Lymph-Borne Melanoma-Derived Proteins as Determinants of Lymph Node Metastasis

项目总结 在肿瘤细胞到来之前，组织已经为转移做好了准备。原发肿瘤通过以下方式驱动这种转变 将肿瘤衍生因子(TDF)，包括细胞外小泡和分泌/脱落蛋白(TSP)释放到 肿瘤相关的血液和淋巴管系统。尽管是最常见的转移部位 对于实体瘤类型，我们对淋巴结(LN)是如何被TDFS转移所启动的知之甚少。我的 初步数据显示，原发性黑色素瘤在其引流的淋巴结中启动重塑，从而增强 相对于对侧、不引流和幼稚的LN的转移性生长。此外，我还有 证明了TDF唯一地激活了这种转移性LN表型，而不是源于 良性真皮或小鼠白蛋白。这一领域的焦点一直集中在肿瘤细胞外小泡的作用上。 在建立这种转移前的利基环境中发挥了作用，然而我的数据进一步表明TSP也足以 激活这个利基市场。使用一种称为BONCAT(双正交非规范)的新的细胞特异性标记策略 氨基酸标记)，我已经确定了几个转移到转移前LN的候选TSP。在这 提议，我将测试假设，即TSP启动重新编程事件在 通过两个特定目标支持转移性生长的引流性LN。第一个目标将使用scRNAseq 识别细胞网络的方法，TSP诱导的驻留LN间质和种植肿瘤之间的串扰 为转移细胞提供营养支持的细胞，以及需要LN淋巴管生成的程度 对于LN转移。第二个目标将调查我已经确定的一种特定的TSP，CSPG4(黑色素瘤- 结合的硫酸软骨素蛋白多糖)。这种蛋白质是由黑色素瘤细胞脱落的，已知黑色素瘤 细胞固有的侵袭作用在人类黑色素瘤患者中升高，然而，尚不清楚这种TSP 有助于在远处部位形成转移前的生态位。这项工作的完成将为 关于tdLN中依赖TSP的转移前生态位发育的信息，并可能发现CSPG4是一种 利基市场发展的新驱动力。拟议的工作将为我提供全面的培训机会 将新的工作台技术与高维数据分析相结合，最终为我的现代 独立从事研究工作。建议的其他培训机会将增强我的专业技能 在写作、演示、教学/指导和网络方面，为我的长期目标奠定了基础 在一家学术机构领导一个研究实验室。我的培训目标得到了我的赞助人(Dr。 阿曼达·隆德；皮肤科和病理学系)和共同赞助者(伊泰·柳井医生； 生物化学和分子药理学以及计算医学研究所所长) 分别具备癌症生物学和免疫学以及测序数据分析和整合方面的专业知识。

PROJECT SUMMARY Tissues are primed for metastasis prior to the arrival of tumor cells. Primary tumors drive this transformation by shedding tumor-derived factors (TDFs), including extracellular vesicles and secreted/shed proteins (TSPs) into tumor-associated blood and lymphatic vasculature. Despite being the most common site of metastasis across solid tumor types, we know very little about how the lymph node (LN) is primed for metastasis by TDFs. My preliminary data demonstrates that primary melanomas initiate remodeling in their draining LNs that enhances metastatic outgrowth relative to contralateral, non-draining and naïve LN controls. Furthermore, I have demonstrated that TDFs uniquely activate this pro-metastatic LN phenotype rather than factors derived from benign dermis or mouse albumin. The focus of this field has been on the role tumor-derived extracellular vesicles play in establishing this pre-metastatic niche, however my data further demonstrates TSPs are also sufficient to activate this niche. Using a novel cell-specific labeling strategy termed BONCAT (biorthogonal non-canonical amino acid tagging), I have identified several candidate TSPs transported to the pre-metastatic LN. In this proposal, I will test the hypothesis that TSPs initiate reprogramming events within resident cell populations of the draining LN that support metastatic growth through two specific aims. The first aim will employ a scRNAseq approach to identify cellular networks, TSP-induced crosstalk between the resident LN stroma and seeded tumor cells, that provide trophic support to metastatic cells and the extent to which LN lymphangiogenesis is required for LN metastasis. The second aim will investigate a specific TSP I have identified, CSPG4 (Melanoma- Associated Chondroitin Sulfate Proteoglycan). This protein is shed by melanoma cells, has known melanoma cell-intrinsic roles of invasion and is elevated in human melanoma patients, yet, it is unknown whether this TSP contributes to pre-metastatic niche formation in distant sites. The completion of this work will provide foundational information on TSP-dependent pre-metastatic niche development in the tdLN, and may uncover CSPG4 as a novel driver of niche development. The proposed work will provide me with comprehensive training opportunities integrating new bench techniques with high dimensional data analysis, ultimately preparing me for a modern independent career as a researcher. Other training opportunities proposed will strengthen my professional skills in writing, presenting, teaching/mentoring, and networking, laying the foundation for my long-term goals of leading a research lab at an academic institution. My training objectives are supported by my sponsor (Dr. Amanda Lund; Departments of Dermatology and Pathology) and co-sponsor (Dr. Itai Yanai; Departments of Biochemistry and Molecular Pharmacology and Director of the Institute for Computational Medicine) who bring expertise in cancer biology and immunology, and sequencing data analysis and integration, respectively.