Hepatic passaging of NRAS-mutant melanoma influences adhesive properties and metastatic pattern.

BACKGROUND: Liver metastasis is a poor prognostic factor for treatment of advanced cutaneous melanoma with either immunotherapy or targeted therapies. In this study we focused on NRAS mutated melanoma, a cohort with high unmet clinical need. METHODS: WT31 melanoma was repeatedly passaged over the liver after intravenous injections five times generating the subline WT31_P5IV. The colonization of target organs, morphology, vascularization and the gene expression profiles of metastases were analyzed. RESULTS: After intravenous injection lung metastasis was significantly decreased and a trend towards increased liver metastasis was detected for WT31_P5IV as compared to parental WT31. Besides, the ratio of lung to liver metastases was significantly smaller. Histology of lung metastases revealed reduced proliferation of WT31_P5IV in relation to WT31 while both size and necrotic areas were unaltered. Liver metastases of both sublines showed no differences in vascularization, proliferation or necrosis. To identify tumor-intrinsic factors that altered the metastatic pattern of WT31_P5IV RNA sequencing was performed and revealed a differential regulation of pathways involved in cell adhesion. Ex vivo fluorescence imaging confirmed that initial tumor cell retention in the lungs was significantly reduced in WT31_P5IV in comparison to WT31. CONCLUSION: This study demonstrates that tumor-intrinsic properties influencing the metastatic pattern of NRAS mutated melanoma are strongly affected by hepatic passaging and the hematogenous route tumor cells take. It has implications for the clinical setting as such effects might also occur during metastatic spread or disease progression in melanoma patients.

Lyve-1 deficiency enhances the hepatic immune microenvironment entailing altered susceptibility to melanoma liver metastasis.

BACKGROUND: Hyaluronan receptor LYVE-1 is expressed by liver sinusoidal endothelial cells (LSEC), lymphatic endothelial cells and specialized macrophages. Besides binding to hyaluronan, LYVE-1 can mediate adhesion of leukocytes and cancer cells to endothelial cells. Here, we assessed the impact of LYVE-1 on physiological liver functions and metastasis. METHODS: Mice with deficiency of Lyve-1 (Lyve-1-KO) were analyzed using histology, immunofluorescence, microarray analysis, plasma proteomics and flow cytometry. Liver metastasis was studied by intrasplenic/intravenous injection of melanoma (B16F10 luc2, WT31) or colorectal carcinoma (MC38). RESULTS: Hepatic architecture, liver size, endothelial differentiation and angiocrine functions were unaltered in Lyve-1-KO. Hyaluronan plasma levels were significantly increased in Lyve-1-KO. Besides, plasma proteomics revealed increased carbonic anhydrase-2 and decreased FXIIIA. Furthermore, gene expression analysis of LSEC indicated regulation of immunological pathways. Therefore, liver metastasis of highly and weakly immunogenic tumors, i.e. melanoma and colorectal carcinoma (CRC), was analyzed. Hepatic metastasis of B16F10 luc2 and WT31 melanoma cells, but not MC38 CRC cells, was significantly reduced in Lyve-1-KO mice. In vivo retention assays with B16F10 luc2 cells were unaltered between Lyve-1-KO and control mice. However, in tumor-free Lyve-1-KO livers numbers of hepatic CD4+, CD8+ and regulatory T cells were increased. In addition, iron deposition was found in F4/80+ liver macrophages known to exert pro-inflammatory effects. CONCLUSION: Lyve-1 deficiency controlled hepatic metastasis in a tumor cell-specific manner leading to reduced growth of hepatic metastases of melanoma, but not CRC. Anti-tumorigenic effects are likely due to enhancement of the premetastatic hepatic immune microenvironment influencing early liver metastasis formation.

Suppression of Endothelial Cell FAK Expression Reduces Pancreatic Ductal Adenocarcinoma Metastasis after Gemcitabine Treatment.

Despite substantial advances in the treatment of solid cancers, resistance to therapy remains a major obstacle to prolonged progression-free survival. Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, with a high level of liver metastasis. Primary PDAC is highly hypoxic, and metastases are resistant to first-line treatment, including gemcitabine. Recent studies have indicated that endothelial cell (EC) focal adhesion kinase (FAK) regulates DNA-damaging therapy-induced angiocrine factors and chemosensitivity in primary tumor models. Here, we show that inducible loss of EC-FAK in both orthotopic and spontaneous mouse models of PDAC is not sufficient to affect primary tumor growth but reduces liver and lung metastasis load and improves survival rates in gemcitabine-treated, but not untreated, mice. EC-FAK loss did not affect primary tumor angiogenesis, tumor blood vessel leakage, or early events in metastasis, including the numbers of circulating tumor cells, tumor cell homing, or metastatic seeding. Phosphoproteomics analysis showed a downregulation of the MAPK, RAF, and PAK signaling pathways in gemcitabine-treated FAK-depleted ECs compared with gemcitabine-treated wild-type ECs. Moreover, low levels of EC-FAK correlated with increased survival and reduced relapse in gemcitabine-treated patients with PDAC, supporting the clinical relevance of these findings. Altogether, we have identified a new role of EC-FAK in regulating PDAC metastasis upon gemcitabine treatment that impacts outcome. SIGNIFICANCE: These findings establish the potential utility of combinatorial endothelial cell FAK targeting together with gemcitabine in future clinical applications to control metastasis in patients with pancreatic ductal adenocarcinoma.

Angiogenic and molecular diversity determine hepatic melanoma metastasis and response to anti-angiogenic treatment.

BACKGROUND: Cutaneous melanoma exhibits heterogeneous metastatic patterns and prognosis. In this regard, liver metastasis, which is detected in ~ 10-20% of stage 4 patients, came to the fore of melanoma research, as it recently evolved as decisive indicator of treatment resistance to immune checkpoint inhibition. METHODS: Hepatic metastases were induced by intrasplenic injection of five different murine melanoma cell lines. The efficiencies of hepatic colonization, morphologic patterns, gene expression profiles and degree of vascularization were analyzed and Sorafenib was applied as anti-angiogenic treatment. RESULTS: WT31 melanoma showed the highest efficiency of hepatic colonization, while intermediate efficiencies were observed for B16F10 and RET, and low efficiencies for D4M and HCmel12. RNAseq-based gene expression profiles of high and intermediate metastatic melanomas in comparison to low metastatic melanomas indicated that this efficiency predominantly associates with gene clusters involved in cell migration and angiogenesis. Indeed, heterogeneous vascularization patterns were found in the five models. Although the degree of vascularization of WT31 and B16F10 metastases differed, both showed a strong response to Sorafenib with a successful abrogation of the vascularization. CONCLUSION: Our data indicate that molecular heterogeneity of melanomas can be associated with phenotypic and prognostic features of hepatic metastasis paving the way for organ-specific anti-angiogenic therapeutic approaches.

Melanoma Extracellular Vesicles Generate Immunosuppressive Myeloid Cells by Upregulating PD-L1 via TLR4 Signaling.

Tumor cell-derived extracellular vesicles (EV) convert normal myeloid cells into myeloid-derived suppressor cells (MDSC), inhibiting antitumor immune responses. Here, we show that EV from Ret mouse melanoma cells upregulate the expression of programmed cell death ligand 1 (PD-L1) on mouse immature myeloid cells (IMC), leading to suppression of T-cell activation. PD-L1 expression and the immunosuppressive potential of EV-generated MDSC were dependent on the expression of Toll-like receptors (TLR). IMC from Tlr4-/- mice failed to increase T-cell PD-L1 expression and immunosuppression with Ret-EV treatment, and this effect was dependent on heat-shock protein 86 (HSP86) as HSP86-deficient Ret cells could not stimulate PD-L1 expression on normal IMC; IMC from Tlr2-/- and Tlr7-/- mice demonstrated similar results, although to a lesser extent. HSP86-deficient Ret cells slowed tumor progression in vivo associated with decreased frequency of tumor-infiltrating PD-L1+CD11b+Gr1+ MDSC. EV from human melanoma cells upregulated PD-L1 and immunosuppression of normal monocytes dependent on HSP86. These findings highlight a novel EV-mediated mechanism of MDSC generation from normal myeloid cells, suggesting the importance of EV targeting for tumor therapy. SIGNIFICANCE: These findings validate the importance of TLR4 signaling in reprogramming normal myeloid cells into functional myeloid-derived suppressor cells.