SPARC激活CAF重塑代谢微环境促进口腔鳞癌复发的机制研究

Recurrence and metastasis are key factors of poor prognosis in oral squamous cell carcinoma (OSCC). And the pattern of invasion (POI) affects the recurrence and metastasis of tumor. It’s reported that cancer associated fibroblasts (CAFs) in tumor microenvironment can promote tumor invasion, but the potential mechanism is unknown. Our preliminary work found SPARC was highly expressed in tumor tissues of OSCC by proteomics, and patients with high SPARC expression showed higher recurrence rate. Moreover, the subcellular localization of SPARC indicated that it was highly expressed in CAFs and related with poorer DOI, all of which suggested that SPARC in CAFs was involved in tumor recurrence. In addition, CAFs with abnormal metabolism augmented tumor invasion by secreting TGFβ to regulate epithelial-mesenchymal transformation (EMT). Furthermore, metabolomics analysis elucidated that glutamate metabolism was significantly activated and in vitro analysis presented SPARC could regulate key enzymes GLS1 and SLC1A3 in CAFs. Thereafter, it is reasonable to consider that SPARC mediates glutamate metabolism reprogramming to active CAFs, and the latter secret TGFβ, which promotes the invasion of tumor cells by EMT, and further influences the recurrence of OSCC. Collectively, the purpose of this study was to:①analyze the relationship between SPARC and recurrence and activated CAFs metabolism in OSCC; ②clarify the effect of SPARC on activation of CAFs metabolism and further role on OSCC function at animals and cells levels; ③reveal the molecular mechanism of SPARC activation of CAFs metabolism affecting the recurrence of OSCC.

复发和转移是口腔鳞癌（OSCC）预后差的关键因素，浸润方式影响其复发和转移。据报道微环境中成纤维细胞（CAFs）可促进肿瘤浸润，但机制不明。前期蛋白组学发现肿瘤组织中分泌蛋白（SPARC）高表达，与患者高复发相关；进一步分析发现SPARC表达定位于CAFs中，与较差的浸润方式相关，提示CAFs中SPARC参与OSCC复发。研究报道代谢异常的CAFs分泌TGFβ调控上皮间质转化促进肿瘤侵袭，代谢组学发现肿瘤组织中谷氨酸代谢显著升高，体外证实SPARC可调控CAFs中谷氨酸分解酶（GLS1）和转运蛋白（SLC1A3）。故推测SPARC调控谷氨酸代谢激活CAF分泌TGFβ引起肿瘤EMT促进其侵袭。本研究拟通过①临床样本分析SPARC对OSCC复发以及活化CAF代谢的关系；②动物和细胞水平明确SPARC激活CAF代谢对OSCC功能的影响；③揭示SPARC激活CAF代谢影响肿瘤复发的分子机制。

复发和转移是口腔鳞癌（OSCC）预后差的关键因素，浸润方式影响其复发和转移。本项目围绕口腔鳞癌浸润前沿处CAF的特征与复发关系、SPARC在口腔鳞癌中表达、SPARC对CAF以及肿瘤代谢的影响，分别从临床标本、细胞、分子和动物水平开展了以下的研究：（1）收集口腔鳞癌样本以及无肿瘤粘膜样本免疫组化分析，浸润前沿处纤维样细胞的增殖能力对口腔鳞癌患者预后的影响；（2）分析SPARC在口腔鳞癌中的表达情况以及与预后的关系；（3）研究高表达的SPARC对口腔鳞癌细胞以及CAF的影响进而促进肿瘤的增殖和侵袭；（4）非靶向和靶向分析口腔鳞癌患者的代谢特征，研究SPARC对口腔鳞癌代谢差异的影响。取得以下研究成果一：通过298例临床口腔鳞癌样本以及98例无肿瘤粘膜样本（包括62例异常增生粘膜和36例正常粘膜）进行免疫组化研究，发现浸润前沿处在纤维样细胞中高表达的Ki67是口腔鳞癌患者预后好的标志；研究成果二：选取7对OSCC患者配对的肿瘤组织、癌旁异常增生组织以及正常粘膜组织，利用iTRAQ标记蛋白组学技术筛选差异蛋白，发现富含半胱氨酸的酸性分泌蛋白 (SPARC)存在显著的差异表达。进一步收集本院98例口腔鳞癌患者组织，免疫组化分析 SPARC结果显示SPARC高表达与口腔鳞癌更差WPOI以及分化相关，且SPARC能够通过PI3K/AKT/PDGFB/PDGFRβ信号轴促进口腔鳞癌细胞的增殖和转移能力；研究成果三：我们发现高表达SPARC的CAFs能够促进肿瘤细胞的侵袭和增殖，同时SPARC能够促进NF向CAF的转化改变肿瘤微环境，进而影响OSCC的复发；研究成果四：我们通过气相质谱联用，非靶向代谢组学筛选出10个氨基酸标记物，并拟合ROC曲线确认其诊断意义，并利用UHPLC-MS/MS联用靶向验证筛选出来的10个氨基酸标记物，其中9个与非靶向筛选结果一致；研究成果五：非靶向代谢组学发现口腔鳞癌患者血清中NNMT与肿瘤复发相关，免疫组化验证进一步发现NNMT表达与淋巴结转移和WPOI相关，能够影响肿瘤细胞的增殖和迁移。这些研究结果为OSCC的诊断和治疗提供了潜在的新分子靶标。