Oral Cancer-derived miR-762 Suppresses T Cell Infiltration and Activation by Horizontally Inhibition of CXCR3 Expression

Oral Cancer-derived miR-762 Suppresses T Cell Infiltration and Activation by Horizontally Inhibition of CXCR3 Expression | 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 Article Oral Cancer-derived miR-762 Suppresses T Cell Infiltration and Activation by Horizontally Inhibition of CXCR3 Expression Hsuan-Yu Peng, Chia-Wei Chang, Ping-Hsiu Wu, Li-Jie Li, Yu-Lung Lin, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4636968/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Oral cancer is an immune cold tumor characterized by an immunosuppressive microenvironment with low cytotoxic activity to eliminate tumor cells. Tumor escape is one of the initial steps in cancer development. Understanding the underlying mechanisms of cancer escape can help researchers develop new treatment strategies. In this study, we found that the oral oncogenic miR-762 can suppress T-cell recruitment and cytotoxic activation in the tumor microenvironment through horizontal transmission from oral cancer cells to adaptive immune T-cells. This horizontal transmission of miR-762 directly suppresses CXCR3 expression in T-cells, inhibiting CXCR3-induced T-cell migration and downstream T-cell cytotoxic activity by disrupting AKT activation. Additionally, miR-762 transmission suppressed T-cell activation marker expression, T-cell proliferation, IL-12 secretion, and T-cell cytotoxicity. In conclusion, our findings reveal a novel miR-762/CXCR3 axis that regulates the immunosuppressive microenvironment in oral cancer and may be a potential RNA-targeted therapeutic approach to restore the anti-tumor immune response in oral cancer immunotherapy. Biological sciences/Cancer/Oral cancer Biological sciences/Cancer/Tumour immunology OSCC microRNA horizontal transmission CXCR3 immune escape Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Cancer is one of the deadliest diseases globally, with an estimated 18.1 million new cases and 10 million deaths annually. In the United States, approximately 1.9 million new cancer cases and about 609,820 deaths are expected each year 1 .. Cancer remains the leading cause of death in Taiwan, presenting a significant public health challenge. Specifically, oral cancer is one of the most lethal cancer in Taiwan, profoundly affecting patients' quality-of-life, increasing medical expenses, and causing considerable socioeconomic impact 2 , 3 . The host immune system plays a crucial role in eliminating cancer cells during the initiation phase of cancer 4 . Effector immune cells, such as effector CD4 + and CD8 + T-cells, γδT cells, and natural killer (NK) cells, are responsible for elimination of cancer 5 . For these effector cells to eliminate tumor cells, immune cells must infiltrate into tumor microenvironment. However, oral cancer employs several strategies to evade the immune system, including the expression of inflammatory cytokines, suppression of cytotoxic CD8 lymphocytes, downregulation of antigen processing machinery, generation of specific inhibitory lymphocytes, and expression of immune checkpoint ligands and receptors. These mechanisms increase oral cancer's resistance to cytotoxic T-cells and inhibit normal T-cell function, facilitating tumor initiation and growth 6 – 8 . Understanding these immune escape mechanisms is essential for developing new treatment strategies for oral cancer. Chemokines-induced immune cell recruitment Chemokines are small cytokines that direct immune cell recruitment. The chemokine family consists of about 50 ligands, 20 G protein-coupled receptors (GPCRs), and 4 atypical chemokine receptors (ACKRs) 9 . They play a key role in cancer immunity by attracting immune cells to the tumor microenvironment (TME). CXCR3, a critical receptor, aids in T and NK cell recruitment and macrophage polarization 10 . Its expression is linked to the progression of cancers like breast, colorectal, and pancreatic. High levels of CXCR3 ligands in pancreatic and colorectal cancers are associated with poor survival, making it a potential prognostic marker 11 . The diverse functions of CXCR3 underscore its potential as a target for immunotherapy and its significance in cancer and immune regulation. Extracellular microRNA as Cancer Microenvironment Coordinator The tumor microenvironment (TME) is a dynamic ecosystem involving cancer cells and host cells like immune cells, fibroblasts, and endothelial cells 12 . In tumor initiation, dysregulated of immune cell distribution shapes the immune landscape of TME 13 . The immune status in TME affects patient prognosis and treatment outcomes for chemotherapy and immunotherapy. Extracellular vesicles (EVs) serve as key communicators between cancer and host cells, carrying proteins and nucleic acids like enzymes, receptors, mRNA, non-coding RNA, and microRNA 14 . The TME EVs harbor proteins and free-nucleic acids, such as enzymes, receptors, mRNA, non-coding RNA, and microRNA, serve as colonial rulers for horizontal transmission between cancer donor and host recipient normal cells 15 . MicroRNAs, known for their stability and gene regulation capabilities, play a significant role in TME regulation 16 . Aberrant microRNA or microRNA cluster expression has been reported in oral cancer tumorigenesis and progression 17 . miR-762, an oncogenic microRNA, contributes to head and neck cancer and is a poor prognostic marker in colon cancer due to its role in WNT signaling 18 . Our group also found that miR-762 is a serum poor prognostic marker and contributing to colon cancer progression through WNT signaling activation 19 . This study found that miR-762 suppresses the immune response in oral cancer by targeting CXCR3 and hindering cytotoxic T-cell recruitment and activation. Materials and methods Cell lines Human acute T-cell leukemia Jurkat cells., human embryonic kidney 293T and human oral cancer cells SCC-4, SCC-9, SCC-15, SCC-25, Cal-27, Cal-33, HSC-2, HSC-3, HSC-3-M3, and HSC-4, Ca9-22, OSC-19, OSC-20, and SAS were purchased from original resource and listed in Table S1 . All cells were cultured in the standard culture condition by following instruction manual and maintained within 3 months. Additionally, all cells were regularly checked for mycoplasma infection and cell morphology to ensure cell health. Activation of Jurkat T cell The Jurkat cells were stimulated with 1.25 µg/ml of anti-CD3 (Clone: OKT3; BioLegend, San Diego, CA, USA) monoclonal antibody (mAb) and 1.25 µg/ml of anti-CD28 (Clone: CD28.2; BioLegend) mAb for 72 hours. Jurkat-CXCR3 cells were stimulated with the same amount of anti-CD3 and anti-CD28 mAb plus 10 ng/ml of IL-12 (R&D Systems, Inc, Minneapolis, MN, USA) for 72 hours. T cell cytotoxicity assay against OSCC cells 2 × 10 4 Jurkat cells were seeded in the upper-transwell chamber and co-transfected with or without miR-762 mimics and CXCR3-CDS expression vector for 72 hours. For T cell cytotoxicity, 1 × 10 4 Cal-27 cells were seeded in the lower-well. After 48 hours, Cal-27 cells were fixed in 70% ice-cold ethanol for 60 minutes then stained with 0.1% crystal violet/20% methanol solution and recorded under a light microscope. RNA extraction and real-time PCR (qRT‐PCR) Total RNA was isolated from cell lines using TRIzol reagent, and cDNA was synthesized by Roche First Strand cDNA Synthesis Kit. All primers were listed in Table S2 . qRT-PCR analysis was used ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and QuantStudio 3 real-time PCR system. For mature miRNA detection, cDNA was prepared using the miScript II RT Kit (Cat: 218161, QIAGEN, Valencia, CA, USA). U6 was used as the internal control for human miRNAs. The expression level was defined based on the threshold cycle, and relative expression levels were calculated as ▵▵Ct after normalization with the reference control. Protein extraction and western blot analysis Cells were lysed in RIPA buffer with protease and phosphatase inhibitor cocktail. 10–30 µg of protein lysates were loaded onto 10% SDS-PAGE and transferred into PVDF membranes. All antibodies were listed in table S3. The membranes were exposed using ECL (Merck-Millipore) for the HRP-coupled secondary antibodies and analyzed using the e-BLOT Touch Imager. Transwell migration assays T cell migration assays were performed using a 3-µm Transwell chamber (SPL 36224, Korea). 2×10 4 Jurkat cells were seeded in the upper-chamber in serum-free medium, and 600 µL of medium containing 2% FBS was added to the lower well. The cells were incubated for 24 hours to allow them to attach. After 24 hours of incubation, migrated cells were fixed on the membrane with 70% ethanol for 60 minutes at 4°C, washed twice with PBS, and the interiors of the inserts were cleaned with wet cotton swabs. Cells were stained with a cell stain solution (0.1% crystal violet, 20% methanol) and visualized under an inverted microscope (200× magnification; Olympus Corporation, Hachioji, Tokyo, Japan). Images were then analyzed and counted using an analytical imaging station software package (Imaging Research, Ontario, Canada). Plasmids and Transfection The pLX304-CXCR3 plasmid was generated by inserting full-length cDNA (CXCR3: NM_001504) into the vector following previously established protocols. The miR-762 mimic (PM) (ID: 15805, Cat: AM17100, Thermo Fisher Scientific) transfection mix was prepared using TransIT-X2 Transfection Reagent (Mirus, Madison, WI, USA) according to the manufacturer's protocol and applied to Jurkat cells. For lentivirus production, 293T cells were co-transfected with pMD2.G, pCMV▵R8.91, and lentiviral transfer plasmids using TransIT-LT1 Transfection Reagent (Mirus, Madison, WI, USA). The culture medium containing lentivirus was harvested 48 hours after transfection. Cells were removed from the culture medium by centrifugation at 500 g for 5 minutes and filtered through a 0.45-µm filter. The lentiviral stocks in the medium, containing polybrene (8 µg/mL), were used to infect the target cells for 6 hours. CCK-8 Assay: Equal numbers of Jurkat cells transfected with miR-762 mimics or non-targeting control (N.C.) for 72 hours were plated in a 96-well plate and allowed to grow for an additional 72 hours. Cell growth was determined using a Cell Counting Kit-8 (CCK-8) (Sigma-Aldrich, St Louis, USA). 10 µL of CCK-8 solution was added to each well of the plate, followed by incubation for 3 hours. The absorbance at 450 nm was measured using a 96-well plate SpectraMax 250 reader (Molecular Devices, CA, USA). Luciferase Reporter Assays: For NF-κB transcriptional activity and CDH1 promoter activity assay, the protocol was followed as per our previous work. For the 3’-UTR luciferase reporter assay, the CXCR3 3’-UTR region was generated by subcloning PCR-amplified full-length human CXCR3 3’-UTR (446 bp from the stop codon) into the modified 3’-UTR CpoI sites of the luciferase in the pGreenfire-CMV firefly luciferase-expressing vector (SBI, Palo Alto, CA, USA), using the primers: Forward: 5’-CGGACCGGGCCGGAATCCGGGCTCCCCTTTC-‘3 and Reverse: 5’-CGGTCCGTCCTGACGATCTTGTTTATT-3’. 293T cells were cultured in 24-well plates and co-transfected with 300 ng of CXCR3 3’-untranslated region (UTR) wild type or mutant type pGreenfire-CXCR3 3’-UTR reporter plasmid, along with 25 nM of miR-762 mimics (PM) or non-targeting control (N.C.), using TransIT-X2 Transfection Reagent (Mirus, Madison, WI, USA) according to the manufacturer’s instructions. The luciferase assay was performed 24 hours post-transfection with the ONE-Glo™ Luciferase Reporter Assay System (Promega, USA) following the manufacturer's protocol. 50 µL of ONE-Glo™ substrate was added into a white plate and the assay was conducted with a SpectraMax iD3 microplate reader. Flow Cytometry: The cells were harvested, washed twice with PBS, fixed in 1% paraformaldehyde (PFA) overnight at 4°C, washed again, and resuspended in flow cytometry buffer. To determine surface markers, these cells were labeled with the surface fluorochrome-conjugated antibodies CD25 and CD69 (BD Biosciences, Franklin Lakes, NJ) for 60 minutes on ice in the dark. The stained cells were analyzed using a Attune NxT Flow Cytometer and the quantitative analysis was performed using FlowJo v10 (Treestar, Ashland, OR). Enzyme-linked immunosorbent assay (ELISA) Cell supernatants were collected and processed using R&D Duoset ELISA kits (R&D Systems, Inc, Minneapolis, MN, USA) according to the manufacturer’s instructions for the detection of cell secretion of IL-12 (Cat# D1200). Statistical analysis All experiments were repeated at least 3 times. Data were presented as the mean ± standard deviation (SD) from repeated independent experiments. Differences between various treatment groups were assessed using the Student’s t test. Between-group differences were considered significant at P < 0.05. Data analyses were performed using GraphPad Prism Ver. 8 (San Diego, CA, USA). Results miR-762 Levels were Significantly Associated with the Poor Prognosis of Head and Neck Cancers Patients We investigated the relationship between miR-762 levels and clinical manifestations in patients with Head and Neck Cancer using data from the Kaplan-Meier plotter database. The high-miR-762 group had significantly shorter overall survival (OS) (Fig. 1 A). The median OS was 77.3 months in the low-miR-762 group versus 37.77 months in the high-miR-762 group. We also found that miR-762 levels are high in OSCC cell lines compared to normal keratinocyte cells (Fig. 1 B). Oral cancer is an immune cold tumor, and OSCC typically has an immunosuppressive microenvironment, involving immune checkpoint proteins, environmental immunosuppressive factors, and an imbalance between immunosuppressive and effective immune cell compositions 4 , 20 . This leads to diminished T-cell numbers and activity in the tumor, resulting in poor prognosis for oral cancer patients 21 . We found that miR-762 is secreted by OSCC cells (Fig. 1 C), with Cal-27 having the lowest secretion of miR-762 among the five OSCC cell lines (Cal-27, SCC-4, HSC-3, SAS, and SCC-15). Secreted miRNAs are highly stable in the TME and may serve as critical intercellular communication molecules, allowing cancer cells to manipulate or diminish surrounding recipient cells through horizontal transfection 22 , 23 . Furthermore, we found that miR-762 not only suppressed NF-κB signaling transactivation (Fig. 1 D) but also E-cadherin promoter transcription activity (Fig. 1 E), which may inhibit immune cell maturation and prevent migration into the TME. These results suggest that miR-762 plays an important role in the OSCC TME with an immune suppression function. We speculate that miR-762 may lead to decreased T-cell activation and function in the OSCC TME. Exogenous miR-762 inhibits the Jurkat cells activation and migration ability To demonstrate the function of extracellular miR-762 in the OSCC tumor microenvironment (TME), particularly in tumor immune evasion through the prevention of T-cell activation, we transfected Jurkat cells with mature miR-762 or scrambled miRNA (NC) for 72 hours. The surface T-cell activation markers, CD25 and CD69, were inhibited by miR-762 transfection (Fig. 2 A). Additionally, the proinflammatory cytokine IL-12, which augments the growth, differentiation, and activation of cytotoxic CD8 T-cells 24 , was reduced by miR-762 manipulation compared to the NC control (Fig. 2 B). This indicates that miR-762 inhibited Jurkat cell cytotoxic activity. Furthermore, miR-762 transfection reduced Jurkat cell proliferation (Fig. 2 C) and transwell migration ability (Fig. 2 D). These results show that miR-762 is an important suppressor of T-cell migration and activation in the TME. Additionally, we enforced miR-762 expression on Cal-27 cells (Fig. 2 E), which have both lower intracellular and secretion levels of miR-762 and harvested the cell culture supernatant as an oral cancer-derived conditioned medium to mimic the TME. The miR-762-containing Cal-27 conditioned medium (Cal-27-miR-762-CM) also suppressed PHA-activated Jurkat T-cell migration and further inhibited the gene expression of T-cell activation markers such as IL2RA, and CD69 (Fig. 2 F). Taken together, these results indicate that miR-762 in the TME can inhibit T-cell recruitment and activity. Exogenous miR-762 inhibits T-cell CXCR3 expression CXCR3 is primarily expressed on T-cells, dendritic cells, and natural killer cells, playing a crucial role in T-cell trafficking and function 25 . Moreover, CXCR3 has three variants, A and B, which exhibit differences in their expression profiles in the TME and have different functions in cancer progression 26 . To determine whether miR-762 could directly target CXCR3, we performed a 3’-untranslated region (3’-UTR) reporter assay in 293T cells. The CXCR3-3’-UTR was significantly suppressed by miR-762 mimic (PM) transfection (Fig. 3 A and B). Additionally, ectopic miR-762 expression directly suppressed CXCR3 mRNA and protein expression in Jurkat cells (Fig. 3 C to E). miR-762 selectively reduced the expression of the T-cell proliferation and migration-promoting variant CXCR3A. To further examine the role of extracellular miR-762 in the oral cancer tumor microenvironment, we applied OSCC conditioned medium to Jurkat T-cells to assess CXCR3 expression. The conditioned medium harvested from miR-762-expressing Cal-27 cells also suppressed CXCR3 expression in Jurkat T-cells (Fig. 3 G and H). These results indicate that oral cancer-secreted miR-762 can suppress T-cell CXCR3 expression in the oral tumor microenvironment. Interestingly, both direct transfection and oral cancer-derived miR-762 conditioned medium stimulated the expression of the CXCR3B variant (Fig. 3 F and H). CXCR3B has a unique receptor function for CXCL4, a chemokine that inhibits cytotoxic T-cell migration and activation. These results suggest that miR-762 may exert a unique T-cell suppression function through the CXCL4/CXCR3B axis, distinct from the traditional CXCR3A function. CXCR3 promotes cytotoxicity T-cell activation To further examine whether CXCR3 contributes to T-cell activation, we evaluated the T-cell activation status following ectopic CXCR3 expression (Fig. 4 A). We found that CXCR3 expression enhanced the expression of T-cell activation genes such as IL2RA (CD25), CD69, and CD71 mRNA (Fig. 4 B). Additionally, we observed that CXCR3 expression increased the expression of T-cell activation surface markers CD69, CD25, and CD154 in Jurkat cells (Fig. 4 C). Moreover, we also found that CXCR3 increased Jurkat T-cell proliferation (Fig. 4 D). IL-12, a T-cell-stimulating cytokine, promotes the growth and function of T-cells and the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T-cells and natural killer (NK) cells 27 . CXCR3 expression also stimulated IL-12 secretion from Jurkat T-cells (Fig. 4 E). Taken together, our results show that CXCR3 enhances Jurkat T-cell cytotoxicity by promoting T-cell polarization, proliferation, and increasing IL-12 secretion, thereby proving that CXCR3 plays a crucial role in T-cell activation. miR-762/CXCR3 suppression axis is critical in environment T-cell activation The molecular mechanisms underlying CXCR3 regulation of T-cell function are not fully understood. Several studies have suggested that the signal transduction of the chemokine receptor CXCR3 involves activating Ras/ERK, Src, and phosphoinositide 3-kinase(PI3K)/AKT pathways 28 , 29 . Additionally, CXCL10/CXCR3 activation can stimulate PI3K/AKT signaling, thereby promoting Th1 cell differentiation and migration. The PI3K/AKT pathways are then stimulated, leading to the promotion of cell survival, proliferation, and cytotoxic activity of CD8 + memory T-cells and NK cells. To clarify the interplay among the miR-762/CXCR3/Akt axis in OSCC immune evasion, we aimed to investigate whether oral cancer miR-762-induced CXCR3 downregulation plays a critical role in T-cell activation. To this end, we generated a CXCR3 expression plasmid lacking the 3’-UTR sequence, thereby preventing inhibition by miR-762. In the miR-762 mimic-transfected Jurkat T-cells, restoring CXCR3 expression (Fig. 5 A) could restore AKT activity (Fig. 5 B). The restored CXCR3 expression further reactivated T-cell polarization (Fig. 5 C and D) and proliferation ability (Fig. 5 E). Moreover, the critical T-cell cytokine IL-12 secretion was also restored by CXCR3 expression (Fig. 5 F). In summary, these results suggest that the miR-762/CXCR3 axis may regulate T-cell activation, polarization, proliferation, and IL-12 secretion in the tumor microenvironment. Horizontal Transmission of miR-762 Promotes Immune Escape in Oral Cancer Finally, we aimed to demonstrate the horizontal transmission of miR-762 between oral cancer cells and T-cells, which regulates T-cell cytotoxicity and promotes oral cancer immune escape. To do this, we used an indirect coculture system (Fig. 6 A). When miR-762 was introduced into low-expressing Cal-27 cells, T-cell migration (Fig. 6 B) and cytotoxicity (Fig. 6 C) were abolished by miR-762 expression. Moreover, this cancer immune escape could be reversed by reintroducing CXCR3 into the T-cells. Taken together, these results suggest that miR-762 attenuates T-cell cytotoxicity against cancer by reducing the expression levels of CXCR3. These findings indicate that T-cell inactivation induced by OSCC is primarily mediated by OSCC-derived miR-762. Discussion Oral cancer employs various immune escape mechanisms, primarily involving inhibitory lymphocytes and other immune cells that release cytokines suppressing cytotoxic CD8 lymphocytes. This study identified that horizontal transmission of miR-762 from oral cancer cells may contribute to immune escape by suppressing CXCR3 expression and activating the CXCR3-AKT signaling pathway in T lymphocytes (Fig. 6 D). Chemokines in the tumor microenvironment play a crucial role in cancer progression by promoting tumorigenesis 30 . However, chemokines also recruit adaptive immune cells, enhancing anti-tumor immunity. For instance, CXCR3, a receptor for chemokines CXCL9, CXCL10, and CXCL11, is more highly expressed in tumor tissues than in normal tissues 31 . In cancer cells, autocrine CXCR3 signaling promotes metastasis and growth through AKT signaling 11 , while CXCL9 and CXCL11 can inhibit tumor growth by facilitating immune cell infiltration 32 . Thus, tumors must find new ways to evade the immune system. Horizontal gene transfer (HGT) is vital in cancer progression 33 , 34 . Unlike genomic DNA or cell-free DNA, microRNAs quickly inhibit gene transcription and translation and are continuously supplied by cancer cells. MicroRNAs can be easily targeted and neutralized by antagonists or complementary anti-sense miRNA sequences 35 . RNA-based medicines, which gained popularity during the COVID-19 pandemic, could offer new therapeutic options 36 . Our discovery of miR-762’s role in oral cancer immune escape via horizontal transmission to immune cells in the tumor microenvironment suggests a new niche for RNA-based cancer therapies. Oral cancer is typically an immune cold tumor, showing low levels of T and NK cell infiltration 37 , 38 . CXCR3 is vital for lymphocyte migration and activation, and miR-762 in the tumor microenvironment (TME) may help maintain an immunosuppressive environment. Besides aiding immune escape during tumor initiation, the immune cell content in TME is crucial for predicting treatment outcomes and patient prognosis 39 . miR-762 distribution in TME prevents immune cell infiltration and worsens responses to conventional cancer treatments. Using anti-sense miR-762 could remove miR-762 from the TME, may enhance immune cell infiltration, and boost anti-tumor cytotoxicity, improving responses to chemotherapy and radiotherapy. In head and neck cancer, CXCR3 enhances lymphatic invasion and cancer growth 40 , while CXCR3A promotes cancer stem-like properties and chemoresistance 41 . These findings show the importance of CXCR3 in oral cancer progression. In late-stage Non-Small Cell Lung Carcinoma (NSCLC) patients, CXCR3 expression is diminished in both CD4 and CD8 T-cells 42 . CXCR3 is highly expressed in adaptive immune cells, aiding in chemotaxis and activation. Reduced CXCR3 expression in NK cells hinders immune cell recruitment to tumors 43 . Suppressing CXCR3 in immune cells can promote cancer progression and immune escape. Interestingly, miR-762 treatment and oral cancer-conditioned medium increase CXCR3B expression, which acts as the CXCL4 receptor 44 . CXCL4/CXCR3B has opposite effects to CXCL10 in T-cells, reducing proinflammatory IFN-gamma and increasing TH2 cytokines 45 . CXCL4 also inhibits activated T-cell proliferation 46 and stimulates Treg cell growth 47 . CXCR3B expression may further oral cancer immune escape and enhance the immunosuppressive environment via miR-762 transmission. In summary, our findings reveal a novel oral cancer escape mechanism through miR-762 transmission, which prevents immune cell infiltration and anti-tumor cytotoxicity. This may offer a new target for modifying the tumor microenvironment and restoring anti-cancer immunity in oral cancer. Declarations Acknowledgments The authors would like to acknowledge the great help and assistance from RNAiCore facility (Academia Sinca) and TMU Core Facility Center. Funding Information This study was supported by the Taipei Medical University Research Center of Cancer Translational Medicine sponsored by Higher Education Sprout Project, Taiwan Ministry of Education. This study was supported by Taipei Medical University Hospital, Taipei Medical University (110TMUH-NE-10) to JWC, Taipei Veterans General Hospital (V111C-004 and V112C-004) to PMC and WMC. Furthermore, MOST111-2314-B038-117 and NSTC112-2314-B038-042 to WMC, MOST111-2314-B038-087 to PHW, NSTC112-2314-B038-041 to HLL, and NSTC112-2320-B038-060-MY2 to YLL from the National Science and Technology Council, Taiwan. Conflict of Interest The authors declare that they have no competing interests. Data availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Data are located in controlled access data storage at Taipei Medical University. Ethics Statement Approval of the research protocol by an Institutional Reviewer Board: N/A. Informed Consent: N/A. Registry and the Registration No. of the study/trial: N/A. Animal Studies: N/A Author Contribution All authors approved to publish the study in this journal. Study conception and design: HYP, CWC, and WMC. Development of methodology: HYP, CWC., PHW, LJL, YLL, HLL, and WMC. Resource and Supervision: MH, JYC, and PMHC. Analysis and interpretation of data: HYP, CWC, LJL, YLL, PHW, and HLL. Writing, reviewing, and/or revision of the manuscript: HYP, CWC, PMHC, and WMC. References Siegel, R. L., Miller, K. D., Wagle, N. S. & Jemal, A. Cancer statistics, 2023. CA Cancer J Clin 73 , 17-48, doi:10.3322/caac.21763 (2023). Chou, C. W., Lin, C. R., Chung, Y. 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Supplementary Files SRWBRawmerge.pdf TableSformiR762.docx Cite Share Download PDF Status: Posted Version 1 posted 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-4636968","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":327671100,"identity":"95853be7-d4f3-4b3f-b097-4173673e682a","order_by":0,"name":"Hsuan-Yu Peng","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hsuan-Yu","middleName":"","lastName":"Peng","suffix":""},{"id":327671102,"identity":"6abbe380-084d-42f0-80d7-b337379541ed","order_by":1,"name":"Chia-Wei Chang","email":"","orcid":"","institution":"Taipei Medical University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chia-Wei","middleName":"","lastName":"Chang","suffix":""},{"id":327671104,"identity":"6737e495-4686-4342-9153-b5c81acca06d","order_by":2,"name":"Ping-Hsiu Wu","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ping-Hsiu","middleName":"","lastName":"Wu","suffix":""},{"id":327671106,"identity":"c19fe18d-5ffd-4cf9-8fd7-d494821f030e","order_by":3,"name":"Li-Jie Li","email":"","orcid":"","institution":"Osaka University","correspondingAuthor":false,"prefix":"","firstName":"Li-Jie","middleName":"","lastName":"Li","suffix":""},{"id":327671108,"identity":"1c57a476-d489-42ce-89f9-43feba3c937b","order_by":4,"name":"Yu-Lung Lin","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yu-Lung","middleName":"","lastName":"Lin","suffix":""},{"id":327671109,"identity":"28b99488-4230-47cf-a393-d59d0d4cab26","order_by":5,"name":"Hsin-Lun Lee","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hsin-Lun","middleName":"","lastName":"Lee","suffix":""},{"id":327671110,"identity":"bdb8d6ba-7ea7-44b8-b2f2-f8d936945224","order_by":6,"name":"Michael Hsiao","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Hsiao","suffix":""},{"id":327671115,"identity":"7b9925f0-aec3-48d6-859f-a526b04e605f","order_by":7,"name":"Jang-Yang Chang","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jang-Yang","middleName":"","lastName":"Chang","suffix":""},{"id":327671116,"identity":"4f89105c-35d5-4951-b579-43b4a4ed9cc6","order_by":8,"name":"Peter Mu-Hsin Chang","email":"","orcid":"","institution":"Taipei Veterans General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"Mu-Hsin","lastName":"Chang","suffix":""},{"id":327671117,"identity":"aa693ddc-33a6-4a7b-be68-12358fab1852","order_by":9,"name":"Wei-Min Chang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYFACxgaGBAYQYmB8ABFJIF4Ls8EBmJYDRNgF0sImQZQWg+PNrRse7rDL45/dfq36Y85hBn72HAPmj214tJw52HYj8UxyscSdM2U3Dm47zCDZ88aA4SAeLWY3EoFa2g4kNtzISQNrMbiRA9SyDY+W+w8hWuYDtRSAtNgT1HKDEaJlw430YwxgWyQIaLE/A3ZYcuLGGznMEme3pfNInHlWcODsP9xaJNuPP7v5s80ucd6N9IcfKrdZy/G3J298UHEGtxYkwGMAJkHEAaI0MDCwPyBS4SgYBaNgFIw0AABSgmVA5MvnswAAAABJRU5ErkJggg==","orcid":"","institution":"Taipei Medical University","correspondingAuthor":true,"prefix":"","firstName":"Wei-Min","middleName":"","lastName":"Chang","suffix":""}],"badges":[],"createdAt":"2024-06-25 13:54:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4636968/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4636968/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60690964,"identity":"ec7fbe1b-8fa3-401a-ad4e-ebb71d91d699","added_by":"auto","created_at":"2024-07-19 15:02:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":242380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-762 is expressed and secreted by OSCC cells. A. \u003c/strong\u003eOverall survival time was significantly associated with miR-762-low grade compared to miR-762-high grades in 522 OSCC samples from the Kaplan-Meier Plotter (https://kmplot.com/analysis/). \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eC\u003c/strong\u003e: qRT-PCR analysis to validate the expression levels of miR-762 in OSCC cell lines and OSCC cell lines conditioned medium. The U6 small nuclear RNA was used as an internal normalized control. \u003cstrong\u003eD\u003c/strong\u003e. NF-kB transactivation assays. \u003cstrong\u003eE.\u003c/strong\u003e CDH1 (E-cadherin) promoter activity assay. The empty luciferase vector used as background control. Values represent mean ± SD (n = 3), with N = 2 replicates. Significance levels were determined using the Student's t-test (**P \u0026lt; 0.01 and ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4636968/v1/5f3ac30dd8d061f68b43a83f.png"},{"id":60690960,"identity":"fe0f3046-dee6-4389-b04a-a6576593479e","added_by":"auto","created_at":"2024-07-19 15:02:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":646475,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe over-expression of miR-762 inhibited the activation of Jurkat cells.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Jurkat cells were transfected with miR-762 or control, co-stimulated with anti-CD3/anti-CD28 antibodies for 72 hours, and cell activation was detected by Flow Cytometry. \u003cstrong\u003eB.\u003c/strong\u003e ELISA analysis of IL-12 in the medium of Jurkat cell was transfected with miR-762 mimics or N.C. miRNA for 72 hours. Data represented means ± SD from 3 independent experiments (each experiment contains two technical replicates).\u003cstrong\u003e C.\u003c/strong\u003e Jurkat cells were transfected with miR-762 or N.C. miRNA for 72 hours, and cell proliferation was determined by the CCK assay. *P \u0026lt; 0.05 versus the N.C. group. \u003cstrong\u003eD.\u003c/strong\u003e Jurkat cells were transfected with miR-762 mimics or non-targeting control miRNA for 72 hours before seeding in the Transwell for 24 hours.\u003cstrong\u003e E.\u003c/strong\u003e Jurkat cells were transfected with miR-762 mimics or non-targeting control miRNA (N.C. miRNA) for 72 hours, and the expression levels of IL-2RA, CD69, and CD71 were detected by qRT–PCR. GAPDH was used as an internal control. \u003cstrong\u003eF. \u003c/strong\u003eRepresentative images and quantified bar charts of Transwell migration assays of Jurkat cells treated with conditioned medium from Cal-27 cells overexpressing miR-762 (miR-762-CM) or control vector-transfected Cal-27 cells (miR-NS-CM) for 24 hours. Values represent mean ± SD (n = 3), with N = 2 replicates. Significance levels were determined using the Student's t-test (**P \u0026lt; 0.01 and ***P \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4636968/v1/69ce588dbd37f61a0e79a16b.png"},{"id":60690961,"identity":"a45dd689-6fe7-4047-97cd-4b20bb3d01f4","added_by":"auto","created_at":"2024-07-19 15:02:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":240543,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCXCR3 is a direct target of miR-762. A. S\u003c/strong\u003echematic representation of the putative miR‐762 binding sequence in the 3′‐UTR of CXCR3 with wild-type form (CXCR3 3′‐UTR WT). \u003cstrong\u003eB. \u003c/strong\u003eLuciferase activities were measured to assess the effect of miR-762 mimics (PM, 50 nM) on constructs containing the wild-type 3′-UTR of CXCR3 in 293T cells. The relative luciferase activity of each sample was measured at 48 hours after transfection and normalized to Renilla luciferase activity. \u003cstrong\u003eC.\u003c/strong\u003e and\u003cstrong\u003e D. \u003c/strong\u003eqRT–PCR analysis showing the expression levels of miR-762 (\u003cstrong\u003eC\u003c/strong\u003e) and CXCR3 (\u003cstrong\u003eD)\u003c/strong\u003e \u003cstrong\u003eE. \u003c/strong\u003eWestern blot analysis of CXCR3 after transfection of miR-762 mimics with 50 nM for 72 hours in Jurkat cells. GAPDH was used as an internal control. \u003cstrong\u003eF.\u003c/strong\u003e the CXCR3 variant analysis from CXCR3 A, CXCR3 alt, and CXCR3 B in Jurkat cell lines after transfection with 50 nM of miR-762 mimics (PM). \u003cstrong\u003eG\u003c/strong\u003e. and \u003cstrong\u003eH\u003c/strong\u003e. The miR-762 expression (\u003cstrong\u003eG\u003c/strong\u003e) and CXCR3 variant analysis (\u003cstrong\u003eH\u003c/strong\u003e) from Jurkat T-cell treated with conditioned medium from Cal-27 cells overexpressing miR-762(CM-miR-762) or control vector-transfected Cal-27 cells (CM-miR-NC) for 72 hours. GAPDH was used as an internal control.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4636968/v1/4ec7fc5802a8b91874635b99.png"},{"id":60690967,"identity":"4240d92c-a27a-4b5a-a16e-eb67c3a1cd93","added_by":"auto","created_at":"2024-07-19 15:02:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":202786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCXCR3 induced the Jurkat T-cell activation and proliferation. A.\u003c/strong\u003e qRT-PCR analysis was conducted to measure the expression levels of CXCR3. B. qRT-PCR analysis of T-cell activation genes after stably expressing CXCR3. GAPDH was utilized as an internal control. \u003cstrong\u003eC.\u003c/strong\u003e CXCR3-expressed Jurkat cells, then co-stimulated with IL-2 (10 ng/ml) for 72 hours, and cell activation was assessed by Flow Cytometry. \u003cstrong\u003eD.\u003c/strong\u003eCell proliferation ability of CXCR3-expressed Jurkat cells. Cell proliferation index was measured CCK-8 assay for 72 hours induction. \u003cstrong\u003eE.\u003c/strong\u003e ELISA analysis of IL-12 in the CXCR3-expressed Jurkat condition medium. Data represent means ± SD from 3 independent experiments, with each experiment containing two technical replicates.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4636968/v1/8c0424bca553f0e608b113fb.png"},{"id":60690966,"identity":"219ce8bd-6da5-4062-8d2f-d47ac3082c24","added_by":"auto","created_at":"2024-07-19 15:02:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":446372,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHorizontal miR-762 inhibits T-cell activation by targeting CXCR3. \u003c/strong\u003eJurkat cells were co-transfected with miR-762 mimics and CXCR3 CDS for 72 hours.\u003cstrong\u003e A.\u003c/strong\u003e CXCR3 mRNA level. \u003cstrong\u003eB. \u003c/strong\u003eCXCR3 protein level and downstream phosphor-Akt activity. Numerical values for protein band intensities are depicted below the gels. The values were quantitated by densitometry and normalized to GAPDH. \u003cstrong\u003eC\u003c/strong\u003e. qRT-PCR analysis of T-cell activation genes. GAPDH was used as an internal control. \u003cstrong\u003eD. \u003c/strong\u003eFlow cytometry analysis of surface CD69 and CD25 expression on \u0026nbsp;miR-762 and CXCR3-expressed Jurkat cells and followed by co-stimulation with anti-CD3/anti-CD28 antibodies for 72 hours. \u003cstrong\u003eE.\u003c/strong\u003e Jurkat T-cell proliferation ability \u003cstrong\u003eF.\u003c/strong\u003e IL12-ELISA analysis in the Jurkat cells medium with miR-762 mimics and CXCR3-CDS expression for 72 hours.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4636968/v1/d111545cd86e95f78d7f0b8a.png"},{"id":60691485,"identity":"e2461ea2-be35-4cdb-9525-6ea19c207099","added_by":"auto","created_at":"2024-07-19 15:10:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":526080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-762 attenuates Jurkat T-cell antitumor cytotoxicity. A. \u003c/strong\u003eSchematic diagram of Jurkat T induced OSCC rejection assay. Representative images (\u003cstrong\u003eB\u003c/strong\u003e) and quantified bar charts of CAL-27 cell viability (\u003cstrong\u003eC\u003c/strong\u003e) in Jukat T antitumor cytotoxicity with miR-762 and CXCR3-CDS expression. Values represent mean ± SD (n = 3).\u003cstrong\u003e D. \u003c/strong\u003eOSCC cells inhibit T-cell activation through inhibition of CXCR3 by horizontal miR-762 transmission.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4636968/v1/a0302baa1fcec04e629926e8.png"},{"id":69497007,"identity":"546ded5c-a41a-49c9-b673-9245a312396e","added_by":"auto","created_at":"2024-11-21 04:17:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3284127,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4636968/v1/150dafa6-ad9a-41a3-96b7-7d17ef0f627d.pdf"},{"id":60691486,"identity":"5907a408-3fa5-4aa2-92c1-2e97804463f3","added_by":"auto","created_at":"2024-07-19 15:10:36","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":818161,"visible":true,"origin":"","legend":"","description":"","filename":"SRWBRawmerge.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4636968/v1/113a3ee53bd6733db507f34c.pdf"},{"id":60690968,"identity":"2c4b4a90-969b-44fe-85b9-c06b6a3c05d9","added_by":"auto","created_at":"2024-07-19 15:02:36","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":22617,"visible":true,"origin":"","legend":"","description":"","filename":"TableSformiR762.docx","url":"https://assets-eu.researchsquare.com/files/rs-4636968/v1/c2c53e920676d9cee84c9490.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oral Cancer-derived miR-762 Suppresses T Cell Infiltration and Activation by Horizontally Inhibition of CXCR3 Expression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCancer is one of the deadliest diseases globally, with an estimated 18.1\u0026nbsp;million new cases and 10\u0026nbsp;million deaths annually. In the United States, approximately 1.9\u0026nbsp;million new cancer cases and about 609,820 deaths are expected each year\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.. Cancer remains the leading cause of death in Taiwan, presenting a significant public health challenge. Specifically, oral cancer is one of the most lethal cancer in Taiwan, profoundly affecting patients' quality-of-life, increasing medical expenses, and causing considerable socioeconomic impact \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The host immune system plays a crucial role in eliminating cancer cells during the initiation phase of cancer \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Effector immune cells, such as effector CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T-cells, γδT cells, and natural killer (NK) cells, are responsible for elimination of cancer \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. For these effector cells to eliminate tumor cells, immune cells must infiltrate into tumor microenvironment. However, oral cancer employs several strategies to evade the immune system, including the expression of inflammatory cytokines, suppression of cytotoxic CD8 lymphocytes, downregulation of antigen processing machinery, generation of specific inhibitory lymphocytes, and expression of immune checkpoint ligands and receptors. These mechanisms increase oral cancer's resistance to cytotoxic T-cells and inhibit normal T-cell function, facilitating tumor initiation and growth \u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Understanding these immune escape mechanisms is essential for developing new treatment strategies for oral cancer.\u003c/p\u003e\n\u003ch3\u003eChemokines-induced immune cell recruitment\u003c/h3\u003e\n\u003cp\u003eChemokines are small cytokines that direct immune cell recruitment. The chemokine family consists of about 50 ligands, 20 G protein-coupled receptors (GPCRs), and 4 atypical chemokine receptors (ACKRs) \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. They play a key role in cancer immunity by attracting immune cells to the tumor microenvironment (TME). CXCR3, a critical receptor, aids in T and NK cell recruitment and macrophage polarization \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Its expression is linked to the progression of cancers like breast, colorectal, and pancreatic. High levels of CXCR3 ligands in pancreatic and colorectal cancers are associated with poor survival, making it a potential prognostic marker \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The diverse functions of CXCR3 underscore its potential as a target for immunotherapy and its significance in cancer and immune regulation.\u003c/p\u003e\n\u003ch3\u003eExtracellular microRNA as Cancer Microenvironment Coordinator\u003c/h3\u003e\n\u003cp\u003eThe tumor microenvironment (TME) is a dynamic ecosystem involving cancer cells and host cells like immune cells, fibroblasts, and endothelial cells \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In tumor initiation, dysregulated of immune cell distribution shapes the immune landscape of TME \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The immune status in TME affects patient prognosis and treatment outcomes for chemotherapy and immunotherapy. Extracellular vesicles (EVs) serve as key communicators between cancer and host cells, carrying proteins and nucleic acids like enzymes, receptors, mRNA, non-coding RNA, and microRNA \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The TME EVs harbor proteins and free-nucleic acids, such as enzymes, receptors, mRNA, non-coding RNA, and microRNA, serve as colonial rulers for horizontal transmission between cancer donor and host recipient normal cells \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. MicroRNAs, known for their stability and gene regulation capabilities, play a significant role in TME regulation \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Aberrant microRNA or microRNA cluster expression has been reported in oral cancer tumorigenesis and progression \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. miR-762, an oncogenic microRNA, contributes to head and neck cancer and is a poor prognostic marker in colon cancer due to its role in WNT signaling \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Our group also found that miR-762 is a serum poor prognostic marker and contributing to colon cancer progression through WNT signaling activation \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This study found that miR-762 suppresses the immune response in oral cancer by targeting CXCR3 and hindering cytotoxic T-cell recruitment and activation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eHuman acute T-cell leukemia Jurkat cells., human embryonic kidney 293T and human oral cancer cells SCC-4, SCC-9, SCC-15, SCC-25, Cal-27, Cal-33, HSC-2, HSC-3, HSC-3-M3, and HSC-4, Ca9-22, OSC-19, OSC-20, and SAS were purchased from original resource and listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. All cells were cultured in the standard culture condition by following instruction manual and maintained within 3 months. Additionally, all cells were regularly checked for mycoplasma infection and cell morphology to ensure cell health.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eActivation of Jurkat T cell\u003c/h2\u003e \u003cp\u003eThe Jurkat cells were stimulated with 1.25 \u0026micro;g/ml of anti-CD3 (Clone: OKT3; BioLegend, San Diego, CA, USA) monoclonal antibody (mAb) and 1.25 \u0026micro;g/ml of anti-CD28 (Clone: CD28.2; BioLegend) mAb for 72 hours. Jurkat-CXCR3 cells were stimulated with the same amount of anti-CD3 and anti-CD28 mAb plus 10 ng/ml of IL-12 (R\u0026amp;D Systems, Inc, Minneapolis, MN, USA) for 72 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eT cell cytotoxicity assay against OSCC cells\u003c/h2\u003e \u003cp\u003e2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e Jurkat cells were seeded in the upper-transwell chamber and co-transfected with or without miR-762 mimics and CXCR3-CDS expression vector for 72 hours. For T cell cytotoxicity, 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e Cal-27 cells were seeded in the lower-well. After 48 hours, Cal-27 cells were fixed in 70% ice-cold ethanol for 60 minutes then stained with 0.1% crystal violet/20% methanol solution and recorded under a light microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and real-time PCR (qRT‐PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from cell lines using TRIzol reagent, and cDNA was synthesized by Roche First Strand cDNA Synthesis Kit. All primers were listed in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. qRT-PCR analysis was used ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and QuantStudio 3 real-time PCR system. For mature miRNA detection, cDNA was prepared using the miScript II RT Kit (Cat: 218161, QIAGEN, Valencia, CA, USA). U6 was used as the internal control for human miRNAs. The expression level was defined based on the threshold cycle, and relative expression levels were calculated as ▵▵Ct after normalization with the reference control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and western blot analysis\u003c/h2\u003e \u003cp\u003eCells were lysed in RIPA buffer with protease and phosphatase inhibitor cocktail. 10\u0026ndash;30 \u0026micro;g of protein lysates were loaded onto 10% SDS-PAGE and transferred into PVDF membranes. All antibodies were listed in table S3. The membranes were exposed using ECL (Merck-Millipore) for the HRP-coupled secondary antibodies and analyzed using the e-BLOT Touch Imager.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eTranswell migration assays\u003c/h2\u003e \u003cp\u003eT cell migration assays were performed using a 3-\u0026micro;m Transwell chamber (SPL 36224, Korea). 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e Jurkat cells were seeded in the upper-chamber in serum-free medium, and 600 \u0026micro;L of medium containing 2% FBS was added to the lower well. The cells were incubated for 24 hours to allow them to attach. After 24 hours of incubation, migrated cells were fixed on the membrane with 70% ethanol for 60 minutes at 4\u0026deg;C, washed twice with PBS, and the interiors of the inserts were cleaned with wet cotton swabs. Cells were stained with a cell stain solution (0.1% crystal violet, 20% methanol) and visualized under an inverted microscope (200\u0026times; magnification; Olympus Corporation, Hachioji, Tokyo, Japan). Images were then analyzed and counted using an analytical imaging station software package (Imaging Research, Ontario, Canada).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePlasmids and Transfection\u003c/h2\u003e \u003cp\u003eThe pLX304-CXCR3 plasmid was generated by inserting full-length cDNA (CXCR3: NM_001504) into the vector following previously established protocols. The miR-762 mimic (PM) (ID: 15805, Cat: AM17100, Thermo Fisher Scientific) transfection mix was prepared using TransIT-X2 Transfection Reagent (Mirus, Madison, WI, USA) according to the manufacturer's protocol and applied to Jurkat cells.\u003c/p\u003e \u003cp\u003eFor lentivirus production, 293T cells were co-transfected with pMD2.G, pCMV▵R8.91, and lentiviral transfer plasmids using TransIT-LT1 Transfection Reagent (Mirus, Madison, WI, USA). The culture medium containing lentivirus was harvested 48 hours after transfection. Cells were removed from the culture medium by centrifugation at 500 g for 5 minutes and filtered through a 0.45-\u0026micro;m filter. The lentiviral stocks in the medium, containing polybrene (8 \u0026micro;g/mL), were used to infect the target cells for 6 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCCK-8 Assay:\u003c/h2\u003e \u003cp\u003eEqual numbers of Jurkat cells transfected with miR-762 mimics or non-targeting control (N.C.) for 72 hours were plated in a 96-well plate and allowed to grow for an additional 72 hours. Cell growth was determined using a Cell Counting Kit-8 (CCK-8) (Sigma-Aldrich, St Louis, USA). 10 \u0026micro;L of CCK-8 solution was added to each well of the plate, followed by incubation for 3 hours. The absorbance at 450 nm was measured using a 96-well plate SpectraMax 250 reader (Molecular Devices, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase Reporter Assays:\u003c/h2\u003e \u003cp\u003eFor NF-κB transcriptional activity and CDH1 promoter activity assay, the protocol was followed as per our previous work. For the 3\u0026rsquo;-UTR luciferase reporter assay, the CXCR3 3\u0026rsquo;-UTR region was generated by subcloning PCR-amplified full-length human CXCR3 3\u0026rsquo;-UTR (446 bp from the stop codon) into the modified 3\u0026rsquo;-UTR CpoI sites of the luciferase in the pGreenfire-CMV firefly luciferase-expressing vector (SBI, Palo Alto, CA, USA), using the primers: Forward: 5\u0026rsquo;-CGGACCGGGCCGGAATCCGGGCTCCCCTTTC-\u0026lsquo;3 and Reverse: 5\u0026rsquo;-CGGTCCGTCCTGACGATCTTGTTTATT-3\u0026rsquo;. 293T cells were cultured in 24-well plates and co-transfected with 300 ng of CXCR3 3\u0026rsquo;-untranslated region (UTR) wild type or mutant type pGreenfire-CXCR3 3\u0026rsquo;-UTR reporter plasmid, along with 25 nM of miR-762 mimics (PM) or non-targeting control (N.C.), using TransIT-X2 Transfection Reagent (Mirus, Madison, WI, USA) according to the manufacturer\u0026rsquo;s instructions. The luciferase assay was performed 24 hours post-transfection with the ONE-Glo\u0026trade; Luciferase Reporter Assay System (Promega, USA) following the manufacturer's protocol. 50 \u0026micro;L of ONE-Glo\u0026trade; substrate was added into a white plate and the assay was conducted with a SpectraMax iD3 microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometry:\u003c/h2\u003e \u003cp\u003eThe cells were harvested, washed twice with PBS, fixed in 1% paraformaldehyde (PFA) overnight at 4\u0026deg;C, washed again, and resuspended in flow cytometry buffer. To determine surface markers, these cells were labeled with the surface fluorochrome-conjugated antibodies CD25 and CD69 (BD Biosciences, Franklin Lakes, NJ) for 60 minutes on ice in the dark. The stained cells were analyzed using a Attune NxT Flow Cytometer and the quantitative analysis was performed using FlowJo v10 (Treestar, Ashland, OR).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eCell supernatants were collected and processed using R\u0026amp;D Duoset ELISA kits (R\u0026amp;D Systems, Inc, Minneapolis, MN, USA) according to the manufacturer\u0026rsquo;s instructions for the detection of cell secretion of IL-12 (Cat# D1200).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were repeated at least 3 times. Data were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) from repeated independent experiments. Differences between various treatment groups were assessed using the Student\u0026rsquo;s t test. Between-group differences were considered significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Data analyses were performed using GraphPad Prism Ver. 8 (San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003emiR-762 Levels were Significantly Associated with the Poor Prognosis of Head and Neck Cancers Patients\u003c/h2\u003e \u003cp\u003eWe investigated the relationship between miR-762 levels and clinical manifestations in patients with Head and Neck Cancer using data from the Kaplan-Meier plotter database. The high-miR-762 group had significantly shorter overall survival (OS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The median OS was 77.3 months in the low-miR-762 group versus 37.77 months in the high-miR-762 group. We also found that miR-762 levels are high in OSCC cell lines compared to normal keratinocyte cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Oral cancer is an immune cold tumor, and OSCC typically has an immunosuppressive microenvironment, involving immune checkpoint proteins, environmental immunosuppressive factors, and an imbalance between immunosuppressive and effective immune cell compositions \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. This leads to diminished T-cell numbers and activity in the tumor, resulting in poor prognosis for oral cancer patients \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. We found that miR-762 is secreted by OSCC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), with Cal-27 having the lowest secretion of miR-762 among the five OSCC cell lines (Cal-27, SCC-4, HSC-3, SAS, and SCC-15). Secreted miRNAs are highly stable in the TME and may serve as critical intercellular communication molecules, allowing cancer cells to manipulate or diminish surrounding recipient cells through horizontal transfection \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Furthermore, we found that miR-762 not only suppressed NF-κB signaling transactivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) but also E-cadherin promoter transcription activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), which may inhibit immune cell maturation and prevent migration into the TME. These results suggest that miR-762 plays an important role in the OSCC TME with an immune suppression function. We speculate that miR-762 may lead to decreased T-cell activation and function in the OSCC TME.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eExogenous miR-762 inhibits the Jurkat cells activation and migration ability\u003c/h2\u003e \u003cp\u003eTo demonstrate the function of extracellular miR-762 in the OSCC tumor microenvironment (TME), particularly in tumor immune evasion through the prevention of T-cell activation, we transfected Jurkat cells with mature miR-762 or scrambled miRNA (NC) for 72 hours. The surface T-cell activation markers, CD25 and CD69, were inhibited by miR-762 transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Additionally, the proinflammatory cytokine IL-12, which augments the growth, differentiation, and activation of cytotoxic CD8 T-cells \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, was reduced by miR-762 manipulation compared to the NC control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This indicates that miR-762 inhibited Jurkat cell cytotoxic activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, miR-762 transfection reduced Jurkat cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and transwell migration ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These results show that miR-762 is an important suppressor of T-cell migration and activation in the TME. Additionally, we enforced miR-762 expression on Cal-27 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), which have both lower intracellular and secretion levels of miR-762 and harvested the cell culture supernatant as an oral cancer-derived conditioned medium to mimic the TME. The miR-762-containing Cal-27 conditioned medium (Cal-27-miR-762-CM) also suppressed PHA-activated Jurkat T-cell migration and further inhibited the gene expression of T-cell activation markers such as IL2RA, and CD69 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Taken together, these results indicate that miR-762 in the TME can inhibit T-cell recruitment and activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eExogenous miR-762 inhibits T-cell CXCR3 expression\u003c/h2\u003e \u003cp\u003eCXCR3 is primarily expressed on T-cells, dendritic cells, and natural killer cells, playing a crucial role in T-cell trafficking and function \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Moreover, CXCR3 has three variants, A and B, which exhibit differences in their expression profiles in the TME and have different functions in cancer progression \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. To determine whether miR-762 could directly target CXCR3, we performed a 3\u0026rsquo;-untranslated region (3\u0026rsquo;-UTR) reporter assay in 293T cells. The CXCR3-3\u0026rsquo;-UTR was significantly suppressed by miR-762 mimic (PM) transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Additionally, ectopic miR-762 expression directly suppressed CXCR3 mRNA and protein expression in Jurkat cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC to E). miR-762 selectively reduced the expression of the T-cell proliferation and migration-promoting variant CXCR3A.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further examine the role of extracellular miR-762 in the oral cancer tumor microenvironment, we applied OSCC conditioned medium to Jurkat T-cells to assess CXCR3 expression. The conditioned medium harvested from miR-762-expressing Cal-27 cells also suppressed CXCR3 expression in Jurkat T-cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and H). These results indicate that oral cancer-secreted miR-762 can suppress T-cell CXCR3 expression in the oral tumor microenvironment. Interestingly, both direct transfection and oral cancer-derived miR-762 conditioned medium stimulated the expression of the CXCR3B variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and H). CXCR3B has a unique receptor function for CXCL4, a chemokine that inhibits cytotoxic T-cell migration and activation. These results suggest that miR-762 may exert a unique T-cell suppression function through the CXCL4/CXCR3B axis, distinct from the traditional CXCR3A function.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCXCR3 promotes cytotoxicity T-cell activation\u003c/h2\u003e \u003cp\u003eTo further examine whether CXCR3 contributes to T-cell activation, we evaluated the T-cell activation status following ectopic CXCR3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We found that CXCR3 expression enhanced the expression of T-cell activation genes such as IL2RA (CD25), CD69, and CD71 mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Additionally, we observed that CXCR3 expression increased the expression of T-cell activation surface markers CD69, CD25, and CD154 in Jurkat cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Moreover, we also found that CXCR3 increased Jurkat T-cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). IL-12, a T-cell-stimulating cytokine, promotes the growth and function of T-cells and the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) from T-cells and natural killer (NK) cells \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. CXCR3 expression also stimulated IL-12 secretion from Jurkat T-cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, our results show that CXCR3 enhances Jurkat T-cell cytotoxicity by promoting T-cell polarization, proliferation, and increasing IL-12 secretion, thereby proving that CXCR3 plays a crucial role in T-cell activation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003emiR-762/CXCR3 suppression axis is critical in environment T-cell activation\u003c/h2\u003e \u003cp\u003eThe molecular mechanisms underlying CXCR3 regulation of T-cell function are not fully understood. Several studies have suggested that the signal transduction of the chemokine receptor CXCR3 involves activating Ras/ERK, Src, and phosphoinositide 3-kinase(PI3K)/AKT pathways \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Additionally, CXCL10/CXCR3 activation can stimulate PI3K/AKT signaling, thereby promoting Th1 cell differentiation and migration. The PI3K/AKT pathways are then stimulated, leading to the promotion of cell survival, proliferation, and cytotoxic activity of CD8\u0026thinsp;+\u0026thinsp;memory T-cells and NK cells.\u003c/p\u003e \u003cp\u003eTo clarify the interplay among the miR-762/CXCR3/Akt axis in OSCC immune evasion, we aimed to investigate whether oral cancer miR-762-induced CXCR3 downregulation plays a critical role in T-cell activation. To this end, we generated a CXCR3 expression plasmid lacking the 3\u0026rsquo;-UTR sequence, thereby preventing inhibition by miR-762. In the miR-762 mimic-transfected Jurkat T-cells, restoring CXCR3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) could restore AKT activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The restored CXCR3 expression further reactivated T-cell polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D) and proliferation ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Moreover, the critical T-cell cytokine IL-12 secretion was also restored by CXCR3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, these results suggest that the miR-762/CXCR3 axis may regulate T-cell activation, polarization, proliferation, and IL-12 secretion in the tumor microenvironment.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eHorizontal Transmission of miR-762 Promotes Immune Escape in Oral Cancer\u003c/h2\u003e \u003cp\u003eFinally, we aimed to demonstrate the horizontal transmission of miR-762 between oral cancer cells and T-cells, which regulates T-cell cytotoxicity and promotes oral cancer immune escape. To do this, we used an indirect coculture system (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). When miR-762 was introduced into low-expressing Cal-27 cells, T-cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and cytotoxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) were abolished by miR-762 expression. Moreover, this cancer immune escape could be reversed by reintroducing CXCR3 into the T-cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, these results suggest that miR-762 attenuates T-cell cytotoxicity against cancer by reducing the expression levels of CXCR3. These findings indicate that T-cell inactivation induced by OSCC is primarily mediated by OSCC-derived miR-762.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOral cancer employs various immune escape mechanisms, primarily involving inhibitory lymphocytes and other immune cells that release cytokines suppressing cytotoxic CD8 lymphocytes. This study identified that horizontal transmission of miR-762 from oral cancer cells may contribute to immune escape by suppressing CXCR3 expression and activating the CXCR3-AKT signaling pathway in T lymphocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Chemokines in the tumor microenvironment play a crucial role in cancer progression by promoting tumorigenesis \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. However, chemokines also recruit adaptive immune cells, enhancing anti-tumor immunity. For instance, CXCR3, a receptor for chemokines CXCL9, CXCL10, and CXCL11, is more highly expressed in tumor tissues than in normal tissues \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In cancer cells, autocrine CXCR3 signaling promotes metastasis and growth through AKT signaling \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, while CXCL9 and CXCL11 can inhibit tumor growth by facilitating immune cell infiltration \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Thus, tumors must find new ways to evade the immune system. Horizontal gene transfer (HGT) is vital in cancer progression \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Unlike genomic DNA or cell-free DNA, microRNAs quickly inhibit gene transcription and translation and are continuously supplied by cancer cells. MicroRNAs can be easily targeted and neutralized by antagonists or complementary anti-sense miRNA sequences \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. RNA-based medicines, which gained popularity during the COVID-19 pandemic, could offer new therapeutic options \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Our discovery of miR-762\u0026rsquo;s role in oral cancer immune escape via horizontal transmission to immune cells in the tumor microenvironment suggests a new niche for RNA-based cancer therapies.\u003c/p\u003e \u003cp\u003eOral cancer is typically an immune cold tumor, showing low levels of T and NK cell infiltration\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. CXCR3 is vital for lymphocyte migration and activation, and miR-762 in the tumor microenvironment (TME) may help maintain an immunosuppressive environment. Besides aiding immune escape during tumor initiation, the immune cell content in TME is crucial for predicting treatment outcomes and patient prognosis \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. miR-762 distribution in TME prevents immune cell infiltration and worsens responses to conventional cancer treatments. Using anti-sense miR-762 could remove miR-762 from the TME, may enhance immune cell infiltration, and boost anti-tumor cytotoxicity, improving responses to chemotherapy and radiotherapy.\u003c/p\u003e \u003cp\u003eIn head and neck cancer, CXCR3 enhances lymphatic invasion and cancer growth \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, while CXCR3A promotes cancer stem-like properties and chemoresistance \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. These findings show the importance of CXCR3 in oral cancer progression. In late-stage Non-Small Cell Lung Carcinoma (NSCLC) patients, CXCR3 expression is diminished in both CD4 and CD8 T-cells \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. CXCR3 is highly expressed in adaptive immune cells, aiding in chemotaxis and activation. Reduced CXCR3 expression in NK cells hinders immune cell recruitment to tumors \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Suppressing CXCR3 in immune cells can promote cancer progression and immune escape. Interestingly, miR-762 treatment and oral cancer-conditioned medium increase CXCR3B expression, which acts as the CXCL4 receptor \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. CXCL4/CXCR3B has opposite effects to CXCL10 in T-cells, reducing proinflammatory IFN-gamma and increasing TH2 cytokines \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. CXCL4 also inhibits activated T-cell proliferation \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e and stimulates Treg cell growth \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. CXCR3B expression may further oral cancer immune escape and enhance the immunosuppressive environment via miR-762 transmission. In summary, our findings reveal a novel oral cancer escape mechanism through miR-762 transmission, which prevents immune cell infiltration and anti-tumor cytotoxicity. This may offer a new target for modifying the tumor microenvironment and restoring anti-cancer immunity in oral cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAcknowledgments\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge the great help and assistance from RNAiCore facility (Academia Sinca) and TMU Core Facility Center.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eFunding Information\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Taipei Medical University Research Center of Cancer Translational Medicine sponsored by Higher Education Sprout Project, Taiwan Ministry of Education. This study was supported by Taipei Medical University Hospital, Taipei Medical University (110TMUH-NE-10) to JWC, Taipei Veterans General Hospital (V111C-004 and V112C-004) to PMC and WMC. Furthermore, MOST111-2314-B038-117 and NSTC112-2314-B038-042 to WMC, MOST111-2314-B038-087 to PHW, NSTC112-2314-B038-041 to HLL, and NSTC112-2320-B038-060-MY2 to YLL from the National Science and Technology Council, Taiwan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eConflict of Interest\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eData availability\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Data are located in controlled access data storage at Taipei Medical University.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eEthics Statement\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproval of the research protocol by an Institutional Reviewer Board: N/A.\u003c/p\u003e\n\u003cp\u003eInformed Consent: N/A.\u003c/p\u003e\n\u003cp\u003eRegistry and the Registration No. of the study/trial: N/A.\u003c/p\u003e\n\u003cp\u003eAnimal Studies: N/A\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAuthor Contribution\u0026nbsp;\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors approved to publish the study in this journal. Study conception and design: HYP, CWC, and WMC. Development of methodology: HYP, CWC., PHW, LJL, YLL, HLL, and WMC. Resource and Supervision: MH, JYC, and PMHC. Analysis and interpretation of data: HYP, CWC, LJL, YLL, PHW, and HLL. Writing, reviewing, and/or revision of the manuscript: HYP, CWC, PMHC, and WMC.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel, R. L., Miller, K. D., Wagle, N. S. \u0026amp; Jemal, A. Cancer statistics, 2023. \u003cem\u003eCA Cancer J Clin\u003c/em\u003e \u003cstrong\u003e73\u003c/strong\u003e, 17-48, doi:10.3322/caac.21763 (2023).\u003c/li\u003e\n\u003cli\u003eChou, C. W., Lin, C. R., Chung, Y. T. \u0026amp; Tang, C. S. Epidemiology of Oral Cancer in Taiwan: A Population-Based Cancer Registry Study. \u003cem\u003eCancers (Basel)\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, doi:10.3390/cancers15072175 (2023).\u003c/li\u003e\n\u003cli\u003eKao, C. 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H. \u0026amp; Visentin, G. P. Platelet Factor 4 Differentially Modulates. \u003cem\u003eJ Immunol\u003c/em\u003e \u003cstrong\u003e174\u003c/strong\u003e, 2680-2686 (2005).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"OSCC, microRNA, horizontal transmission, CXCR3, immune escape ","lastPublishedDoi":"10.21203/rs.3.rs-4636968/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4636968/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOral cancer is an immune cold tumor characterized by an immunosuppressive microenvironment with low cytotoxic activity to eliminate tumor cells. Tumor escape is one of the initial steps in cancer development. Understanding the underlying mechanisms of cancer escape can help researchers develop new treatment strategies. In this study, we found that the oral oncogenic miR-762 can suppress T-cell recruitment and cytotoxic activation in the tumor microenvironment through horizontal transmission from oral cancer cells to adaptive immune T-cells. This horizontal transmission of miR-762 directly suppresses CXCR3 expression in T-cells, inhibiting CXCR3-induced T-cell migration and downstream T-cell cytotoxic activity by disrupting AKT activation. Additionally, miR-762 transmission suppressed T-cell activation marker expression, T-cell proliferation, IL-12 secretion, and T-cell cytotoxicity. In conclusion, our findings reveal a novel miR-762/CXCR3 axis that regulates the immunosuppressive microenvironment in oral cancer and may be a potential RNA-targeted therapeutic approach to restore the anti-tumor immune response in oral cancer immunotherapy.\u003c/p\u003e","manuscriptTitle":"Oral Cancer-derived miR-762 Suppresses T Cell Infiltration and Activation by Horizontally Inhibition of CXCR3 Expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-19 15:02:30","doi":"10.21203/rs.3.rs-4636968/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a84c0a22-cdc4-4a75-b5ec-3f20aff753a6","owner":[],"postedDate":"July 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34679927,"name":"Biological sciences/Cancer/Oral cancer"},{"id":34679928,"name":"Biological sciences/Cancer/Tumour immunology"}],"tags":[],"updatedAt":"2024-11-21T04:08:50+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-19 15:02:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4636968","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4636968","identity":"rs-4636968","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}