Nuovi approcci farmacologici per superare l'immunosoppressione in tumori trattati con gli inibitori degli immunocheckpoints.

ICIs, have demonstrated unprecedented clinical efficacy for the treatment of metastatic melanoma (MM) and microsatellite instability-high colorectal cancer. However, in cold tumors, both MM and CRC, inhibitory immune checkpoints limit antitumor immunity and confer resistance to ICIs. Emerging evidence has highlighted that anti-angiogenic drugs have the potential benefit of improving tumor immunogenicity and reduce immunosuppression in the tumor microenvironment (TME), thus transforming “immunologically cold tumors” into “hot tumors,” and therefore may have a key role in overcoming resistance to ICIs-based therapy. The aim of my PhD thesis was to gain mechanistic insights into the potential pharmacological synergism of combination therapy with antiangiogenic inhibitors and PD1 inhibitors in CRC and MM, employing both in vivo and ex vivo tumor models. In detail, by investigating the antitumor effects of antiangiogenic agent sunitinib in combination with anti-PD1 in syngenic mouse CRC in vivo model, I discovered that effectiveness of treatment relies on mechanisms leading to immune and vascular normalization of the TME, which ultimately led to tumor growth inhibition. The treatments reduced both micro vascular density (CD31+ cells) and VEGF in TME of tumors, indicating that they hindered the development of a pro-angiogenic tumor milieu, probably because they caused the depletion of pro-angiogenic and immunosuppressive myeloid cells, which release VEGF. Accordingly, the infiltration of immunosuppressive cell populations, including Treg, MDSCs and M2-macrophages was reduced, thereby contributing to create an immunity supportive microenvironment. Further investigations performed in CRC patient-derived organoids and human cell lines highlighted that sunitinib, both as single agent and in combination with the anti-PD1, inhibited the chemotaxis of M2-like versus M1-like macrophages and reduced the release of signalling molecules that drive macrophages polarization toward M2 immunosuppressive macrophages, through the inhibition of UPR signalling pathways IRE/XBP-1 and ATF6 in TAMs. Furthermore, such drugs combination enhanced anti-tumor immune response by unleashing T cell cytotoxicity against CRC organoids. Regarding MM, the combination of anti-angiogenic therapy and ICIs has been demonstrated a valid therapeutic strategy that can enhance anti-tumor immunity and further expand the landscape of the treatments for such disease. During the second part of my PhD thesis, I focused on the identification of immunosuppressive targets in the tumor immune microenvironment (TIME) of MM potentially related to resistance to anti-PD1, and on the investigation of lenvatinib ability to suppress such immunosuppressive targets. In organotypic tissue cultures (OTCs) derived from 9 patients with MM, who displayed resistance to anti-PD1, the combination with lenvatinib and pembrolizumab resulted in a significantly greater antitumor efficacy as respect to single agent in almost all OTCs. Consistently, the combined treatment induced the activation of caspase-3/7, leading to apoptosis of tumor cells. By analysing culture supernatants of OTCs before and after treatment(s), I found that combined treatment reduced the release of soluble immune checkpoints PD1, TIM-3, LAG-3 and BTLA to a greater extent than each drug alone, highlighting that the antitumor potential of such combination relies on the inhibition of signalling pathways supporting the accumulation of exhausted T cells. Nevertheless, I also observed a significant reduction of perforin release by cytotoxic T cells, which suggest a potential reduction of T-cell killing activity, while reducing exhaustion. In recent years, the generation of PDOs/OTCs from patient tumor biopsies allows the investigation of personalized pharmacological treatments, unveiling resistant pathways and identifying possible new anti-cancer therapies for patients, through a personalized medicine approach.