Objectives: Stromal cells, including endothelial cells (ECs), are critical elements of the tumor microenvironment (TME), releasing factors that facilitate tumor growth. Although the role of the TME in the growth and spread of primary tumors has been investigated, less is known about the role the TME might play in regulating the progression of metastatic tumor foci. Based on extensive preliminary data we hypothesize that during the late stages of metastatic tumor progression, vascular endothelial PECAM-1 modulates the TME to induce a proliferative tumor cell gene expression profile that promotes metastatic tumor growth. To test this, studies are proposed that will (i) determine the molecular basis for the involvement of endothelial PECAM-1 in tumor metastasis to the lung (Specific Aim 1); (ii) identify PECAM-1-dependent, endothelial-derived mediators that regulate metastatic tumor growth and progression in the lung (Specific Aim 2); and (iii) characterize the influence of endothelial PECAM-1 on the gene profile of metastatic tumors in the lung (Specific Aim 3). Methodology: Specific Aim 1. Determine the molecular basis for the involvement of endothelial PECAM-1 in tumor metastasis to the lung. PECAM-1-null endothelial cells from murine lung will be transduced with wild type PECAM-1 or PECAM-1 mutated in its ability to mediate ligand binding or intracellular signaling. The resulting cells will then be used in tumor-endothelial co-culture studies to assess the effects of perturbing PECAM-1 function on tumor cell proliferation. Specific Aim 2. Identify PECAM-1-dependent, endothelial-derived mediators that regulate metastatic tumor growth and progression in the lung. In vitro cell proliferation will be studied in tumor cells cultured in conditioned media derived from tumor-endothelial co-cultures, for which the levels of expression of suspected, PECAM-1-regulated, endothelial-derived factors have been altered. The in vivo significance of any factor implicated as a PECAM-1-regulated secreted protein by these studies will then be confirmed by assessing lung metastasis in mice injected with tumors over-expressing the factor of interest. Specific Aim 3. Characterize the influence of endothelial PECAM-1 on the gene profile of metastatic tumors in the lung. The gene expression levels in tumor cells of candidate, PECAM-1- regulated, growth-promoting genes, as well as the effects of up- or down-regulating them, will be determined in intravenous and spontaneous models of lung metastasis, using wild type mice treated with anti-PECAM-1 antibody and PECAM-1-null mice. Clinical Relationship: As the vast majority of cancer deaths are caused by metastatic disease, developing a more complete understanding of the events involved in tumor metastasis will be critical to developing novel treatments for patients with advanced cancer. Impact/Significance: The late progression of micro-metastatic tumor foci to macroscopic, clinically apparent tumors and the role that the TME might play in that process has not been vigorously investigated. PECAM-1 expressed on vessels may be a mediator of the late progression of metastatic tumors through a modulation of the TME. This represents an unanticipated, but potentially very important new function for PECAM-1 that may have significant implications for the mechanistic understanding and treatment of late-stage cancer.

We hypothesize that during the late stages of metastatic progression endothelial PECAM-1 modulates the tumor microenvironment to induce a proliferative tumor cell gene profile and thus tumor growth. To evaluate this hypothesis, studies will be done to (i) determine the molecular basis of endothelial PECAM-1's involvement in mediating the tumor metastasis to the lung; (ii) identify PECAM-1-dependent, endothelial- derived mediators that regulate metastatic tumor growth and progression in the lung; and (iii) characterize the influence of endothelial PECAM-1 on the gene profile of metastatic tumors.