Understanding Esophageal Adenocarcinoma Progression Using Inflammatory and Neoplastic Extracellular Matrix Hydrogels

项目总结/摘要 尽管已知微环境在癌细胞进展中的重要性[1]， 概括微环境的工具在很大程度上保持不变：胶原蛋白凝胶和基质胶 分别在过去50年和33年中占据癌症生物学文献的主导地位。2011年，申请人 作为赞助机构，Badylak实验室开发了第一个“器官特异性”细胞外基质（ECM） 来自脱细胞组织的水凝胶，特别是膀胱基质（UBM）水凝胶[3]。ECM是一种组织- 由所有活细胞分泌的一组特殊的结构和功能蛋白质，主要由胶原蛋白组成 而且还含有糖蛋白（层粘连蛋白、纤连蛋白）、蛋白聚糖、糖胺聚糖（GAG）和生长 这些因素使细胞具有时空线索[1]。ECM现在被认为是管理组织的关键- “动态互易性”的特定功能：细胞与周围基质之间的双向串扰 以决定细胞行为[4]。来自脱细胞组织的ECM支架，如UBM，已被证明保留了细胞外基质。 生物活性、大部分超微结构和组成以及天然基质的大部分机械完整性 [5]的文件。器官特异性ECM水凝胶已被创建用于各种器官。拟议的研究将 是疾病特异性ECM水凝胶的第一个证明，特别是正常，化生和肿瘤 食管腺癌（EAC）ECM水凝胶。此外，我们的小组最近发现， 从ECM生物支架分离的外泌体。外来体是一种进化上保守的机制， 细胞间信号传导，并且尚未表征为生物支架的分子机制。[第十一届] 这将是第一项表征来自正常和患病食管ECM的外泌体的研究 细胞外基质（eECM），特别是指导食管干细胞分化和免疫细胞活化。 这项工作的中心假设是，炎症和肿瘤eECM提供了物理和生物学功能。 上皮细胞向间充质细胞转化的生化信号， 发育途径（Sox，Wnt），并反过来促进EAC肿瘤发生。假设正常， 化生和肿瘤ECM水凝胶将1）指导食管干细胞的疾病特异性分化 类器官中的细胞2）将巨噬细胞活化为独特的M1/M2亚型，3）含有疾病特异性 外泌体具有在巨噬细胞和干细胞中诱导EMT的不同能力;如由基因决定的 表达、免疫组织化学和分泌产物的分析。这项工作将提供更好的 了解“病变ECM”指导干细胞行为的机制， 免疫细胞，并可能告知未来的工作，使用“正常”ECM来调节肿瘤进展， 使癌细胞恢复正常。此外，ECM水凝胶和ECM外泌体将提供新的 在肿瘤工程和药物发现的背景下，为癌症生物学家提供生物工程工具。这些发现 这项工作的一部分将改善EAC患者的再生医学策略， 食管癌（BE），并确定这种日益毁灭性的癌症形式的临床生物标志物。 .

PROJECT SUMMARY/ABSTRACT Despite the known importance of the microenvironment in cancer cell progression [1], cancer biology tools to recapitulate the microenvironment have largely remained unchanged: collagen gels and Matrigel have dominated the cancer biology literature for the past 50 and 33 years, respectively. In 2011, the applicant's sponsoring institution, the Badylak laboratory developed the first “organ-specific” extracellular matrix (ECM) hydrogels from decellularized tissue, specifically urinary bladder matrix (UBM) hydrogels [3]. ECM is the tissue- specific set of structural and functional proteins secreted by all living cells, and consists primarily of collagen but also contains glycoproteins (laminin, fibronectin), proteoglycans, glycosaminoglycans (GAGs) and growth factors that present cells with spatiotemporal cues [1]. ECM is now viewed as essential to govern tissue- specific function by “dynamic reciprocity”: the bidirectional crosstalk between a cell and its surrounding matrix to dictate cell behavior [4]. ECM scaffolds from decellularized tissue, like UBM, have been shown to retain the bioactivity, majority of ultrastructure and composition, and most of the mechanical integrity of the native matrix [5]. Organ-specific ECM hydrogels have since been created for a variety of organs. The proposed study would be the first demonstration of disease-specific ECM hydrogels, specifically normal, metaplastic, and neoplastic esophageal adenocarcinoma (EAC) ECM hydrogels. Furthermore, our group has recently identified and isolated exosomes from ECM biologic scaffolds. Exosomes are an evolutionarily conserved mechanism for intercellular signaling, and have not been characterized as a molecular mechanism for biological scaffolds.[11] This will be the first study to characterize exosomes derived from normal and diseased esophageal ECM (eECM), specifically to direct esophageal stem cell differentiation and immune cell activation. The central hypothesis of this work is that inflammatory and neoplastic eECM provide physical and biochemical cues of epithelial-to-mesenchymal transition, which activate deregulated esophageal developmental pathways (Sox, Wnt) and in turn promote EAC tumorigenesis. It is hypothesized normal, metaplastic, and neoplastic ECM hydrogels will 1) direct disease-specific differentiation of esophageal stem cells in organoids 2) activate macrophages to distinctive M1/M2 subtypes and 3) contain disease-specific exosomes with differential capacity to induce EMT in macrophages and stem cells; as determined by gene expression, immunohistochemistry, and analysis of secreted products. This work will provide a better understanding of the mechanism by which “diseased ECM” directs behavior of stem cells and recruited immune cells, and may inform future work of using a “normal” ECM to modulate neoplastic progression and direct cancer cells back to normalcy. Furthermore, ECM hydrogels and ECM exosomes would provide new bioengineering tools for cancer biologists in the context of tumor engineering and drug discovery. The findings of this work will improve regenerative medicine strategies for patients with EAC and the precursor Barrett's Esophagus (BE), and identify clinical biomarkers in this increasingly devastating form of cancer. .