Bone Marrow MSCs/Pericytes: Gatekeepers Controlling Skeletal Metastasis.

描述（由申请人提供）：转移是癌症生存的主要预后指标，也是癌症死亡率的主要因素。骨骼是各种恶性肿瘤中癌症扩散的首选器官之一。目前的靶向癌症疗法被设计为不仅改变癌细胞中的特定生物学功能，而且改变肿瘤基质/微环境的组分，如血管。与周细胞限制癌细胞从原发性肿瘤传播的既定作用相反，这些基质组分在癌症转移期间在靶器官的作用尚不清楚。本研究提案的主要目的是了解骨髓（BM）微环境的功能，特别是其血管成分，在建立骨转移中的作用，基于一种新的假设，即BM间充质干细胞（BM-MSC）作为血管周围细胞（周细胞），作为控制癌细胞侵入骨的守门人。这一新的假设为治疗策略提供了临床相关信息，这些治疗策略的创新目的是通过关闭转移细胞转移到正常骨中的大门来减少循环癌细胞的植入。为了在实验上做到这一点，将使用血管系统的扰动周细胞覆盖的遗传模型（PDGF-Bret/ret小鼠），其中内皮细胞和周细胞之间的物理结合由于改变的PDGF-B信号传导而被破坏，来测试循环的骨营养性黑素瘤和乳腺癌细胞的BM植入。这一模式将有助于：第一，确定BM窦状网络的完整性/稳定性以及BM-MSC在其血管周围小生境中的存在如何参与癌细胞的外渗（SA 1）;第二，鉴定BM侵入期间这种癌细胞/组织基质关系所需的细胞相互作用和分子途径。将使用体外和体内模型（SA 2）进一步检测这些相互作用。在SA 1中，生物发光将用于跟踪循环细胞的BM植入、其作为转移性肿瘤的时间进展以及导致骨质溶解的解剖学/功能相关性（由？CT成像），还将通过组织学、组织形态计量学和血清标志物进行评估。使用特定的细胞标记物的免疫组织化学分析将揭示移植的黑色素瘤细胞和BM基质的改变的血管成分之间的相互作用的细节。在SA 2中，将使用遗传修饰的MSC、黑色素瘤和乳腺癌细胞的体内和体外试验进行机制评估。除了显著的直接临床影响外，这项拟议的工作预计还将为未来的项目提供基础，解决可能影响骨骼转移的BM-MSC的其他已知作用，例如对其他骨恶性肿瘤（包括前列腺，肾脏和肺部）的局部调节，从而扩大发现和结论的意义。

DESCRIPTION (provided by applicant): Metastasis is a leading prognostic indicator for cancer survival and a major contributor to cancer mortality. The skeleton is one of the preferred organs for cancer dissemination in various malignancies. Current targeted cancer therapies are designed to alter not only specific biological functions in cancer cells, but also components of the tumor stroma/microenvironment such as blood vessels. Contrary to the established role of pericytes limiting cancer cell dissemination from the primary tumor, the role(s) of these stromal components at the target organ during cancer metastasis are not understood. The main objective of this research proposal is to understand the function of the Bone Marrow (BM) microenvironment, and specifically of its vascular components, in the establishment of skeletal metastasis, based on a novel hypothesis that suggests that BM Mesenchymal Stem Cells (BM-MSCs), as perivascular cells (pericytes), function as gatekeepers controlling cancer cell invasion to the bone. This new hypothesis provides clinically relevant information for therapeutic strategies that innovately aim at reducing the engraftment of circulating cancer cells by closing the gate through which the metastatic cell transit into the normal bone. To experimentally do so, a genetic model of perturbed pericyte coverage of the vasculature (PDGF-Bret/ret mice), in which the physical association between endothelial cells and pericytes is disrupted due to altered PDGF-B signaling, will be used to test the BM engraftment of circulating osteotropic melanoma and breast cancer cells. This model will help: first, to establish how the integrity/stability of te BM sinusoidal network, and the presence of BM-MSCs in their perivascular niche, participate in the extravasation of cancer cells (SA1); and second, to identify the cellular interactions and molecular pathways required for this cancer cell/tissue stroma relationship during BM invasion. These interactions will be further tested using in vitro and in vivo models (SA2). In SA1, Bioluminescence will be used to track the BM engraftment of circulating cells, their progression in time as metastatic tumors and the anatomical/functional correlation of resulting osteolysis (analyzed by ?CT imaging), which will be also assessed by histology, histomorphometry and serum markers. Immunohistochemical analysis using specific cellular markers will reveal the details of the interaction between engrafted melanoma cells and the altered vascular components of the BM stroma. In SA2 a mechanistic assessment will be performed using in vivo and in vitro assays with genetically modified MSCs, melanoma and breast cancer cells. In addition to the significant direct clinical impact, this proposed work is expected also to provide the foundation for future projects addressing other known roles of BM-MSCs that may influence skeletal metastasis, such as regulation of locally of other osteotropic malignancies including prostate, renal and lung, thus broadening the significance of the findings and conclusions.