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Abstract

The approval of immune checkpoint inhibition (ICI) for advanced melanoma patients heralded a new era in melanoma therapy. ICI is approved as palliative and adjuvant treatment for stage IV and stage III patients, respectively. Neoadjuvant ICI is currently not approved but is investigated in clinical trials for stage II and stage III patients. Liver metastasis, which is detected in ~10-20% of stage IV patients, gained special attention, as it recently evolved as important indicator of treatment resistance to ICI. In this study, the pre-metastatic immunological conditioning of the murine hepatic vascular niche is characterized and compares different ICI treatment regimens (i.e. palliative, adjuvant and neoadjuvant) regarding their efficiency to prevent and treat liver metastasis formation. Hepatic metastases in mice were induced either by intravenous or intrasplenic injection of melanoma cell lines WT31 and B16F10 to assess treatment responses among palliative, adjuvant and neoadjuvant ICI. In the neoadjuvant setting, melanoma cells were also injected intracutaneously to simulate primary cutaneous melanomas. The immune cell composition and activation was comparatively analyzed within the primary tumor, the blood and the liver with FACS, IF/IHC, in situ hybridization and cytokine assays. Hepatic metastasis was similar in extent in mice with intracutaneous melanomas compared to PBS injected controls indicating that a primary melanoma did not induce a pre-metastatic niche with a strong pro- or anti-tumoral phenotype. Naïve adjuvant therapy starting on day 0 showed reduced liver metastases in comparison to late palliative therapy starting on day 9. This was accompanied by increased hepatic infiltration of CD3+ CD8+ T cells in the naïve adjuvant therapy. Neoadjuvant therapy in the presence of a primary cutaneous melanoma showed even less hepatic metastases in comparison to adjuvant therapy. Primary tumors showed a T cell inflamed phenotype in neoadjuvant therapy and an immune excluded phenotype in adjuvant therapy. This was paralleled by increased CD4+ and CD8+ T cells in the peripheral blood in the neoadjuvant therapy. Additionally, hepatic CD4+ T-bet+ T cells significantly increased in neoadjuvant therapy while CD4+ Gata3+ T cells decreased in comparison to adjuvant therapy. Our data indicate that the choice of the therapeutic regimen is an important factor influencing the susceptibility of the hepatic vascular niche to liver metastasis and also therapy response to immune checkpoint inhibition. Neoadjuvant ICI was superior to adjuvant ICI regarding the prevention of liver metastasis formation. Furthermore, the liver showed a more Th1 driven immune response. Increased numbers of CD4+ Gata3+ T cells in the livers of mice in the adjuvant setting indicate a more Th2-driven immune response. Therefore, neoadjuvant ICI may be an excellent option for CM to prevent the spread to distant organs and to help improving the outcome of patients with distant metastases.