A Influência do Secretoma do Cancro da Mama Triplo Negativo na Reprogramação Pré-Metastática do Osso

Bone-related events caused by breast cancer bone metastasis substantially compromise patients’ survival and quality of life. Since TNBC lacks hormone receptors and HER2-targeted therapeutic options, progress in the treatment of TNBC bone metastasis has been very slow. Therefore, it is crucial to identify the players and the molecular mechanisms that drive bone metastases in TNBC patients. Our major GOAL is thus to identify TNBC membrane predictive biomarkers associated with bone metastatic progression. To address this important challenge, preliminary analysis of the membrane proteome of metastatic bone-tropic TNBC cells has been performed by our group and potential highly enriched biomarkers have been already identified, namely NGFR and ICAM1 (Annex_Preliminary data_Fig.4). Interestingly, since a NGFR-ICAM1 mechanism has been recently described by Peinado et al.[9], underlying melanoma metastasis to lymph nodes (Annex_Supporting Data), we decided to investigate, for the first time, whether this mechanism can also be hijacked by TNBC cells to specifically metastasize to the bone. To achieve this general goal, we will address the following complementary objectives: 1) to determine the in vivo impact of bone-tropic NGFR/ICAM1-sEVs in bone PMN formation and bone metastases (TASK_1); 2) to study, in vitro, the uptake of these bone tropic-derived sEV by bone resident cells (TASK_2); and 3) to evaluate NGFR/ICAM1 expression in human samples, namely in a retrospective series of primary tumors and matched metastases, as well as in CTCs and circulating sEV isolated from patient liquid biopsies (TASK_3). In TASK_1, experiments will be carried out at i3S, with the close support of HPeinado (consultant of this project), who has a large expertise in sEV distribution and on their effect on different PMNs (including bone), using pre-clinical models (Annex_Supporting Data). In detail, NGFR/ICAM1 bone-tropic-derived sEV will be widely characterized (Task_1.1) and tested in vivo for their bone metastatic capacity (Task_1.2.). Bone metastatic lesions will be histologically characterized (Task_1.3.). In TASK_2, a breast cancer bone metastasis-on-a-chip microfluidic device, already established at i3S (Annex_Supporting Data), will be used to study the effect of sEV on different constituents of the bone PMN, namely endothelial cells and osteoclasts (Task_2.1.). Additionally, several in vitro assays will be used to validate the functional impact of these NGFR/ICAM1 bone-tropic-derived sEV on angiogenesis, tumor-endothelial cell adhesion, proliferation and migration (Task_2.2). In TASK_3, in a strong collaboration with IPOP, we will evaluate and characterize the expression profiles of NGFR and ICAM1 in human breast cancer samples. A series of primary tumors and matched metastases will be first evaluated, where correlations with the metastatic site and patient prognosis will be determined (Task_3.1.). Subsequently, liquid biopsies collected from stage III/IV metastatic breast cancer patients will be collected to study NGFR and ICAM1 expression in isolated circulating sEV and CTCs (Task_3.2.). (Annex_Experimental Plan)