TECHNICAL ABSTRACT The skeleton is a preferred organ for cancer dissemination from various tumors malignancies. The main objective of this research proposal is to understand the function of the Bone Marrow microenvironment, and specifically of its perivascular components, in the establishment of skeletal metastasis. The studies that are proposed aim to test the innovative hypothesis that Mesenchymal Stem Cells (MSCs), as perivascular cells (pMSCs), function as gatekeepers controlling tumor 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. We propose that the inhibition of one or combination of tumor cells and pMSCs specific genes would prevent or attenuate the metastatic process in vivo. To experimentally dissect the various cellular and extracellular matrix components controlling extravasation, we have designed and validated a unique in vivo extraskeletal humanized bone marrow niche-mimicking platform (humanized Ossicle). This platform is centered around the use of a porous, calcium phosphate ceramic into which human bone marrow-derived MSCs (hBM-MSC) are loaded and then implanted into the back of immune deficient mice. Bone is fabricated onto the walls of the ceramic by hBM-MSCs anchored at these locations and, at the centers; the host-derived blood vessels have pMSCs within the marrow space. We have documented that when melanoma is injected into the blood stream of the mouse, the bones become black (melanin), as is the bone in habitat. If we manipulate the expression of molecules are we propose involved in the process, such as CXCL12 or MCAM in the pMSCs, the mouse bone is black and the habitat is white. This black-and-white result sets the stage for Specific Aim 1 where we dissect the participation of pMSCs and the perivascular basement membrane in driving Skeletal Metastasis. In Specific Aim 2, we propose to determine the participation of HSC-niche ligand molecules expressed on cancer cells in driving SM. We have established a baseline using melanoma, which has the highest rate of lethality once it metastasizes into bone. This proposal will also be focused on the study of molecular mechanisms using breast cancer cell lines. A logical extension of this platform is to provide a predictive diagnostic for the patients' control over metastasis for different osteotropic cancers. In addition to the significant direct clinical impact, this proposed work is expected also to provide the platform for future projects addressing the roles of pMSCs that may influence other skeletal metastasis, such as regulation of local antitumor immune response, cancer cells dormancy and tumor angiogenesis, and to serve as template for the study of other osteotropic malignancies (prostate and lung cancer), thus broadening the significance of the findings and conclusions.

PROJECT NARRATIVE /RELEVANCE Bone is a preferred organ for cancer dissemination (metastasis) which most of the times is a terminal condition. We propose to understand the mechanism(s) behind the establishment of bone metastasis based on a novel hypothesis suggesting that perivascular cells in the bone marrow control cancer cell invasion to the bone. This new hypothesis provides clinically relevant information for therapeutic strategies that innovately aim at reducing bone metastasis by closing the gate through which the metastatic cancer cell transit into the bone, thus, providing a paradigm for improving the devastating outcomes of bone metastasis.