Assessing TGF Beta1-Mediated ECM Remodeling in Metastatic Oral Squamous Cell Carcinoma

摘要 口腔鳞状细胞癌（OSCC）患者由于远处转移而具有令人沮丧的生存率， 逃避初级保健。转化生长因子β（TGFβ）是众所周知的转移驱动因子，肿瘤转移的调节因子， 免疫细胞活性和细胞外基质（ECM）基因调节因子。在乳腺癌中，僵硬的ECM通常 这归因于过量的I型胶原沉积和交联，其促进癌细胞转移和癌症 干细胞（CSC）扩增。在口腔鳞状细胞癌中，对于硬ECM还是软ECM尚未达成共识， 增加转移潜能和CSC扩增。口腔鳞癌细胞外基质成分对免疫细胞的影响 贩运情况也不清楚。我们的实验室已经产生了来自角蛋白15+干细胞的鼠OSCC细胞系 Smad 4缺失和KrasG 12 D突变的细胞。尽管具有相同的遗传背景，但这些细胞系具有 不同的转移能力，表明微环境因素或细胞内在差异可能介导 转移RNAseq分析显示，转移性OSCC细胞具有增加的层粘连蛋白和层粘连蛋白的水平。 结合整合素，但下调I型胶原基因。此外，当OSCC细胞在硬ECM上培养时， 尽管在软ECM上培养的那些显示出增加的侵袭，但在软ECM上培养的那些显示出增加的CSC特征。治疗 具有TGFβ抑制剂的转移性OSCC细胞减少迁移和侵袭。综合免疫分析 使用流式细胞术显示转移性肿瘤的CD 8 + T细胞浸润数量减少， 淋巴细胞（TIL）与非转移性OSCC肿瘤相比。这些CD 8 + TILs有助于稳健的 抗肿瘤免疫应答和免疫检查点抑制剂（ICI）治疗的成功。识别 ICI和TGFβ抑制剂的应答者和这些治疗的致敏无应答者持续作为主要的 障碍。确定OSCC细胞如何与ECM相互作用以促进其扩散并增强电流 预后标志物对于改善患者护理和延长生存期都至关重要。这项提案的目的是 使用独特的小鼠模型和人类患者样本来确定ECM成分和刚性如何影响 OSCC CSC转移，以提高新兴的靶向治疗剂抑制OSCC转移的疗效 结果该建议的假设是，在OSCC中升高的TGFβ信号传导诱导了OSCC中TGFβ表达的增加。 ECM中整合素表达和层粘连蛋白/胶原蛋白失衡，改变ECM硬度并调节OSCC 转移、OSCC CSC、CD 8 + TIL运动性和ICI反应性。我将1）评估TGFβ依赖性， 层粘连蛋白和相关整合素在OSCC转移中的功能; 2）确定层粘连蛋白如何促进ECM 刚性和随后对OSCC CSC特征和运动性的影响;以及3）评估是否抑制层粘连蛋白 沉积增强ICI。这项研究将提供深入了解如何增强层粘连蛋白结合整合素表达 层粘连蛋白沉积促进OSCC转移、CSC特征和CD 8 + TIL排斥。它还将 鉴定ICI的预测性预后生物标志物和治疗转移性OSCC的治疗靶点。

Abstract Oral squamous cell carcinoma (OSCC) patients have a dismal survival rate due to distant metastases that escape primary care. Transforming growth factor beta (TGFβ) is a well-known driver of metastasis, modulator of immune cell activity, and regulator of extracellular matrix (ECM) genes. In breast cancer, a stiff ECM is generally attributed to excess type I collagen deposition and crosslinking that promotes cancer cell metastasis and cancer stem cell (CSC) expansion. In OSCC, a consensus has not been reached on whether a stiff or soft ECM increases metastatic potential and CSC expansion. The impact of OSCC ECM composition on immune cell trafficking is also unknown. Our laboratory has produced murine OSCC cell lines derived from Keratin15+ stem cells with Smad4 loss and KrasG12D mutation. Despite having the same genetic background, these cell lines have different metastatic ability, suggesting microenvironmental factors or cell intrinsic differences may mediate metastasis. RNAseq analysis revealed that metastatic OSCC cells have increased levels of laminins and laminin- binding integrins but downregulated type I collagen genes. Additionally, while OSCC cells cultured on stiff ECM demonstrate increased invasion, those cultured on a soft ECM display increased CSC characteristics. Treating metastatic OSCC cells with a TGFβ inhibitor reduced migration and invasion. Comprehensive immune profiling using flow cytometry revealed that metastatic tumors have decreased numbers of CD8+ T infiltrating lymphocytes (TILs) compared to non-metastatic OSCC tumors. These CD8+ TILs are instrumental for a robust anti-tumor immune response and the success of immune checkpoint inhibitor (ICI) therapy. Identifying responders to ICI and TGFβ inhibitors and sensitizing non-responders to these therapies persist as a major obstacle. Defining how OSCC cells interact with the ECM to promote their dissemination and enhancing current prognostic markers are both crucial to improve patient care and prolong survival. The goal of this proposal is to use unique murine models and human patient samples to define how ECM components and rigidity influence OSCC CSCs metastasis to improve the efficacy of emerging, targeted therapeutics to inhibit OSCC metastatic outgrowth. The hypothesis of this proposal is that elevated TGFβ signaling in OSCC induces increased integrin expression and a laminin/collagen imbalance in the ECM, altering ECM stiffness and modulating OSCC metastasis, OSCC CSCs, CD8+ TIL motility and ICI responsiveness. I will 1) evaluate TGFβ-dependency and function of laminins and associated integrins in OSCC metastasis; 2) determine how laminins contribute to ECM rigidity and subsequent impact on OSCC CSC characteristics and motility; and 3) assess if inhibiting laminin deposition enhances ICI. This study will provide insight into how enhanced laminin-binding integrin expression and laminin deposition facilitates OSCC metastasis, CSC characteristics, and CD8+ TIL exclusion. It will also identify predictive prognostic biomarkers for ICI and therapeutic targets for treating metastatic OSCC.