Tumor-derived extracellular vesicles in the pre-metastatic niche.

Metastasis is the leading cause of death in cancer patients, and its early detection and prevention are currently difficult. Thus, a better understanding of the earliest stages of metastasis, is an important and presently unmet challenge. The objective of this application is to gain a better understanding of the mechanisms leading to the formation of the pre-metastatic niche (PMN), an early step in the metastatic process, with the goal of finding targets for intervention. The focus of this highly collaborative grant is on the role of tumor-derived extracellular vesicles (TEV) in melanoma (MEL) and neuroblastoma (NB). Recently published1 and preliminary data revealed that TEV are primarily, but not exclusively, captured by macrophages (MF) in metastasis-tropic organs prior to their colonization by tumor cells, and that they trigger an inflammatory reaction in MF that includes the production of interleukin (IL)-18 and IL-1β and activation of the inflammasome. We also found that TEV-capturing cells in the PMN are heterogenous, including MF and non-MF cells, with the contribution of non-MF cells, including myeloid derived suppressive cells (MDSC), increasing as the PMN evolves toward a metastatic niche (MN). Using newly murine models of orthotopic metastasis to track endogenously released TEV and their capture in the PMN developed in our laboratory, state of the art single cell analysis methods, and a unique collection of patient samples, we will test the hypothesis that TEV capture by resident MF in the PMN triggers via the inflammasome the release of inflammatory cytokines/chemokines, which signal for the recruitment of bone marrow -derived myeloid cells (BMDMC) and MDSC to the PMN, supporting the subsequent colonization of the PMN by metastatic tumor cells. In AIM 1, we will perform spatiotemporal analysis of the PMN formation and its evolution into MN, identifying at the single cell level subpopulations of TEV-capturing cells defined by their RNA and protein expression. We will use pharmacological and loss-of-function approaches targeting the activation of the inflammasome in the PMN to determine whether the development of the PMN is disrupted and metastatic colonization inhibited. In AIM 2, we will perform kinetics analysis of the colonization of the PMN by BMDMC. We will determine whether TEV-capturing MF send signal(s) that promote the differentiation of BMDMC into MDSC through the release of IL-1β and IL-18 or other cytokines. We will demonstrate the role of IL-18 and IL-1β on PMN formation and metastatic colonization using pharmacological inhibition of IL-18 or mice deficient in IL-1β or IL-18. In AIM 3, we will use a unique collection of retrospective and prospective liver tissues from children with NB to characterize by Imaging Mass Cytometry the PMN in human samples, comparing the results with those from murine models. We expect to identify mechanisms involved in the formation of the PMN and MN, providing a strong premise for development of intervention approaches that could prevent the initiation of metastasis. Considering the current absence of therapeutic agents preventing metastasis, the project, if successful, could have a significant impact for MEL, NB, and other cancers.