The molecular mechanisms dictating metastasis remain unknown, contributing to the limited efficacy of existing treatments as well as to reduced survival rates in patients with metastatic prostate cancer (mPCa), compared to those with localized tumors. Thus, to improve mPCa patients' diagnosis and prognosis, our study aims to uncover key metastatic regulators using high-troughput CRISPR/Cas9 screenings and elucidate organ specific metastasis mechanisms.

To identify novel factors promoting the acquisition of metastatic traits, we have developed an in vivo gain of function (GoF) genome-wide screening using orthotopic inoculation of low-metastatic PCa cells (LNCaP) infected with a genome-wide SAM library. To study temporal and spatial components of metastasis, we examined 5 tissues (bone marrow (BM), lung, liver, lymph nodes and brain) at either 4 (early metastasis-EM)- or 6 (late metastasis-LM) weeks post orthotopic inoculation of PCa cells. Functional studies using CRISPR/Cas9-modified PCa cell lines have identified critical regulators of BM dissemination and colonization.

We have identified distinct pathways driving PCa metastasis depending on the target tissue, as well as an enrichment of specific gRNAs in LM compared to EM. These results highlight the influence of both the microenvironment and time-dependent factors on metastatic evolution. Since the BM is the most common metastatic destination in mPCa, we have tried to identify a molecular signature driving bone spread. By analyzing the gRNAs significantly enriched across time in the BM that weren’t altered in the primary tumor, we have identified 4 candidates: FGF8, SLC24A5, CCDC110 and CEP70. Analysis of patient cohort databases revealed that FGF8, SLC24A5, CCDC110 and CEP70 overexpression is associated with increased tumor aggressiveness and reduced patient survival. FGF8 has been previously described as a metastatic promoter thereby validating our system. Functional in vitro assays demonstrated that both genetic depletion and silencing of FGF8 reduces the migratory and invasive capacities of PCa cells, whereas treatment with recombinant FGF8 enhances PCa cells’ metastatic traits.

Our study has provided us with a complete overview of the whole metastatic cascade in vivo, revealing the different tropisms that disseminated tumor cells acquired in response to the host microenvironment. Moreover, we have identified new potential drivers of BM dissemination, which could represent promising biomarkers and therapeutic targets to improve outcomes for patients with bone-metastatic prostate cancer.

Grants PID2024-157599OB-I00, CNS 2023-144109, PID2022-136959OB-I00, PID2022-137717OB-C21 from MCIN/AEI /10.13039/501100011033 (cosponsored by the European FEDER Program) and by the European union Next Generation EU/ PRTR. IdlGl is supported by the Spanish Ministry of Education (FPU fellowship FPU23/03823).