关键词: 口腔鳞状细胞癌, 细胞外囊泡, GW4869, 免疫原性, 抗肿瘤免疫反应

Abstract: Objective    To investigate the effects of pretreatment with extracellular vesicles（EVs）derived from mouse squamous cell carcinoma（SCC）cells on the growth of orthotopic xenograft tumor，and to explore the immunomodulatory effects and potential molecular mechanisms underlying the antitumor activity. Methods    The study was conducted at Jiangsu Ailingfei Biotechnology Co.，Ltd. from January to September 2025. SCC7 cell-derived EVs were isolated via ultracentrifugation and characterized by transmission electron microscopy，nanoparticle tracking analysis，and Western blotting. Thirty six-week-old female mice were chosen and randomly assigned to three groups（10 mice per group）：a control group，a GW4869（a selective neutral sphingomyelinase inhibitor）treatment group，and an EVs pretreatment group. Mice in the EVs group，GW4869 group，and control group received intrabuccal injections of 10 µg EVs，GW4869，and PBS respectively，once every two days for four consecutive weeks prior to SCC7 cell transplantation. Tumor formation and growth were dynamically monitored and recorded on days 7，9，11，13，15，17，19，and 21 after tumor transplantation. At the seventh week of the experiment，all mice were anesthetized，and orbital blood samples were collected. The serum levels of interleukin-6（IL-6），IL-10，IL-17A，interferon-gamma（IFN-γ），transforming growth factor-beta 1（TGF-β1），programmed death-ligand 1（PD-L1），and tumor necrosis factor-alpha（TNF-α）were determined by enzyme-linked immunosorbent assay. Following euthanasia via overdose anesthesia，orthotopic tumors and bilateral cervical draining lymph nodes were completely dissected，and their volumes were measured. Hematoxylin and eosin（HE）staining was performed to evaluate the area proportions of liquefactive necrosis and inflammatory cell infiltration in tumor tissues. Fluorescent quantitative reverse transcription polymerase chain reaction was used to detect the mRNA expression of cluster of differentiation（CD）86，CD206，IL-6，IL-10，TGF-β1，and TNF-α. Immunofluorescence was applied to detect and statistically analyze the mean fluorescence intensity of CD206 and CD8，the co-localization area of F4/80+CD206+，and the number of CD8+ T cells. Results    The SCC7 cell-derived EVs exhibited typical cup-shaped bilayer membrane structures，with particle sizes mainly distributing between 100 - 150 nm，and positively expressed the classical EV biomarkers TSG101，CD63，and CD9. Significant differences existed in time-dependent body weight changes and tumor volume growth among the three groups of mice（all P < 0.05）. After euthanasia，tumor formation analysis showed that both the control group and GW4869 group successfully developed orthotopic oral squamous cell carcinoma（OSCC）xenografts，whereas the tumor incidence rate in the EVs group was only 60%（6/10），with a statistically significant difference in tumor incidence among the three groups（χ2 = 9.231，P = 0.010）. Compared with the control and GW4869 groups，mice in the EVs group presented smaller tumor volumes and cervical lymph node volumes，as well as reduced proportions of tumor liquefactive necrosis and inflammatory infiltration areas. In terms of immune and inflammatory indicators，the EVs group showed lower mean fluorescence intensity of CD206 and smaller co-localization area of F4/80+CD206+ cells. The EVs group also showed lower serum concentrations of IL-6 and IL-10，and lower relative mRNA expression levels of CD206 and IL-10. In contrast，the EVs group displayed elevated serum concentrations of IL-17A and IFN-γ，increased relative mRNA expression of CD86 and TNF-α，and higher CD8 fluorescence intensity and CD8+ T cell number. The differences in the above-mentioned items were all statistically significant（all P < 0.05）. Except for tumor volume，no significant differences were observed in all the above immunological and inflammatory indicators between the control group and GW4869 group（all P > 0.05）. Conclusion    Pretreatment with SCC7 cell-derived EVs can effectively suppress the growth and metastasis of orthotopic OSCC xenografts in mice，promote macrophage polarization toward the M1 anti-inflammatory phenotype and inhibit the polarization toward M2，increase intratumoral CD8+ T cell infiltration，and regulate peripheral inflammatory cytokine expression. This study provides a novel research direction for the prevention and treatment of OSCC.

Key words: oral squamous cell carcinoma, extracellular vesicles, GW4869, immunogenicity, anti-tumor immune response