assigned to four groups, PBS group, anti-ITGA5 antibody (5 mg/kg) treatment, anti-PD-1 antibody (5 mg/kg) treatment, or anti-ITGA5 antibody plus anti-PD-1 antibody (Fig. 6 A). The tumor growth curves showed that combination therapy produced markedly greater anti-tumor efficacy than either monotherapy (Fig. 6 B), demonstrating a strong synergistic interaction between ITGA5 blockade and immune reactivation. To determine the clinical relevance of these findings, we analyzed proteomic data from the Cancer Immunology Data Engine (CIDE) database. Elevated ITGA5 levels were associated with poor survival in hepatocellular carcinoma patients receiving anti–PD-1 therapy (Fig. 6 C). This association extended across tumor types: urothelial cancer (Fig. 6 D), renal cancer (Fig. 6 E) and non-small cell lung cancer (Fig. 6 F). These clinical associations, together with our in vivo findings, position MEPCE as a central mediator of immune evasion and a promising therapeutic target for overcoming immunotherapy resistance. DISCUSSION Our study identifies integrin α5 (ITGA5) as a critical regulator of the immunosuppressive tumor microenvironment (TME) in cervical cancer. We demonstrated that elevated ITGA5 expression in tumor vasculature correlates with poor clinical outcomes. ITGA5 mediated immune evasion through suppression of MHC-I/II-dependent antigen presentation, resulting in impaired CD8 + T cell infiltration and accelerated T cell exhaustion. Crucially, modulating endothelial ITGA5 expression directly reshaped the functional profile of co-cultured T cells, suggesting a novel mechanism of vascular-driven immune suppression. In the tumor microenvironment, beyond professional antigen presenting cells (APCs), other cell types including epithelial cells, vascular endothelial cells, fibroblasts, and even cancer cells are also capable of presenting tumor antigens via both MHC class I and class II molecules 25 . These cell types within the TME are often referred to as “amateur APCs”. Amateur APCs vastly outnumber the professional APCs and may play a significant role in initiating anti-tumor immune responses 26 , 27 . Studies have indicated that TVECs dysregulated specific cell surface markers, reduced antigen presentation capacity, and impaired recruitment of immune cells, collectively contributing to immunosuppression 28 , 29 . Notably, Marelli-Berg et al. demonstrated that when VECs expressed MHC class II and were co-cultured with antigen-specific effector memory CD4 + T cells (CD45RO + ), these T cells migrate more rapidly across a monolayer of VECs 30 . In vitro experiments demonstrated that tumor endothelial cells with high ITGA5 expression induced CD4⁺ T cell exhaustion and impaired the cytotoxic function of CD8⁺ T cells through direct cell-cell contact. We hypothesized that ITGA5-high endothelial cells contribute to the establishment of an immunosuppressive tumor microenvironment (TME) through two potential mechanisms. First, ITGA5-high endothelial cells exhibited reduced ICAM-2 expression. Gene Set Enrichment Analysis (GSEA) further revealed suppression of the focal adhesion pathway, suggesting that elevated ITGA5 expression may promote an endothelial anergy phenotype. Such a dysfunctional state might impair immune cell adhesion to the endothelium, thereby limiting immune cell extravasation and contributing to an immunosuppressive tumor microenvironment (TME). Second, ITGA5-high tumor vascular endothelial cells (TVECs) displayed decreased expression of antigen-presenting molecules, which might attenuate T cell recruitment to the TME and ultimately result in reduced T cell infiltration. Notably, our in vivo experiments suggested that ITGA5 blockade might enhance the antitumor efficacy of PD-1 blockade, indicating that endothelial ITGA5 may serve as a potential therapeutic target to improve immune checkpoint blockade responses. Several limitations of this study should be acknowledged. First, the translational relevance of our findings is constrained by methodological considerations. The simplified cell culture systems employed here do not fully recapitulate the complex cellular interactions and mechanical cues present in human tumors. Second, the therapeutic potential of ITGA5 targeting requires further validation in preclinical models that more accurately reflect human tumor-immune interactions prior to clinical translation. The therapeutic relevance of ITGA5 in cancer immunotherapy is largely determined by its cell-type-specific and tissue-restricted expression. Single-cell transcriptomic analysis revealed that ITGA5 is predominantly expressed in tumor-associated endothelial cells, with expression substantially higher than in normal cervical endothelium, indicating that therapeutic targeting of ITGA5 may primarily affect the tumor endothelium. Functionally, endothelial ITGA5 restricts T cell infiltration and effector activity while promoting tumor angiogenesis and progression. Therefore, inhibiting ITGA5 could simultaneously enhance antitumor immunity and suppress tumor vascularization, supporting its potential as a promising therapeutic target. Declarations Funding Lu Shen was funded by Basic Research Program of Jiangsu (BK20230222). Peng Lin was funded by Basic Research Program of Jiangsu (BK20250446). Ethics approval and consent to participate All study procedures strictly adhered to the Declaration of Helsinki. Ethics approval and consent to perform animal studies were approved by the Institutional Animal Care and Use Committee of the Nanjing medical university. The Ethics Committee of the Affiliated Suzhou Hospital of Nanjing Medical University approved all human-associated tissues used in this research (KL901461). The informed consent has been signed by all patients before their tissues were acquired. Data availability The data supporting the findings of this study are available within the article. Additional data related to this research may be requested from the corresponding authors. Consent for publication All listed authors consent to the submission. CRediT authorship contribution statement Lu Shen: Writing-original draft, Investigation, Conceptualization, Methodology. XiaoQiu Dai: Investigation, Methodology, Formal analysis. Yue Liu: Investigation, Methodology. Yayun Zhang: Formal analysis. Xiaoxue Xi: Data Curation. Xiao Wu: Data Curation. Chen Wang: Conceptualization, Methodology. Peng Lin: Methodology, Writing-review & editing, Supervision. Declaration of competing interest The authors declare no competing financial interests. ACKNOWLEDGMENTS We are grateful to all the participants who contributed to this study. References B. Han, R. Zheng, H. Zeng et al., Cancer incidence and mortality in China, 2022. J. Natl. Cancer Cent. 4 (1), 47–53 (2024). 10.1016/j.jncc.2024.01.006 K.S. Tewari, Cervical Cancer. N. Engl. J. Med. 392 (1), 56–71 (2025). 10.1056/NEJMra2404457 M. Xu, C. Cao, P. Wu, X. Huang, D. Ma, Advances in cervical cancer: current insights and future directions. Cancer Commun. 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Commun. 14 (1), 60 (2026). 10.1186/s40478-026-02244-8 K.A. Muczynski, D.M. Ekle, D.M. Coder, S.K. Anderson, Normal human kidney HLA-DR-expressing renal microvascular endothelial cells: characterization, isolation, and regulation of MHC class II expression. J. Am. Soc. Nephrol. 14 (5), 1336–1348 (2003). 10.1097/01.asn.0000061778.08085.9f F.X. Mauvais, van P. Endert, Cross-presentation by the others. Semin. Immunol. 67 , 101764 (2023). 10.1016/j.smim.2023.101764 L.B. Darragh, S.D. Karam, Amateur antigen-presenting cells in the tumor microenvironment. Mol. Carcinog. 61 (2), 153–164 (2022). 10.1002/mc.23354 M.J. Schuijs, H. Hammad, B.N. Lambrecht, Professional and ‘Amateur’ Antigen-Presenting Cells In Type 2 Immunity. Trends Immunol. 40 (1), 22–34 (2019). 10.1016/j.it.2018.11.001 H. Won Jun, H. Kyung Lee, I. Ho Na et al., The role of CCL2, CCL7, ICAM-1, and VCAM-1 in interaction of endothelial cells and natural killer cells. Int. Immunopharmacol. 113 , 109332 (2022). 10.1016/j.intimp.2022.109332 A.U. Kabir, C. Zeng, M. Subramanian et al., ZBTB46 coordinates angiogenesis and immunity to control tumor outcome. Nat. Immunol. 25 (9), 1546–1554 (2024). 10.1038/s41590-024-01936-4 F.M. Marelli-Berg, L. Frasca, L. Weng, G. Lombardi, R.I. Lechler, Antigen recognition influences transendothelial migration of CD4 + T cells. J. Immunol. 162 (2), 696–703 (1999) Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 28 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers invited by journal 07 Apr, 2026 Editor assigned by journal 03 Apr, 2026 Submission checks completed at journal 03 Apr, 2026 First submitted to journal 27 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9241604","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":620219944,"identity":"ebf36db3-8cbf-4919-9c43-cccddc6073c0","order_by":0,"name":"Lu Shen","email":"","orcid":"","institution":"The Affiliated Suzhou Hospital of Nanjing Medical University, Nanjing Medical University, Suzhou Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Shen","suffix":""},{"id":620219945,"identity":"d6291539-eed0-455d-afd2-760704e59cf6","order_by":1,"name":"XiaoQiu Dai","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"XiaoQiu","middleName":"","lastName":"Dai","suffix":""},{"id":620219946,"identity":"162736ca-323b-40a4-bd6d-ff01abf7d634","order_by":2,"name":"Yue Liu","email":"","orcid":"","institution":"The Affiliated Suzhou Hospital of Nanjing Medical University, Nanjing Medical University, Suzhou Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Liu","suffix":""},{"id":620219947,"identity":"9d7eb54c-f009-4587-ae25-2268eff29ef3","order_by":3,"name":"Yayun Zhang","email":"","orcid":"","institution":"The Affiliated Suzhou Hospital of Nanjing Medical University, Nanjing Medical University, Suzhou Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yayun","middleName":"","lastName":"Zhang","suffix":""},{"id":620219948,"identity":"baf9576c-b26a-4499-b926-8701efbb3213","order_by":4,"name":"Xiaoxue Xi","email":"","orcid":"","institution":"The Affiliated Suzhou Hospital of Nanjing Medical University, Nanjing Medical University, Suzhou Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxue","middleName":"","lastName":"Xi","suffix":""},{"id":620219949,"identity":"7444ef57-41a3-4c51-831a-8cac20ef9e3e","order_by":5,"name":"Xiao Wu","email":"","orcid":"","institution":"The Affiliated Suzhou Hospital of Nanjing Medical University, Nanjing Medical University, Suzhou Municipal Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Wu","suffix":""},{"id":620219950,"identity":"d455d544-5fe7-4956-9591-a71f55d98e0a","order_by":6,"name":"Chen Wang","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Wang","suffix":""},{"id":620219951,"identity":"90a0200a-5d43-44a7-b538-493bbb95e558","order_by":7,"name":"Peng Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYBACPmYg8cGAFC1sQC2MM0jTAsTMPKToYGBj5z322KagLpqB/fDTjT8Y7PKIcBhfunGOweHcBp40s9s8DMnFRGjhMZPOMTiQ2yDBYHabgeFAYgNRWiwM6oBa2L/d/EG0FgYDZqAWHrMbPERqMTfsAfqljSen7DaPQTJhLfz8Z8we/PhTl9vPfnzbzR8VdoS1MECiBkYSGadsxCkbBaNgFIyCkQsAwrcwOWZzsLUAAAAASUVORK5CYII=","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":true,"prefix":"","firstName":"Peng","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2026-03-27 07:38:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9241604/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9241604/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106961227,"identity":"c22cd6c5-5b8f-42c0-af93-d11dd0c56870","added_by":"auto","created_at":"2026-04-15 09:24:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":909577,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEndothelial ITGA5 correlates with poor prognosis in cervical cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eUMAP plot of single-cell RNA sequencing datasets (GSE236738, GSE208653, GSE197461, and GSE171894). \u003cstrong\u003e(B) \u003c/strong\u003eMarker genes of 10 distinct cell clusters. \u003cstrong\u003e(C) \u003c/strong\u003eITGA5 surface expression heterogeneity across identified cell clusters. \u003cstrong\u003e(D)\u003c/strong\u003e Correlation analysis between ITGA5 expression and endothelial markers (CDH5 and PECAM1), as well as angiogenic and stemness-associated genes (VEGFR2 and CD34). \u003cstrong\u003e(E)\u003c/strong\u003e Multi-color immunohistochemistry (mIHC) showed the colocalization of ITGA5 with CD31 in both cervical cancer tissue and normal cervix tissue. \u003cstrong\u003e(F) \u003c/strong\u003eKaplan‒Meier curves showing survival based on ITGA5 mRNA expression in four cancer types patients from the TCGA dataset.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9241604/v1/e8167cb2c507fd0f82a7d4ad.png"},{"id":106960828,"identity":"53cc6a54-56c9-41ff-8525-2755563f8bff","added_by":"auto","created_at":"2026-04-15 09:23:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":949513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eITGA5-expressing endothelial cells influence T cell infiltration and function.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eCorrelation between ITGA5 and several immune cell types. \u003cstrong\u003e(B)\u003c/strong\u003e The percentage of different cell populations based on ITGA5 expression in endothelial cells. \u003cstrong\u003e(C,D)\u003c/strong\u003eUMAP visualization showing gene expression. Pseudotime analysis revealing the differentiation trajectory and functional state evolution of CD4+ T cells based on endothelial ITGA5 expression. \u003cstrong\u003e(E)\u003c/strong\u003e Multiplex immunofluorescence revealed the expression and co-localization of CD4, CD8, PD-1, ITGA5, and CD31 in cervical carcinoma and chronic cervicitis tissue.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9241604/v1/7d667be9c76727d8660459d6.png"},{"id":106808732,"identity":"2e429f48-91fa-4864-9178-48596f02ba07","added_by":"auto","created_at":"2026-04-13 15:59:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":365582,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eITGA5 knockout in endothelial cells impair the ability of antigen processing and presentation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eVolcano plot revealed the differentially expressed genes in ITGA5-low from ITGA5-high endothelial cells. \u003cstrong\u003e(B)\u003c/strong\u003e GSEA analysis identified the signaling pathways activated in ITGA5-low endothelial cells. \u003cstrong\u003e(C)\u003c/strong\u003e Gene knockout via the CRISPR/sgRNA system. \u003cstrong\u003e(D)\u003c/strong\u003e Western blotting validated ITGA5 knockout efficiency. \u003cstrong\u003e(E)\u003c/strong\u003e qRT-PCR showed the changes in mRNA level of MHC molecules in endothelial cells after ITGA5 knockout. \u003cstrong\u003e(F)\u003c/strong\u003e Flow cytometry confirmed surface protein expression of both MHC-I (HLA-ABC) and MHC-II (HLA-DRB) following ITGA5 knockout.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9241604/v1/3c484bbc1ff5b2b9553cdff3.png"},{"id":107480037,"identity":"6abdf88f-3e26-464b-acd6-4364126efbf5","added_by":"auto","created_at":"2026-04-22 02:03:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":405965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eITGA5 knockout in endothelial cells influence T cell function.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic diagram of the co-culture system between endothelial cells and T cells. \u003cstrong\u003e(B)\u003c/strong\u003e GSEA analysis revealed the activated pathways in T cells co-cultured with ITGA5-low endothelial cells. \u003cstrong\u003e(C)\u003c/strong\u003e Flow cytometry analyzed the surface expression of exhaustion markers and intracellular effector molecules in CD4\u003csup\u003e+\u003c/sup\u003e T cells following co-culture with endothelial cells expressing different levels of ITGA5. \u003cstrong\u003e(D) \u003c/strong\u003eFlow cytometry analyzed the surface expression of exhaustion markers and intracellular effector molecules in CD8\u003csup\u003e+\u003c/sup\u003e T cells following co-culture with endothelial cells expressing different levels of ITGA5.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9241604/v1/e467f9ceaadb36dcdea6178d.png"},{"id":106808734,"identity":"4592a31a-7261-4f2c-9a06-db2dffc6ad60","added_by":"auto","created_at":"2026-04-13 15:59:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":242306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSoluble factors from endothelial cells do not alter T-cell phenotype.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eFlow cytometry analyzed the surface expression of activation markers in CD4\u003csup\u003e+ \u003c/sup\u003eand CD8\u003csup\u003e+\u003c/sup\u003e T cells following co-culture with endothelial cells expressing different levels of ITGA5. \u003cstrong\u003e(B) \u003c/strong\u003eFlow cytometry analyzed the surface expression of exhaustion markers and intracellular effector molecules in CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells following co-culture with endothelial cells expressing different levels of ITGA5.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9241604/v1/4f753c1586d1f0eb4701966c.png"},{"id":106808735,"identity":"0c861ed1-de84-4a1f-b144-d8a66ccd176a","added_by":"auto","created_at":"2026-04-13 15:59:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":151917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eITGA5 inhibition potentiates PD-1–mediated antitumor responses in vivo.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eExperimental schematic of \u003cstrong\u003e(B)\u003c/strong\u003e. \u003cstrong\u003e(B) \u003c/strong\u003eTumor volume following engraftment of TC-1 cells in mice\u003cstrong\u003e \u003c/strong\u003eand subsequent treatment withcombination therapy group or either monotherapy. Right panels show individual tumor growth curves for each mouse in the indicated treatment groups. \u003cstrong\u003e(C-F) \u003c/strong\u003eKaplan-Meier survival analysis of patients receiving immune checkpoint blockade therapy from the CIDE database stratified by ITGA5 expression. (C) Overall survival in melanoma patients. (D) Overall survival in urothelial carcinoma patients. (E) Progression-free survival in renal cell carcinoma patients. (F) Progression-free survival in non-small cell lung cancer patients.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9241604/v1/a55cee0844d3f1997bd923ae.png"},{"id":107482095,"identity":"0e754cd2-a059-49b7-987f-1503921ea33f","added_by":"auto","created_at":"2026-04-22 02:21:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3099537,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9241604/v1/520d1bc6-fa6f-4797-a86c-d03979a4c8de.pdf"},{"id":106808729,"identity":"e36bd975-9bec-419f-8377-fa0e185b2ca9","added_by":"auto","created_at":"2026-04-13 15:59:28","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":7097481,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9241604/v1/0a68952e93561af03e5d5ea0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tumor Endothelial ITGA5 Expression Induces T cell Dysfunction in Cervical Cancer","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCervical cancer remains the most prevalent gynecological malignancy, with a mortality rate of 5.57 per 100,000 women\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Advanced-stage disease carries a poor prognosis, with recurrence rates of 28%\u0026ndash;64% in patients with stage IIB\u0026ndash;IV disease\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Current therapeutic options for cervical cancer, including conventional chemotherapy, radiotherapy, anti-VEGFA-targeted therapy, and immune checkpoint blockade targeting PD-1/PD-L1, provide limited survival benefit and rarely achieve durable remission\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. A fundamental contributor to cervical cancer lethality is the presence of tumor vascular endothelial cells (TVECs) which originated from normal vascular endothelial cells surrounding the tumor\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. TVECs drive cervical cancer progression by promoting angiogenesis, metastasis and conferring resistance to chemotherapy and anti-angiogenic therapy\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Furthermore, TVECs play a pivotal role in orchestrating cervical cancer immunosuppressive microenvironment and promoting immunotherapy resistance\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Studies have shown that vascular endothelial cells can modulate T cell responses independently of professional antigen-presenting cell activation. This immunomodulatory function is achieved through the expression of co-stimulatory receptors, co-inhibitory receptors, adhesion molecules, and secretion of cytokines\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIntegrins are a family of heterodimeric cell-surface receptors that interact with diverse ligands and regulate a wide range of physiological processes, including cell migration, invasion, and proliferation\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Accumulating evidence indicates that integrin signaling also plays important roles in tumor progression. For instance, our previous study showed that cervical cancer cells maintained a pro-angiogenic ITGA5/AKT/VEGFA signaling axis\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Beyond their roles in tumor cells, integrins are also critical regulators of immune responses, including the maintenance of T-cell function and the modulation of inflammatory signaling\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Consistently, targeting integrin α5 in fibroblasts has been shown to enhance anti-tumor immunity and improve the efficacy of immune checkpoint blockade in colorectal cancer\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, a functional assessment of integrins in TVECs is still lacking. To address this gap, we performed a comprehensive assessment of integrin expression and function in cervical cancer-derived TVECs. We identify ITGA5 as a key regulator of T-cell activity within the cervical cancer microenvironment, providing a potential target for enhancing immunotherapy responsiveness.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and cell culture\u003c/h2\u003e \u003cp\u003eWe purchased the human umbilical vein cell line, EA.hy926 from Procell Inc. (Wuhan, China). The endothelial cell line was cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) (Gibco, Cat#C11995500BT) supplemented with 10% FBS (Gibco, Cat#A5256701) and 1% penicillin/streptomycin (Beyotime, Cat#C0222). Primary T cells were cultured in (RPMI) 1640 medium (Gibco, Cat#C11875500BT) supplemented with 10% FBS, 50 IU/ml interleukin-2 (IL-2) (Novoprotein, Cat#C013), 5 ng/mL interleukin-7 (IL-7) (Novoprotein, Cat#C086) and 5 ng/mL interleukin-15 (IL-15) (Novoprotein, Cat#C016). All cells were cultured in a humidified incubator at a constant temperature of 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. All cell lines were authenticated using Short Tandem Repeat (STR) profiling and tested annually for mycoplasma contamination via PCR-based detection of cellular supernatants. Cells were used within 20 passages.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIsolation and expansion of human T cells and ethics\u003c/h3\u003e\n\u003cp\u003ePeripheral blood was obtained from healthy volunteers who provided informed consent. All procedures involving human participants were conducted in accordance with the Declaration of Helsinki and were approved by the Ethics Committee of Suzhou Hospital Affiliated to Nanjing Medical University (KL901461). All blood samples were handled following the required ethical and safety procedures. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood by Ficoll density gradient centrifugation (Meilunbio, Cat#PWL100-1). T cells were isolated from PBMCs using CD3 magnetic beads (T\u0026amp;L Biotechnology, Cat#TL-622) and then stimulated with anti-CD3(T\u0026amp;L Biotechnology, Cat#GMP-TL101-0500) and anti-CD28 antibody (T\u0026amp;L Biotechnology, Cat#GMP-TL102-0500) for 48 h. We employed human CD4 or CD8 positive selection magnetic beads to isolate pure populations of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells for further functional assays.\u003c/p\u003e\n\u003ch3\u003eCo-culture experiment\u003c/h3\u003e\n\u003cp\u003eOne day in advance, EA.hy926 cells were plated in 48-well plates at a density of about 50%. The next day, the supernatant was changed by adding cervical cancer cell supernatant at a 1:1 ratio and incubated for 48 h. For direct co-culture, we co-incubated EA.hy926 cells with CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell at a ratio of 2:1 for 48 h. For indirect co-culture, CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells were incubated for 48 h with a 1:1 mixture of supernatant collected from EA.hy926 cells. To delineate the functional and phenotypic changes induced by co-culture, we performed transcriptome sequencing on the purified CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell populations. To avoid the confounding effects of exogenous cytokines, the culture medium for co-culture experiments was devoid of IL-2, IL-7, and IL-15.\u003c/p\u003e\n\u003ch3\u003eMultiplex Immunofluorescence Staining\u003c/h3\u003e\n\u003cp\u003eFFPE sections were deparaffinized in xylene and subsequently rehydrated through a series of graded ethanol solutions. Antigen retrieval was conducted in EDTA buffer (pH 8.0) with microwave heating (medium power 8 min, standing 8 min, medium-low power 7 min). After cooling and PBS washing, sections were blocked with 3% BSA for 30 min at room temperature. Primary antibodies diluted in PBS were applied to the tissue sections and incubated overnight at 4\u0026deg;C: ITGA5 (Abcam, Cat#Ab150361, 1:400), CD31 (Huilan Biotech, Cat#ABB00001, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e:100), CD4 (Huilan Biotech, Cat#HL18254, 1:250), CD8 (Huilan Biotech, Cat#HL06846, 1:100), PD-1 (Huilan Biotech, Cat#HL20641, 1:75), PD-L1 (Huilan Biotech, Cat#HL21176, 1:250). Co-staining of CD4, CD8 and PD-1 as well as PD-L1, ITGA5 and PECAM was performed using a four-color mIHC fluorescence kit (Huilanbio Biological Technology, Shanghai, China) based on tyramide signal amplification (TSA) technology according to the manufacture\u0026rsquo;s instructions. After washing with PBS, the sections were incubated with the appropriate fluorophore-conjugated secondary antibodies for 50 min at room temperature in the dark. Following PBS washes, nuclei were stained with DAPI for 10 min. Slides were mounted with anti-fade medium and imaged under a Nikon fluorescence microscope. The excitation wavelength of DAPI was 330\u0026ndash;380 nm, the emission wavelength was 440 nm; the excitation wavelength of CD8 and PD-L1 was 488 nm, the emission wavelength was 520 nm; the excitation wavelength of CD4 and ITGA5 was 647 nm, the emission wavelength was 690 nm; the excitation wavelength of PD-1 and PECAM was 555 nm, the emission wavelength was 570 nm.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eFlow Cytometry\u003c/h3\u003e\n\u003cp\u003eAll flow cytometry experiments were performed using standard sample processing and staining protocols. Assays were performed on Navios EX (Beckman Coulter, USA) or BD LSRFortessa (BD Biosciences, USA). Live cells were identified as staining negative for DAPI. Raw data were analyzed with the FlowJo software v.10.8.1. The following antibodies were used for flow cytometry: ITGA5 (also known as CD49e, APC, Biolegend, Cat#328012), HLA-ABC (FITC, eBioscience, Cat#11\u0026minus;9983\u0026minus;42), HLA-DRB1 (FITC, eBioscience, Cat#E-AB-F1111C), CD3 (BV421, RY610, BD Biosciences, Cat#566697, 571134), LAG3 (BV480, PE, BD Biosciences, Biolegend, Cat#746609, 369306), PD\u0026minus;1 (BB515, BD Biosciences, Cat#565936), CD4 (BB700, BD Biosciences, Cat#568370), TIM3 (PE/APC, BD Biosciences, Biolegend, Cat#565570, 345012), CD62L (PE-CF594, BD Biosciences, Cat#562330), CCR7 (APC, BD Biosciences, Cat#566762), CD69 (PE/Cy7, BD Biosciences, Cat#557745), CD8 (APC/Cy7, BD Biosciences, Cat#557834), CTLA4 (PE/Cy7, Biolegend, Cat#349914), IL\u0026minus;2 (BV421, Biolegend, Cat#500327), Perforin (BV711, Biolegend, Cat#308130), IFN-γ (PE/Cy7, BD Biosciences, Cat#560741), Granzyme B (Alexa Fluor 700, BD Biosciences, Cat#560213), Zombie Aqua Fixable Viability Kit (Biolegend, Cat#423101).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReal-time quantitative polymerase chain reaction\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using TRlzol reagent (Thermofisher, Cat#15596018) and reverse transcribed into cDNA with the HiScript III qRT SuperMix kit (Vazyme, R323-01). qPCR was performed with Novostart SYBR qPCR Supermix Plus (Novoprotein, E096-01A) in a LightCycler 480 (ROCHE) instrument. The sequences of the primers were as follows:\u003c/p\u003e\u003cp\u003eHuman ITGA5 (Forward primer, 5\u0026rsquo;-TTACGGGACTCAACTGCACC-3\u0026rsquo;, Reverse primer, 5\u0026rsquo;-AGCCTGAAACACTCAGCCTC-3\u0026rsquo;), Human PD-L1 (Forward primer, 5\u0026rsquo;-GGACAAGCAGTGACCATCAAG-3\u0026rsquo;, Reverse primer, 5\u0026rsquo;-CCCAGAATTACCAAGTG AGTCCT-3\u0026rsquo;), Human HLA-A (Forward primer, 5\u0026rsquo;-AGATACACCTGCCATGTGCAGC-3\u0026rsquo;, Reverse primer, 5\u0026rsquo;-GATCACAGCTCCAAGGAGAACC-3\u0026rsquo;), Human HLA-B (Forward primer, 5\u0026rsquo;-CTGCTGTGATGTGTAGGAGGAAG-3\u0026rsquo;, Reverse primer, 5\u0026rsquo;-GCTGTGAGAG ACACATCAGAGC-3\u0026rsquo;), Human HLA-DRB1 (Forward primer, 5\u0026rsquo;-GAGCAAGATGCTGAGTGGAGTC-3\u0026rsquo;, Reverse primer, 5\u0026rsquo;-CTGTTGGCTGAAGTC CAGAGTG-3\u0026rsquo;), Human HLA-DRA (Forward primer, 5\u0026rsquo;-AGCTGTGGACAAAGCCAACCTG-3\u0026rsquo;, Reverse primer, 5\u0026rsquo;-CTCTCAGTTCCACAGG GCTGTT-3\u0026rsquo;)\u003c/p\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction and virus transfection\u003c/h2\u003e \u003cp\u003eRestriction enzymes used in this study were purchased from New England Biolabs (NEB). Plasmids were constructed using NEBuilder HiFi DNA Assembly Master Mix with polymerase chain reaction (PCR) products and backbone restriction digests. For guide plasmid cloning, protospacer oligos were annealed and then inserted using BsmBI golden gate assembly into lentiCRISPR v2-U6-sgRNA plasmids (Addgene, Cat#52961) as previously reported. SgRNAs used in this study were: sgNT 5\u0026rsquo;-CTCTGCTGCGGAAGGATTCG-3\u0026rsquo;, sgITGA5#1 5\u0026rsquo;-GGGCTTCAACTTAGACGCGG-3\u0026rsquo;, sgITGA5#2 5\u0026rsquo;-GGGGCAACAGTTCGAGCCCA-3\u0026rsquo;. For the generation of stable ITGA5-knockout cells, EA.hy926 cells were seeded in 96-well plates (3,000\u0026ndash;4,000 cells/well) and transduced at 20\u0026ndash;30% confluence with lentivirus carrying either ITGA5-targeting or control sgRNA at an MOI of 10, using HitransG/P as an enhancer. After 24 h, the medium was replaced with fresh complete medium for 48 h, followed by selection with puromycin (1:5,000) for 2 weeks. Knockout efficiency was confirmed by Western blot before downstream assays were performed at 7\u0026ndash;10 days post-transduction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePublic database data analysis\u003c/h2\u003e \u003cp\u003eGene expression data from the cervical cancer-related datasets GSE171894, GSE197461, GSE236738, and GSE208653 were obtained from the GEO database, comprising 4 normal cervical tissue samples, 2 precancerous lesion samples, and 33 cervical cancer tissue samples. Using R language, we integrated the cells from all datasets and performed cell clustering. The mRNA expression levels of integrin alpha 5 (ITGA5) were analyzed, and samples with expression levels below the average were defined as the ITGA5-low group, while those above the average were designated as the ITGA5-high group. Using the GEPIA2 database, we examined the correlation between ITGA5 and vascular endothelial markers, as well as the overall survival rates of individuals with different endothelial ITGA5 expression levels. The TIMER database was employed to analyze the correlation between ITGA5 and the infiltration degree of various immune cell populations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnimal studies\u003c/h2\u003e \u003cp\u003eSix-week-old female C57BL/6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were randomly assigned to four groups (n\u0026thinsp;=\u0026thinsp;4 per group) and subcutaneously injected with 3 \u0026times; 10⁶ TC-1 murine cervical cancer cells to establish syngeneic tumors. Beginning on day 5 post-implantation, mice were treated with anti-ITGA5 antibody, anti-PD-1 antibody, a combination of both antibodies, or PBS as control until day 17. Tumor dimensions were measured every two days with calipers, and tumor volumes were calculated using the formula: volume = (length \u0026times; width\u0026sup2;) / 2.\u003c/p\u003e \u003cp\u003eThe antibodies used for animal studies are listed as follows: ATN\u0026minus;161 (integrin α5β1 Antagonist, Selleck, Cat#S8454), In vivo MAb anti-mouse PD\u0026minus;1 (CD279) antibody (Bioxcell, Cat#BE0273).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 10 (GraphPad Software), R version 4.5.1 and GSEA version 4.4.0 were taken for statistical analyses. For comparisons between two groups, we used the two-tailed Student\u0026rsquo;s t test. Signifcant differences were determined as: *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHigh expression of ITGA5 in TVEC correlates with poor prognosis in cervical cancer\u003c/h2\u003e \u003cp\u003eIn our previous study, immunohistochemistry analysis of cervical cancer tissues revealed that ITGA5 was not only expressed in tumor cells but also in stromal compartments, particularly in vascular endothelial cells\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. This observation suggests a potential role for endothelial integrins in shaping the tumor microenvironment (Supplementary Fig.\u0026nbsp;1A), consistent with previous reports\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. To further characterize integrin expression in endothelial cells, we analyzed integrin profiles in cervical cancer using publicly available single-cell RNA sequencing datasets. Specifically, we integrated four datasets from the GEO database (GSE236738, GSE208653, GSE197461, and GSE171894), comprising 4 normal cervical tissue samples, 2 precancerous lesion samples, and 33 cervical cancer tissue samples. Unbiased clustering analysis of all cells revealed 10 distinct populations, annotated by their unique marker gene expression profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,B). Among the integrin family members, ITGA5, ITGA6, and ITGB1 were the most highly expressed in endothelial cells (Supplementary Fig.\u0026nbsp;1B). Notably, ITGA5 was preferentially enriched in endothelial cells, characterized by the canonical markers PECAM1, VWF, and IGFBP7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Importantly, analysis of the GEPIA database revealed that ITGA5 was the only integrin significantly associated with poor prognosis in cervical cancer patients (Supplementary Fig.\u0026nbsp;1C). Based on these findings, we focused on elucidating the role of endothelial ITGA5 in cervical cancer progression.\u003c/p\u003e \u003cp\u003eFurther analysis revealed that ITGA5 expression was positively correlated with endothelial markers CDH5 and PECAM1. Notably, ITGA5 expression was also positively correlated with VEGFR2 and CD34, markers enriched in tumor vascular endothelial cells (TVECs) relative to normal endothelial cells (NECs), supporting preferential upregulation of ITGA5 in TECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). To validate these findings in clinical specimens, multi-color immunohistochemistry (mIHC) with CD31 and ITGA5 antibody confirmed colocalization of ITGA5 with CD31, with increased ITGA5 expression observed in endothelial cells within cervical cancer tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). To further assess the clinical significance of TVEC ITGA5, we analyzed TCGA and GTEx datasets and found that high ITGA5 normalized by PECAM1 expression reflecting endothelial cells with elevated ITGA5 levels, was significantly associated with reduced overall survival in patients with cervical squamous cell carcinoma (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Moreover, elevated ITGA5 normalized by PECAM1 expression correlated with poor prognosis across multiple cancer types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Collectively, these findings demonstrate that ITGA5 is highly expressed in tumor-associated endothelial cells and is associated with poor clinical outcomes, highlighting a potential role for endothelial ITGA5 in cervical cancer progression.\u003c/p\u003e \u003cp\u003e \u003cb\u003eITGA5-expressing endothelial cells impede T cell infiltration and induce T cell dysfunction.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIntegrins have been shown to promote tumor progression by generating immune tolerance via the inhibition of immune cell responses\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. We therefore investigated whether ITGA5 modulates the cervical cancer microenvironment. Utilizing the TIMER2 database, we analyzed the correlation between ITGA5 expression and immune cell infiltration. TIMER2 analysis revealed an inverse correlation between \u003cem\u003eITGA5\u003c/em\u003e mRNA expression and T cells infiltration, particularly CD8\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Cervical cancer samples were further stratified into ITGA5-low and ITGA5-high groups based on the median expression level in the scRNA-seq dataset. In the ITGA5 high group, the infiltration levels of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells were significantly reduced, whereas no significant difference were observed in the infiltration of B cells, macrophages, dendritic cells, and other immune cell populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To further explore the functional states of T cells, we performed pseudotime analysis to reconstruct the differentiation trajectories and functional state evolution of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells based on ITGA5 expression level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC,D). Compared to ITGA5 low group, both CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells in ITGA5-high group exhibited a more rapid and pronounced trajectory toward functional exhaustion. To validate these findings at the tissue level, we performed multiplex immunofluorescence staining of cervical cancer tissue microarrays. Tumor tissue exhibited enrichment of PD-1\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells and ITGA5\u003csup\u003e+\u003c/sup\u003e CD31\u003csup\u003e+\u003c/sup\u003e populations compared to normal cervical tissues, along with an increase proportion of PD-1\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Collectively, these results indicate that elevated ITGA5 expression is associated with an immunosuppressive microenvironment in cervical cancer, characterized by impaired T cell infiltration and increased T cell dysfunction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eITGA5 knockout in endothelial cells enhance MHC expression\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular mechanisms underlying endothelial ITGA5 regulated T cell infiltration, we reanalyzed publicly available scRNA-seq datasets. Compared with ITGA5 high endothelial cells, genes involved in antigen processing and presentation pathway (\u003cem\u003eHLA-DRB5\u003c/em\u003e, \u003cem\u003eHLA-DQA1\u003c/em\u003e and \u003cem\u003eHLA-DQB1., et al\u003c/em\u003e) were enriched in ITGA5 low endothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Consistently, gene set enrichment analysis (GSEA) revealed that antigen processing and presentation via both MHC class I and MHC class II pathways were indeed enriched in ITGA5 low endothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Supporting this observation, previous studies have demonstrated that human renal vascular endothelial cells express the major histocompatibility complex class II (HLA-DR) molecule and are capable of presenting antigens to CD4\u003csup\u003e+\u003c/sup\u003e T cells\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. To experimentally validate whether ITGA5 regulates antigen processing and presentation pathway, we generated a CRISPR-mediated ITGA5 knockout in a human umbilical vein endothelial cell line (EA.hy926) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC,D). Compared with control cells, ITGA5-deficient endothelial cells exhibited increased mRNA expression of \u003cem\u003eHLA-A\u003c/em\u003e, \u003cem\u003eHLA-B\u003c/em\u003e, \u003cem\u003eHLA-DRA\u003c/em\u003e and \u003cem\u003eHLA-DRB1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Consistent with this transcriptional changes, flow cytometry analysis revealed a corresponding increase in the surface expression of both MHC-II (HLA-DRB) and MHC-I (HLA-ABC) proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Collectively, these results demonstrate that ITGA5 suppresses antigen presentation in endothelial cells by downregulating MHC class I and class II expression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eITGA5 knockout in endothelial cells enhances T cell function in a contact-dependent manner.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether tumor-associated endothelial cells regulate T cell function, we performed a direct in vitro co-culture assay. Endothelial cells were first stimulated with tumor cell conditioned medium for 48 h and then co-cultured with Healthy donor-derived T cells for an additional 48 h. Following co-culture, T cells were isolated using magnetic-activated cell sorting (MACS) and separated into CD4⁺ and CD8⁺ subsets for subsequent flow cytometric analysis and transcriptomic sequencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). GSEA revealed that CD8⁺ T cells co-cultured with ITGA5 deficient endothelial cells exhibited significant enrichment of genes involved in T cell mediated cytotoxicity, compared with CD8⁺ T cells co-cultured with control endothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Consistent with these transcriptomic changes, flow cytometry analysis showed that both CD4⁺ and CD8⁺ T cells co-cultured with ITGA5 deficient endothelial cells displayed reduced surface expression of exhaustion markers (LAG3 and TIM3) and increased production of effector cytokines (IL-2 and IFN-γ), compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC,D). Together, these results demonstrate that ITGA5 deficiency in tumor-associated endothelial cells enhances T cell effector function and alleviates T cell exhaustion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSecreted factors from endothelial cells are insufficient to modulate T cell phenotype\u003c/h2\u003e \u003cp\u003eTo evaluate the effects of secreted factors, we established an indirect co-culture system by treating healthy donor-derived T cells with conditioned medium collected from endothelial cells expressing different levels of ITGA5 and cervical cancer cells. Flow cytometry analysis revealed no differences in the CD62L\u003csup\u003e+\u003c/sup\u003e CCR7\u003csup\u003e+\u003c/sup\u003e central memory T cell (TCM) population following stimulation with endothelial conditioned medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In addition, the expression of early activation marker CD69 and classical exhaustion markers (PD-1, TIM-3, and LAG-3) remained unchanged in T cells regardless of ITGA5 expression in endothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These results indicate that modulation of T cell function by endothelial cells requires direct cell\u0026ndash;cell contact, rather than being mediated solely by secreted factors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eITGA5 blockade enhances the anti-tumor efficacy of PD-1 immunotherapy in vivo\u003c/h2\u003e \u003cp\u003eTo evaluate the therapeutic potential of ITGA5 blockade in vivo, we established a syngeneic cervical cancer model by subcutaneously injecting 3.5\u0026nbsp;million TC-1 murine cervical cancer cells into C57BL/6J mice. Following 5days after transplantation, mice were randomly assigned to four groups, PBS group, anti-ITGA5 antibody (5 mg/kg) treatment, anti-PD-1 antibody (5 mg/kg) treatment, or anti-ITGA5 antibody plus anti-PD-1 antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The tumor growth curves showed that combination therapy produced markedly greater anti-tumor efficacy than either monotherapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), demonstrating a strong synergistic interaction between ITGA5 blockade and immune reactivation. To determine the clinical relevance of these findings, we analyzed proteomic data from the Cancer Immunology Data Engine (CIDE) database. Elevated ITGA5 levels were associated with poor survival in hepatocellular carcinoma patients receiving anti\u0026ndash;PD-1 therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). This association extended across tumor types: urothelial cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), renal cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) and non-small cell lung cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). These clinical associations, together with our in vivo findings, position MEPCE as a central mediator of immune evasion and a promising therapeutic target for overcoming immunotherapy resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur study identifies integrin α5 (ITGA5) as a critical regulator of the immunosuppressive tumor microenvironment (TME) in cervical cancer. We demonstrated that elevated ITGA5 expression in tumor vasculature correlates with poor clinical outcomes. ITGA5 mediated immune evasion through suppression of MHC-I/II-dependent antigen presentation, resulting in impaired CD8\u0026thinsp;+\u0026thinsp;T cell infiltration and accelerated T cell exhaustion. Crucially, modulating endothelial ITGA5 expression directly reshaped the functional profile of co-cultured T cells, suggesting a novel mechanism of vascular-driven immune suppression.\u003c/p\u003e \u003cp\u003eIn the tumor microenvironment, beyond professional antigen presenting cells (APCs), other cell types including epithelial cells, vascular endothelial cells, fibroblasts, and even cancer cells are also capable of presenting tumor antigens via both MHC class I and class II molecules\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. These cell types within the TME are often referred to as \u0026ldquo;amateur APCs\u0026rdquo;. Amateur APCs vastly outnumber the professional APCs and may play a significant role in initiating anti-tumor immune responses\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Studies have indicated that TVECs dysregulated specific cell surface markers, reduced antigen presentation capacity, and impaired recruitment of immune cells, collectively contributing to immunosuppression\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Notably, Marelli-Berg et al. demonstrated that when VECs expressed MHC class II and were co-cultured with antigen-specific effector memory CD4\u003csup\u003e+\u003c/sup\u003e T cells (CD45RO\u003csup\u003e+\u003c/sup\u003e), these T cells migrate more rapidly across a monolayer of VECs\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn vitro experiments demonstrated that tumor endothelial cells with high ITGA5 expression induced CD4⁺ T cell exhaustion and impaired the cytotoxic function of CD8⁺ T cells through direct cell-cell contact. We hypothesized that ITGA5-high endothelial cells contribute to the establishment of an immunosuppressive tumor microenvironment (TME) through two potential mechanisms. First, ITGA5-high endothelial cells exhibited reduced ICAM-2 expression. Gene Set Enrichment Analysis (GSEA) further revealed suppression of the focal adhesion pathway, suggesting that elevated ITGA5 expression may promote an endothelial anergy phenotype. Such a dysfunctional state might impair immune cell adhesion to the endothelium, thereby limiting immune cell extravasation and contributing to an immunosuppressive tumor microenvironment (TME). Second, ITGA5-high tumor vascular endothelial cells (TVECs) displayed decreased expression of antigen-presenting molecules, which might attenuate T cell recruitment to the TME and ultimately result in reduced T cell infiltration. Notably, our in vivo experiments suggested that ITGA5 blockade might enhance the antitumor efficacy of PD-1 blockade, indicating that endothelial ITGA5 may serve as a potential therapeutic target to improve immune checkpoint blockade responses.\u003c/p\u003e \u003cp\u003eSeveral limitations of this study should be acknowledged. First, the translational relevance of our findings is constrained by methodological considerations. The simplified cell culture systems employed here do not fully recapitulate the complex cellular interactions and mechanical cues present in human tumors. Second, the therapeutic potential of ITGA5 targeting requires further validation in preclinical models that more accurately reflect human tumor-immune interactions prior to clinical translation.\u003c/p\u003e \u003cp\u003eThe therapeutic relevance of ITGA5 in cancer immunotherapy is largely determined by its cell-type-specific and tissue-restricted expression. Single-cell transcriptomic analysis revealed that ITGA5 is predominantly expressed in tumor-associated endothelial cells, with expression substantially higher than in normal cervical endothelium, indicating that therapeutic targeting of ITGA5 may primarily affect the tumor endothelium. Functionally, endothelial ITGA5 restricts T cell infiltration and effector activity while promoting tumor angiogenesis and progression. Therefore, inhibiting ITGA5 could simultaneously enhance antitumor immunity and suppress tumor vascularization, supporting its potential as a promising therapeutic target.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLu Shen was funded by Basic Research Program of Jiangsu (BK20230222).\u003c/p\u003e\n\u003cp\u003ePeng Lin was funded by Basic Research Program of Jiangsu (BK20250446).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll study procedures strictly adhered to the Declaration of Helsinki. Ethics approval and consent to perform animal studies were approved by the Institutional Animal Care and Use Committee of the Nanjing medical university. The Ethics Committee of the Affiliated Suzhou Hospital of Nanjing Medical University approved all human-associated tissues used in this research\u0026nbsp;(KL901461). The informed consent has been signed by all patients before their tissues were acquired.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available within the article. Additional data related to this research may be requested from the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll listed authors consent to the submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLu Shen: Writing-original draft, Investigation, Conceptualization, Methodology. XiaoQiu Dai: Investigation, Methodology, Formal analysis. Yue Liu: Investigation, Methodology. Yayun Zhang: Formal analysis. Xiaoxue Xi: Data Curation. Xiao Wu: Data Curation. Chen Wang: Conceptualization, Methodology. Peng Lin: Methodology, Writing-review \u0026amp; editing, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to all the participants who contributed to this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eB. Han, R. Zheng, H. Zeng et al., Cancer incidence and mortality in China, 2022. J. Natl. 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Immunol. \u003cb\u003e162\u003c/b\u003e(2), 696\u0026ndash;703 (1999)\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ceon","sideBox":"Learn more about [Cellular Oncology](http://link.springer.com/journal/13402)","snPcode":"13402","submissionUrl":"https://submission.nature.com/new-submission/13402/3","title":"Cellular Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cervical cancer, tumor vascular endothelial cells, integrin ITGA5, immunosuppressive tumor microenvironment, T cell dysfunction","lastPublishedDoi":"10.21203/rs.3.rs-9241604/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9241604/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTumor vascular endothelial cells (TVECs), which originate from normal vascular endothelial cells surrounding the tumor, play a critical role in shaping the immunosuppressive tumor microenvironment and contributing to resistance to immunotherapy in cervical cancer. However, the molecular mechanisms underlying TVEC-mediated regulation of anti-tumor immunity remain elusive. Here, we performed a comprehensive assessment of integrin family proteins in cervical cancer-derived TVECs and identified ITGA5 as a key regulator of T cell activity within the tumor microenvironment. Loss of ITGA5 in TVECs markedly enhanced antigen presentation by upregulating both MHC class I and class II pathways, thereby promoting T cell activation and cytotoxic function. Importantly, therapeutic blockade of ITGA5 significantly enhanced the efficacy of anti\u0026ndash;PD-1 treatment, and the combination of ITGA5 antibody and PD-1 blockade synergistically inhibited tumor growth in orthotopic murine models. Collectively, these findings identify ITGA5 as a previously unrecognized immune regulator in tumor vasculature and suggest that targeting endothelial ITGA5 may represent a promising strategy to improve immunotherapy efficacy in cervical cancer.\u003c/p\u003e","manuscriptTitle":"Tumor Endothelial ITGA5 Expression Induces T cell Dysfunction in Cervical Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 15:59:23","doi":"10.21203/rs.3.rs-9241604/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-28T12:36:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7552410815375057120706927779136492285","date":"2026-04-07T17:12:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291570026139134655438613670804724308631","date":"2026-04-07T09:25:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-07T09:02:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-03T05:54:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-03T05:53:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular Oncology","date":"2026-03-27T07:33:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ceon","sideBox":"Learn more about [Cellular Oncology](http://link.springer.com/journal/13402)","snPcode":"13402","submissionUrl":"https://submission.nature.com/new-submission/13402/3","title":"Cellular Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d391203b-cd87-4481-b522-35127fe40c4d","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T15:59:23+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 15:59:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9241604","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9241604","identity":"rs-9241604","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}