Tumour microenvironment, extracellular vesicles and their effect on prostate cancer progression and docetaxel resistance

Abstract

Prostate cancer is the fourth most common cancer in Europe and the third leading cause of death in males. The standard of care as initial treatment for prostate cancer is, typically, androgen-deprivation therapy. Although successful, in the long run 80% of the patients relapse due to the development of a more aggressive form of prostate cancer known as castration-resistant prostate cancer. Ultimately this is treated with chemotherapy, with docetaxel being the gold-standard chemotherapeutic agents. However, the use of chemotherapy in prostate cancer is problematic. In fact, it inevitably develops resistance to chemotherapy and in this case, not many other treatment options are left. The main focus of this project was to unravel the importance of the tumour microenvironment, especially EVs, in the spreading of aggressive behaviours within prostate cancer. EVs, being released inside the tumour microenvironment and containing precious information from the cells of origin, are able to shuttle and transfer aggressive behaviours to receiving cells. In prostate cancer, initially our group and subsequently others, have shown that EVs are able to transfer aggressive traits including the transfer of chemotherapy resistance, the reprogramming of tumour microenvironment, the ability to form pre-metastatic niche and increased metastatic tendency. For these reasons, we initially evaluated the entire cellular secretome derived from two docetaxel-resistant prostate cancer cells in order to better understand the role played by both EVs and soluble factors. We found that although EVs impact docetaxel resistance transfer, cellular proliferation and migration, secretomes? soluble factors play a role as well, demonstrating their complex interplay with EVs within the tumour microenvironment. In order to further understand the effect of EVs on the transfer of aggressive behaviours on receiving cells and taking into consideration the enormous EVs heterogenicity, we moved on to investigating four proposed EVs inhibitors with the purpose to identify a possible pharmacological agent that blocking a specific EVs sub-population would reduce the transfer of the aggressive traits. In order to do this, we had to first optimise an EVs separation protocol able to recover as many clean/pure EVs as possible. We thus evaluated and combined different techniques, namely tangential flow filtration, gradient ultracentrifugation, size-exclusion chromatography and ultracentrifugation washing, in order to meet our requirements. Once optimised the EVs separation protocol, we started to evaluate the effect of our four EVs biogenesis/release inhibitors, used alone or in combination, on the two docetaxel-resistant prostate cancer cells, PC3RD and DU145RD. We found that on both cell lines, calpeptin and GW4869 reduced the shedding of small vesicles, while manumycin A and Y27632 increased the release of small vesicles. GW4869 treatment proved to significantly increase the release of bigger vesicles. Once tested on receiving cell lines, vesicles that continued to be released following Y27632 and manumycin A treatment proved to reduce cellular proliferation and cellular migration of cancer cells, but had no effect on normal prostate cells used in this thesis as control. We finally investigated a potential connection between chemotherapy and immunosuppressive tumour microenvironment focusing our attention on two enzymes associated with T-cell suppression, IDO1 and CD73. We observed a strong downregulation of both CD73 and IDO1 when seven different cell lines, representative of different cancers and cancer sub-types, were treated with high dose of docetaxel. We thus focus our attention back on prostate cancer and we demonstrated the accumulation of both enzymes inside EVs. The EVs enzymes cargo was not affected following docetaxel treatment. This suggests that EVs, more than cells per se, might play a fundamental role in the immunosuppressive microenvironment that characterise most cancer. In conclusion our results demonstrate that EVs play fundamental roles within the tumour microenvironment of advanced castration-resistant prostate cancer as carriers of fundamental enzymes that potentially redirect cells toward an immunosuppressive phenotype. These vesicles have the potential, once received by neighbouring cancer cells, to increase their aggressive traits. Pharmacological inhibition of specific EVs biogenesis/release pathways could be beneficial for the prevention of cancer progression.