BMP7 in melanoma niche morphogenesis and homeostasis

总结 黑色素瘤可以说是人类癌症中最致命的，部分原因是它有转移的倾向， 以及它对传统抗癌疗法的抵抗力。治疗失败的一个关键因素是 肿瘤异质性，特别是具有干细胞样特性的亚群，称为黑色素瘤 起始细胞（MIC）。我们的长期目标是了解MIC和 它们的基质协作以促进生长和进展，作为开发有效治疗的手段。 战略布局在目前的项目中，我们专注于定义微环境的“生态位”，CD 133 +/ABCB 5 + MIC作为阐明维持代谢和代谢的复杂细胞和分子相互作用的门户， 我们的初步数据表明：1）CD 133 +/ABCB 5 + MIC在空间上是 以与“血管生成拟态（VM）"相同的模式排列; 2）在血管生成拟态中如此形成的血管样通道， VM本身是CD 144（VE-钙粘蛋白）+，与真实的内皮结构密切相关， 符合所谓的“血管周围龛”; 3）黑色素瘤细胞表达骨形态发生蛋白7 （BMP 7）及其拮抗剂Noggin与肿瘤进展相关，并且还共定位于VM区域; 4）BMP 7上调基质微环境中的血管生成因子VEGF，但同时 诱导Noggin缺陷型黑素瘤细胞的选择性凋亡。总的来说，我们的研究结果支持中央 假设CD 133 +/ABCB 5 + MIC相关的BMP 7可能不仅通过 促进“生态位”形态发生（通过协调VM和血管生成），但也平衡代谢 需求（通过刺激VEGF依赖的血管生成和诱导选择性凋亡）。 竞争性、Noggin缺陷、非起始群体）。使用多标记免疫荧光和免疫- 引导激光捕获显微切割，然后进行qRT-PCR，结合功能循环分析，我们 进一步阐明黑色素瘤细胞CD 133、ABCB 5、BMP 7之间的时空关系 和CD 144表达，以及微循环的类型/模式（例如黑素瘤VM通道形成与 血管生成）（目的1a）。CD 133+黑色素瘤亚群在“生态位”中的动态变化及其功能影响 将在原位异种移植模型中使用诱导型RNAi介导的敲低来测试Aim 1b 在不同的节奏。最后，在“小生境”中分析CD 133 +/ABCB 5 + MIC相关的BMP 7分子信号， 形态发生（目标2a）和肿瘤异质性/稳态（目标2b），我们将采用 功能的方法来评估BMP 7-VEGF/BMP 7-Noggin轴，在组织背景下，在独特的三个- 体外三维（3D）器官型培养物和体内黑素瘤异种移植模型。界定机制 通过MIC相关的BMP 7信号有助于“利基”发展和维护， 通过靶向其基质依赖性直接或间接治疗性消除MIC的新机会。

Summary Melanoma is arguably the most virulent among human cancers, in part due to its propensity to metastasize, and its resistance to conventional anti-cancer therapies. One key factor responsible for treatment failure relates to tumor heterogeneity, particularly subpopulations that possess stem cell-like properties, known as melanoma initiating cells (MICs). Our long-term goal is to understand the molecular mechanisms, whereby MICs and their stroma collaborate to promote growth and progression, as a means to develop effective therapeutic strategies. In the current project, we focus on defining the microenvironmental "niche", of CD133+/ABCB5+ MICs as a gateway to elucidate the complex cellular and molecular interplay that maintains the metabolic and replicative integrity of MICs. Our preliminary data indicate that : 1) the CD133+/ABCB5+ MICs are spatially arranged in a pattern identical to that of "vasculogenic mimicry (VM)"; 2) the vessel-like channels so formed in VM are themselves CD144 (VE-cadherin)+ and intimately associated with authentic endothelial structures, in keeping with so-called "perivascular niches"; 3) melanoma cell expression of bone morphogenetic protein 7 (BMP7) and its antagonist Noggin is associated with tumor progression and also co-localizes to areas of VM; and 4) BMP7 upregulates the angiogenic factor VEGF in the stromal microenvironment, but at the same time induces selective apoptosis in Noggin-deficient melanoma cells. Collectively, our findings support the central hypothesis that CD133+/ABCB5+ MIC-associated BMP7 may not only support the maintenance of MICs by facilitating "niche" morphogenesis (through coordinated VM and angiogenesis), but also balance metabolic demands (through stimulating VEGF-dependent angiogenesis and inducing selective apoptosis in the competitive, Noggin-deficient, non-initiating population). Using multi-label immunofluorescence and immuno- guided laser capture microdissection followed by qRT-PCR, together with functional circulation analysis, we propose to further delineate the temporal/spatial relationship between melanoma cell CD133, ABCB5, BMP7 and CD144 expression, and the types/ patterns of microcirculation (e.g. melanoma VM channel formation vs. angiogenesis) in vivo (Aim 1a). The dynamics and functional impacts of CD133+ melanoma subsets in "niche" formation (Aim 1b) will be tested in an orthotopic xenograft model using inducible RNAi-mediated knockdown at varying tempos. Finally, to dissect the CD133+/ ABCB5+ MIC-associated BMP7 molecular signals in "niche" morphogenesis (Aim 2a) and tumor heterogeneity/ homeostasis (Aim 2b), we will employ loss- and gain-of- function approaches to assess the BMP7-VEGF/ BMP7-Noggin axes, in a tissue context in unique three- dimensional (3D) organotypic cultures in vitro and melanoma xenograft models in vivo. Defining mechanisms through which MIC-associated BMP7 signals contribute to "niche" development and maintenance offers a novel opportunity to therapeutically eliminate MICs directly or indirectly by targeting their stromal dependency.