Investigating the role of senescence in the development of incurable prostate cancer bone metastases within a human 3D in vitro model of aging bone

Prostate cancer (PCa) is a leading cause of morbidity and mortality in men and although recent advances extended overall survival (OS), men with advanced PCa invariably develop metastases, an incurable condition often affecting the bone. To treat PCa, therapies including chemotherapy and androgen-deprivation therapy are commonly used. However, these therapies trigger senescence both at the tumor site and throughout the body. In addition, with increasing age, senescent cells accumulate in multiple tissues including bone. Senescent cells are characterized by a stable cell cycle arrest and by the secretion of senescence-associated secretory phenotype (SASP), which has been recently shown to be a driver for breast cancer metastasis to bone. In the context of PCa, our project partner already demonstrated that senescent PCa cells foster metastasis formation in lung and lymph nodes. Furthermore, the elimination of these senescent cells with senolytic drugs allowed to block metastasis formation. Although the use of senolytics represents a promising therapeutic approach, no data are available on the role of senescence and efficacy of senolytics in PCa bone metastasis. This is partly due to the lack of preclinical models able to recapitulate the process of bone metastases formation, since no genetically engineered mouse models (GEMM) can mimic the formation of human PCa bone metastases and available in vitro models are too simplified. The hypothesis of the present proposal is that the accumulation of senescent cells in the metastatic bone microenvironment can drive the progression of PCa bone metastases, and to test it we will develop a microphysiological platform recapitulating aged bone microenvironment, based on our already developed 3D in vitro bone models. With the same platform, we will then test the application of clinically-relevant therapies based on senolytics, to verify if their application can improve current therapeutic regimens for PCa bone metastasis. The project is structured into 4 aims. Firstly, we will define the senescence profile of cells harvested from human bone samples derived from young and old donors, through single-cell transcriptomic analyses. In parallel, we will design and microfabricate a microphysiological platform hosting up to 64 independent minitissues replicating the bone microenvironment. These minitissues will be generated using bone cells from old and young donors and will be characterized through high throughput imaging and secretome analyses. Once fully characterized, PCa cells from cell lines and patient biopsies will be introduced into the bone microenvironments to investigate bone metastasis formation in presence or not of senescent bone cells. In parallel, we will test the introduction of senescent or non-senescent PCa cells to assess the effects of senescent tumor cells in the bone microenvironment. An ex vivo validation using tissue biopsies from human PCa bone metastases will allow to verify the correlation between the presence of senescence and PCa metastasis. As a final aim, we will screen the effect of selected senolytic therapies in metastasis formation, to verify the possibility to counteract senescence effects also in bone. The project will allow to elucidate the role of senescence in the bone microenvironment as a trigger of bone metastases from prostate cancer. Furthermore, it will test the possibility to counteract senescence effects with the use of senolytics, helping to define new therapeutic protocols for metastatic patients. The possibility to deeply and individually characterize bone senescence will result in a precise definition of the patient profile, thus foreseeing a future personalized approach to treatment. Finally, the availability of an in vitro platform including bone-mimicking 3D human minitissues, would allow the study of PCa bone metastases in a relevant pre-clinical model, opening the possibility to test new drugs in a more efficient and safe way.