Project 2: Contribution of the Stromal Microenvironment to Early Dissemination

项目2 -概要 转移在黑色素瘤早期开始，并且与肿瘤细胞的表型和功能变化一致， 周围基质细胞和细胞外基质成分。在基质细胞类型中，癌症 相关的成纤维细胞（CAF）作为特别相关的细胞群出现。虽然他们是 遗传稳定，CAFs是表型和功能多样性和CAFs的不同子集被发现， 支持或抑制胰腺癌模型中的癌生长。尽管这些发现表明 不同的CAF组也可能存在于其他癌症中，但它们在大多数其他癌症类型中尚未被定义。 此外，由于特异性CAFs可能是有价值的预后标志物或治疗靶点，因此它正成为 更好地了解它们的发展和监管越来越重要。在这里，我们建议调查 黑色素瘤细胞和成纤维细胞之间的信号如何在肿瘤内产生异质性， 异质性释放了黑色素瘤细胞亚群中的转移行为。为了验证这个假设，我们 开发了基因工程同类小鼠模型，使我们能够从基因上操纵黑素细胞， 干细胞启动肿瘤，忠实地重演人类黑色素瘤的病理，并独立地 抑制黑色素瘤细胞或CAF中的特定基因。这些模型将独特地使我们能够研究如何 特异性信号传导机制影响CAF在黑色素瘤发展和转移中的功能。我们提出 将我们的小鼠模型与单细胞RNA测序、空间转录组学、ATAC-seq和ChIP-seq相结合， 方法来转录定义不同的CAF状态。我们还将沿着使用它们和黑色素瘤 从有和没有转移性进展的患者中采集标本，以绘制特定CAF及其与 其他细胞类型与高度多重免疫组织化学（Aim 1）。接下来，我们将利用这些工具和数据， 发现细胞状态特异性受体-配体相互作用，并研究IGF 1-IGF 1 R信号传导在黑色素瘤之间的作用 细胞和CAF-我们初步研究确定的一种模型途径-影响肿瘤内异质性， 体内转移进展（目的2）。 项目2将利用核心B和C的病理、技术和分析资源。它将 协同项目1，以确定黑色素瘤细胞是否以及如何与独特的CAF相互作用，以及如何与CAF相互作用。 这些相互作用允许转移性传播。项目2还将通过提供新的信息来加强项目3 调节肿瘤引流淋巴结并使免疫细胞耐受 转移性黑色素瘤细胞一旦我们在分子水平上理解了这些过程， 临床特征，我们可以开始告知患者选择早期黑色素瘤的辅助治疗 并促进靶向治疗的合理发展，预防或治疗早期转移性播散， 分期黑色素瘤和其他癌症类型。

PROJECT 2 – SUMMARY Metastasis initiates early in melanoma, and coincides with phenotypic and functional changes in tumor cells, surrounding stromal cells, and extracellular matrix components. Amongst the stromal cell types, cancer associated fibroblasts (CAFs) are emerging as a particularly relevant cell population. Although they are genetically stable, CAFs are phenotypically and functionally diverse and distinct subsets of CAFs were found to support or inhibit cancerous growth in pancreatic cancer models. Although these findings suggest functionally diverse sets of CAFs could also exist in other cancers, they have yet to be defined in most other cancer types. Furthermore, because specific CAFs might be valuable prognostic markers or therapeutic targets, it is becoming increasingly important to better understand their development and regulation. Here, we propose to investigate how signals between melanoma cells and fibroblasts create heterogeneity within tumors and how this heterogeneity unleashes metastatic behaviors in a subset of melanoma cells. To test this hypothesis, we developed genetically engineered congenic mouse models that allow us to genetically manipulate melanocyte stem cells to initiate tumors that faithfully recapitulate the pathology of human melanoma and to independently inhibit specific genes in melanoma cells or CAFs. These models will uniquely enable us to investigate how specific signaling mechanisms influence CAF functions in melanoma development and metastasis. We propose to couple our mouse models with single-cell RNA sequencing, spatial transcriptomics, ATAC-seq, and ChIP-seq approaches to transcriptionally define distinctive CAF states. We will also use them along with melanoma specimens from patients with and without metastatic progression to map specific CAFs and their proximity to other cell types with highly multiplexed immuno-histochemistry (Aim 1). Next, we will use these tools and data to discover cell state specific receptor-ligand interactions and study how IGF1-IGF1R signaling between melanoma cells and CAFs –one model pathway our preliminary studies identified – influences intratumor heterogeneity and metastatic progression in vivo (Aim 2). Project 2 will leverage the pathological, technological, and analytical resources of Cores B and C. It will synergize with Project 1 to determine whether and how melanoma cells interact with distinctive CAFs and how these interactions allow for metastatic dissemination. Project 2 will also enhance Project 3 by shedding new light onto CAF-secreted cytokines that modulate tumor draining lymph nodes and make immune cells tolerant to metastasizing melanoma cells. Once we understand these processes on a molecular level and link them to clinical characteristics, we can begin to inform patient selection for adjuvant therapy in early-stage melanoma and catalyze a rational development of targeted therapies that prevent or treat metastatic dissemination in early- stage melanoma and other cancer types.