Bone Marrow MSCs/Pericytes: Gatekeepers Controlling Skeletal Metastasis.

描述(由申请人提供)：转移是癌症存活率的主要预后指标，也是癌症死亡率的主要因素。骨骼是癌症在各种恶性肿瘤中扩散的首选器官之一。目前的靶向癌症治疗不仅旨在改变癌细胞的特定生物学功能，而且还旨在改变肿瘤间质/微环境的成分，如血管。与已确定的周细胞限制癌细胞从原发肿瘤扩散的作用相反，这些间质成分在肿瘤转移过程中在靶器官的作用(S)尚不清楚。本研究的主要目的是基于一种新的假设，即骨髓间充质干细胞(BM-MSCs)作为血管周围细胞(周细胞)，作为控制癌细胞向骨的侵袭的守门人，了解骨髓微环境(BM)，特别是其血管成分在骨转移中的作用。这一新的假说为治疗策略提供了临床上的相关信息，这些治疗策略创新地旨在通过关闭转移细胞进入正常骨骼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，将使用一个血管系统周细胞被扰乱覆盖的遗传模型(PDGF-Bret/ret小鼠)，其中内皮细胞和周细胞之间的物理联系因PDGF-B信号改变而中断，将用于测试循环中的嗜骨黑色素瘤和乳腺癌细胞的骨髓植入。该模型将有助于：第一，确定TE BM正弦网络的完整性/稳定性，以及BM-MSCs在其血管周围壁龛中的存在，如何参与癌细胞的外渗(SA1)；第二，确定这种癌细胞/组织间质关系在BM侵袭过程中所需的细胞相互作用和分子途径。这些相互作用将使用体外和体内模型(SA2)进行进一步测试。在SA1中，生物发光将被用来跟踪循环细胞的骨髓植入，它们作为转移性肿瘤的时间进展，以及由此产生的骨溶解的解剖/功能相关性(通过CT成像分析)，这也将通过组织学、组织形态计量学和血清标志物来评估。使用特定细胞标记物的免疫组织化学分析将揭示植入的黑色素瘤细胞与骨髓基质中改变的血管成分之间相互作用的细节。在SA2中，将使用转基因MSCs、黑色素瘤和乳腺癌细胞的体内和体外测试来进行机制评估。除了重大的直接临床影响外，这项拟议的工作还有望为未来的项目提供基础，解决BM-MSCs可能影响骨转移的其他已知角色，如局部调节其他嗜骨性恶性肿瘤，包括前列腺癌、肾脏和肺，从而扩大研究结果和结论的意义。

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.