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

Cutaneous melanoma (CM) is a phenotypic and molecular heterogeneous disease arising from melanocytes of the skin and preferentially spreads to the skin, lymph nodes, lung, liver and brain [1]. In recent years, the treatment of metastatic CM has been revolutionized by immune checkpoint inhibition (ICI) and targeted therapies (BRAF/ MEK inhibitors (BRAFi/MEKi)). Meanwhile, a median survival of 60 months is achieved by combined ICI [2]. And even for BRAF-mutated melanoma treated with combined BRAFi/MEKi durable treatment responses are seen in subpopulations with favorable prognostic factors such as normal or low LDH and low volume of disease [3]. Despite this huge progress in melanoma therapy, liver metastasis of CM is well documented as predictor of poor response to ICI [4] or targeted therapy of BRAF-mutated melanoma [5]. Therefore, it is important to develop novel treatment options for melanoma patients with advanced disease suffering from liver metastases.

Recent studies have provided insight into mechanisms of global therapy resistance mediated by hepatic metastasis. In murine models of colorectal carcinoma (CRC) and melanoma, subcapsular injection of MC38 CRC or B16F10 melanoma cell lines into the liver abolished the response of corresponding subcutaneous tumors to ICI. The authors tie this to infiltration of regulatory T-cells into the subcutaneous tumors which in turn recruit CD11b⁺ monocytes [6]. Most recently, these findings were extended by the fact that liver metastases of CRC and melanoma recruit tumor-specific CD8⁺ T-cells from the periphery, which underwent apoptosis, and, as consequence, induced systemic immunosuppression and reduced response to ICI [7].

Current clinical research focuses on sequential and combined treatment approaches of ICI and targeted therapies [8]. Moreover, additional compounds targeting components of the tumor microenvironment such as macrophages, fibroblasts or blood vessels are tested in preclinical studies to improve therapeutic options for melanoma patients suffering from liver metastasis.

In general, liver metastases most commonly arise from CRC, pancreatic, lung or mammary carcinoma. Regarding CRC or pancreatic cancer, the liver is the first organ in line of the vascular tree and therefore could be considered as sieve for disseminating tumor cells. In contrast, this does not apply for lung or mammary carcinoma, uveal melanoma (UM) or CM. Paget postulated that both tumor-intrinsic factors (“seeds”) as well as the microenvironment of the target organ (“soil”) need to be taken into account to understand organotropic metastasis [9]. UM is therefore often seen as paragon of this hypothesis, as it shows a remarkable proclivity for liver colonization as in nearly 90% of metastatic patients hepatic lesions are detected [10].

Research has often focused on phenotypic characteristics of liver metastasis in these tumor entities. Morphologic characterization identified pushing, replacement and desmoplastic histological growth patterns (HGPs) of breast cancer, colorectal, UM and CM liver metastases [11‐13]. In CM almost 55% of liver metastases are pure desmoplastic and associated with an improved prognosis as compared to any replacement HGP. The pushing HGP occurs in around 12% of cases and leads to rapid mortality [13]. Interestingly, the desmoplastic HGP of CRC correlates with an increased response to anti-angiogenic therapy and strong immune cell infiltration [14, 15].

In regard to tumor cell-intrinsic mechanisms regulating organotropic liver metastasis epithelial-to-mesenchymal transition controlled by miR-200c and the PKCζ/ADAR2 axis is a decisive step to hepatic metastasis of CRC [16]. Moreover, SMAD3 mutations in CRC liver metastasis are associated with the poorest prognosis [16‐18]. In breast cancer, human epidermal growth factor receptor 2 (HER2)-enriched subtypes significantly correlate with increased hepatic metastasis [19, 20]. At the molecular level reduced BMP-SMAD1/5 signaling is related to decreased distant metastasis free and decreased overall survival of patients [21]. This pathway controls breast cancer metastasis in an organ-specific manner as pharmacological inhibition by tacrolimus with or without addition of a MEK inhibitor reduces metastasis to liver and bone while lung and brain metastasis are not affected. In UM, loss of the tumor suppressor BRCA1-associated protein 1 (BAP1) is the key event to metastasis and correlates with disease outcome [22, 23].

In CM, mutations of both NRAS and BRAF correlate with brain and liver metastasis [24]. However, this correlation is weak as NRAS or BRAF mutations are found in approximately 80% of melanomas and cannot be used as reliable clinical predictor of hepatic metastasis. So far, no mutational drivers of organ-specific hepatic metastasis have been described for CM. However, a liver passaged B16 melanoma subline shows increased expression of integrin alpha2 and enhanced liver but not lung metastasis [25]. Besides, tumor-intrinsic MSX1 expression regulating neural crest-like reprogramming is associated with preferential metastasis to the liver. Yet it is not known whether this also occurs in an immunocompetent setting as this study was performed in immunodeficient mice only [26].

Tumor intrinsic features associated with hepatic colonization of CM have not been characterized in detail. Therefore, this study comparatively analyzed five genetically different murine melanoma cell lines, reflecting the genetic heterogeneity of melanomas, and their molecular and phenotypic features underlying the different efficiencies of liver metastasis formation in mice.

Since CM differ in underlying driver mutations as well as their clinical and morphologic features, five murine melanoma cell lines with heterogenous driver mutations were selected (Table 1) to evaluate their ability to colonize the liver. These cell lines showed a highly variable efficiency of liver metastasis after intrasplenic injection (Fig. 1A–C; Additional file 1: Figure S1A; Additional file 2: Table S1, S2A,B). WT31 melanoma reliably led to development of hepatic metastases and high numbers of metastatic nodules even at low cell concentrations. In comparison, B16F10 luc2 and RET showed a medium colonization efficacy and lower numbers of liver metastases. D4M and HCmel12 melanoma cells formed liver metastases at the highest cell concentrations injected indicating the lowest efficiency of hepatic colonization. Macroscopically, most of the tested melanoma cell lines developed black or grey round tumor nodules, except for D4M, which formed white, pinpoint-like lesions. In cell culture, cell pellets of WT31 melanoma were the most pigmented ones (Additional file 1: Figure S1B). Due to the strong and reliable liver colonization efficiency of WT31 melanoma after spleen injection, WT31 cells were also injected intravenously (Fig. 1D; Additional file 1: Figure S1A; Additional file 2: Table S3A, B). Interestingly, WT31 cells not only managed to colonize the liver even after intravenous (i.v.) injection, but metastases were also detected in the lungs, the brain, the bones, the kidneys and the spleen (Table 2; Additional file 1: Figure S1C).

Histomorphologically, all melanoma metastases showed a pushing type HGP with no desmoplastic rim (Fig. 2A). Sirius Red and PAS stainings did neither reveal relevant fibrosis nor alterations of glycogen deposition (Additional file 1: Figure S2A, B). Histological measurements revealed that D4M formed the smallest metastases (0.06 mm² ± 0.01) and WT31 the largest (1.92 mm² ± 0.9) (Fig. 2B). The percentage of necrotic metastases strongly differed between the cell lines as D4M and WT31 showed the fewest necrotic cores (D4M: 6.7% ± 6.7; WT31: 18.2% ± 12.2), while B16F10 luc2, RET and HCmel12 presented significantly more necrotic areas (B16F10 luc2: 86.7% ± 9.1; RET: 85.7% ± 14.3; HCmel12: 66.7% ± 14.2) (Fig. 2B). Among the latter three, B16F10 luc2 (16.1% ± 2.2) showed the largest necrotic areas as compared to RET (5.7% ± 1.6) and HCmel12 (6.1% ± 1.7) (Fig. 2B). Therefore, apoptosis and proliferation were assessed by cleaved Caspase3 (cCasp3) and Ki-67 staining. Here, the metastases formed by B16F10 luc2 (9.2% ± 2.7) and HCmel12 (7.6% ± 3.6) revealed the most cCasp3-positive cells at the metastatic center (Fig. 2C, E). Besides, the highest proliferative index was found in RET melanoma metastases (76.7% ± 4.4) (Fig. 2D, E). Furthermore, tumor cell density was the highest in hepatic lesions of D4M (7493 cells per mm² ± 147.5) indicating a smaller cell size (Fig. 2E).

To gain molecular and mechanistic insights into the pathways and processes influencing hepatic metastatic efficiency, all melanomas were analyzed by RNA-seq of cultured cells at the same confluency as used for in vivo experiments. High and intermediate metastatic melanoma cell lines (WT31, B16F10 luc2 and RET) (HIM-melanoma) were therefore compared to the ones with low metastatic efficiency (D4M or HCmel12) (LM-melanoma). To select for commonly regulated genes and pathways, only significant genes with the same direction of regulation were considered among HIM-melanoma. First, uniformly regulated significant genes of HIM-melanoma were compared in relation to D4M identifying a gene set of 6386 regulated significant genes (Fig. 3A). Second, the same process was repeated with HCmel12 melanoma as reference identifying 4249 uniformly regulated significant genes (Fig. 3A). Last, these two gene sets were then matched to identify 1995 commonly regulated significant genes of HIM-melanoma in comparison to LM-melanoma (Fig. 3B). Subsequent pathway analyses of gene ontology biological processes (GOBP) (Fig. 3C) revealed significant regulation of processes involved in cell migration or angiogenesis. Moreover, gene enrichment analysis of HALLMARK pathways (Fig. 3D) demonstrated significant involvement of epithelial mesenchymal transition, oxidative phosphorylation or TNF-α signaling among others. The ten genes with the strongest up- and downregulation of this gene set were validated by qPCR (Fig. 3E; Additional file 1: Figure S3A, Additional file 2: Table S5). In HCmel12 melanoma, the downregulation of a melanocyte differentiation gene set is associated with local tumor growth and lung metastasis [40]. But, among the cell lines used here a similar relation of this gene set with liver colonization could not be detected (Additional file 1: Figure S3B).

Overall, comparative gene expression analysis including heat maps of GOBP demonstrated that differences between the cell lines with different propensity for hepatic colonization are especially found in the processes of cell migration (Additional file 1: Figure S4) and angiogenesis (Fig. 3F, Additional file 1: Figure S5).

To further investigate the angiogenic heterogeneity of the cell lines, the total vessel area of the metastases was analyzed (Fig. 4A). WT31 melanoma metastases were the most vascularized ones (11.8% ± 1.1), while hepatic metastases of D4M presented with the fewest vessels (1.7% ± 0.3), followed by B16F10 luc2 (4.9% ± 0.5) (Fig. 4C). To assess the phenotype of intratumoral blood vessels, markers for continuous endothelial cells (ECs), such as Endomucin (Fig. 4A, D) and CD31 (Fig. 4B, E; Additional file 1: Figure S6A), or markers of Liver sinusoidal endothelial cells (LSEC), Lyve-1 (Fig. 4A, D), CD32b (Fig. 4B, E; Additional file 1: Figure S6A) or Stab2 (Additional file 1: Figure S7A), were analyzed. Strong expression of continuous EC markers and reduced expression of LSEC markers was seen in all hepatic metastases indicating a predominantly capillarized phenotype of intratumoral vessels (Fig. 4D, E). However, the percentages of Lyve1⁺ or CD32b⁺ intratumoral vessels were significantly higher in HCmel12 in comparison to the other melanoma metastases (Lyve1⁺: 59.9% ± 6.3; CD32b⁺: 9.2% ± 3.4) indicating a mixed sinusoidal and capillarized molecular phenotype in a minor fraction of HCmel12 vessels (Fig. 4D, E). There was a strong and significant correlation of increasing metastasis size with vascular density of the different melanomas (Fig. 4F). Altogether vascularization tended to correlate with increasing efficiency and intratumoral vessels showed a predominant continuous endothelial cell phenotype.

To further characterize vessel maturation and differentiation in the formed metastases, the deposition of extracellular matrix proteins surrounding the intratumoral endothelium was assessed. Strong subendothelial deposition of Collagen IV (Fig. 5A), Collagen I (Additional file 1: Figure S7B), Collagen III (Additional file 1: Figure S7C), Fibronectin (Fig. 5B) and Laminin-α4-integrin (Lama4) (Fig. 5C) was observed in all metastases indicating a continuous phenotype with basement membrane formation. Likewise, Desmin⁺ periendothelial cells were similarly present in all metastases indicating similar coverage by pericytes (Fig. 5D). Remarkably, Collagen IV expression outside the perivascular space was seen in metastases of WT31, HCmel12 and D4M (Fig. 5A). In highly vascularized WT31 and HCmel12 these Collagen IV ⁺ structures appeared sleeve-like. This indicates high angiogenic activity with so called empty sleeves as they lack direct association with CD31⁺ EC [41]. Collagen IV⁺ CD31⁻ empty vessel sleeves were far more frequent in WT31 and HCmel12 in comparison to the other cell lines suggesting highly active but partially inefficient angiogenesis (Fig. 5E; Additional file 1: Figure S6B). Overall, high vascularization and angiogenic activity appeared to relate with larger hepatic metastatic size and lower propensity to tumor necrosis.

To assess whether these different patterns of vascularization also translate into differing treatment responses to anti-angiogenic treatment, highly vascularized WT31 and poorly vascularized B16F10 luc2 were treated with Sorafenib, an anti-angiogenic multi-kinase inhibitor, or a corresponding solvent control (vehicle). In both models Sorafenib led to central pseudocystic, hemorrhagic degradation of metastases with a thin residual rim of viable tumor cells in around 80–90% of metastases (Fig. 6A–C, Additional file 1: Figure S8A). Likewise, WT31 and B16F10 luc2 showed a significantly decreased tumor cell area and almost complete abolishment of intratumoral blood vessels after Sorafenib treatment (Fig. 6B–D). However, comparison between WT31 and B16F10 luc2 indicated that treatment with Sorafenib is indeed less effective in poorly vascularized B16F10 luc2. The reduction of the viable tumor cell area was significantly larger in WT31 (70.9% vs. 52.3% reduction) and similar the induction of pseudocystic degradation was significantly higher in WT31 (73% vs. 60.3%) indicating stronger dependence on vascularization than B16F10 luc2 (Fig. 6E, F). In addition, there was a trend to significance (p = 0.1970) that the fraction of metastases showing degradation was higher in WT31 (97.2% ± 2.8) than B16F10 luc2 (84.92% ± 8.1) (Additional file 1: Figure S8C). In summary, these data show that hepatic metastases of highly and poorly vascularized murine melanomas significantly respond to anti-angiogenic treatment with Sorafenib. Of note, this response is even stronger in highly vascularized lesions in comparison to poorly vascularized ones.

Hepatic metastasis was recently shown to be a decisive negative factor for the response to immunotherapy in patients with melanoma[7]. Therefore, there is an urgent need for preclinical models to study the underlying mechanisms driving organ-specific melanoma metastasis to the liver and to develop novel therapeutic approaches. Genetic mouse models to study spontaneous melanoma metastasis are scarce [42] and hepatic metastasis usually only occurs at a very low frequency [27, 29, 43, 44]. Thus, genetic models are currently not feasible in this context. Spleen injection of various tumor cells including melanoma cells is an established method to investigate liver colonization [31, 45]. Here we show that this model can reliably be used to comparatively study efficiency, morphologic and molecular features as well as tumor angiogenesis and treatment responses of hepatic metastases of different melanoma cells.

WT31 melanoma cells stood out with the highest efficiency of hepatic colonization after intrasplenic injection. In addition, they consistently established metastases to the liver and other organs in a CM-like pattern after intravenous injection. These two techniques provide ideal and reliable settings to study hepatic melanoma colonization either with or without simultaneous metastasis to other organs. WT31 is the only cell line investigated by us with a human NRAS mutation [28]. Although human BRAF- and NRAS-mutated melanomas have been associated with an increased risk for liver metastases, this association was rather weak [24] and until now it has not been shown that one of these main driver mutations is a key determinant of hepatic metastasis. This notion is also supported by the fact that D4M, which harbor a BRAF mutation, showed the lowest efficiency and smallest metastatic sizes in our analysis. Nonetheless, the mutational landscape of melanoma clearly influences organ-specific metastasis as KRAS driver mutations were recently associated with brain colonization as first site of metastasis [46]. Although, the mutational landscape of human melanoma with liver metastases has not been specifically characterized, it can be assumed that hepatotropism of melanoma is controlled in multifactorial fashion and involves non-genetic and epigenetic traits.

To more comprehensively study the molecular differences that may underly the heterogeneity of hepatic melanoma metastasis in our study, all melanoma cell lines were analyzed by RNAseq. Differential transcriptomic analysis of high and intermediate metastatic (HIM-) melanoma compared to low metastatic (LM-) melanoma showed the most marked differences in pathways involved in cell migration, angiogenesis, EMT, oxidative phosphorylation or TNF-α signaling. Cell adhesion and migration are the first steps of organ colonization in the metastatic cascade. In this regard, integrin alpha2 is associated with enhanced hepatic colonization while integrin alpha4 is associated with lymph node metastasis in B16 melanoma [25, 47, 48]. Although integrin alpha2 was inconsistently expressed among cell lines of HIM- and LM-melanoma, WT31 melanoma was the only cell line with strong integrin alpha4 expression which may contribute to its high metastatic efficiency. MSX1 which has been shown to increase hepatic melanoma metastasis in an immune-deficient setting [26] was only expressed in D4M melanoma and therefore could not account for the differences observed by us (data not shown). Overall, cell migration is likely a major determinant of organ-specific metastatic efficiency and appears to be controlled by complex gene sets. This notion is also supported by the fact that expression of gene sets involved in cell adhesion can be used as predictors of lymph node metastasis [49]. Therefore, there is an urgent need for studies identifying crucial gene sets in melanoma patients with liver metastasis to select patients at risk early in the course.

Angiogenesis also stood out as a differentially regulated process between HIM- and LM-melanoma. Angiogenesis is well known as determinant of hepatic metastasis in general [50]. In CRC the stromal subtype with an angiogenic signature shows enhanced liver metastasis [17]. The critical role of angiogenesis for hepatic metastasis of CRC is further supported by a molecular analysis of liver metastases and their corresponding primary tumors [51]. Liver metastases of breast cancer, CRC, UM and CM share three common main HGPs with different tumor vessels: desmoplastic, replacement and pushing type [11‐14]. Desmoplastic and pushing type HGP strongly rely on de-novo angiogenesis of tumor vessels whereas replacement HGP co-opts the pre-existing sinusoidal liver vasculature. Paku et al. postulated cooperation of smooth muscle actin-positive cells and fusion of partly capillarized sinusoids as hallmark of angiogenesis in pushing type HGPs [52]. In our model pushing type HGPs were found in all metastases and supportive connective tissue was detected in all types supporting the hypothesis of Paku. Transdifferentiation of liver sinusoidal endothelial cells to a capillarized phenotype is also observed in tumor endothelium of murine and human hepatocellular carcinoma [53]. Since the melanoma cell lines with higher vascularization developed larger hepatic metastases, efficiency of hepatic melanoma colonization and outgrowth of metastases are strongly regulated by angiogenic processes.

Aside from angiogenesis itself, vessel maturity which is regulated by vessel pruning and regression is an important process involved in tumor blood supply [41]. Detection of empty vessel sleeves as sign for vessel pruning and regression showed that both WT31 and HCmel12 melanoma metastases have high angiogenic activities. Although HCmel12 exhibited a partly sinusoidal phenotype of tumor EC, the vessel morphology did not imply co-option as seen in replacement HGP.

The tumor vasculature can be disrupted by anti-angiogenic agents such as bevacizumab, an anti-VEGF antibody, or Sorafenib, a multi-kinase-inhibitor [14, 54]. First approaches with such therapeutic agents were disappointing and clinical studies in patients with advanced melanoma were usually terminated in early phases [55]. However, clinical studies with Sorafenib did not investigate treatment responses in subgroups of patients with hepatic metastasis. In addition, in these patients it may be necessary to stratify for HGPs or tumor vascularization patterns and to treat in combination or sequentially with current standard of care treatments. In our study, Sorafenib induced reliable pseudocystic degeneration in high metastatic melanoma (WT31) and in intermediate metastatic melanoma (B16F10 luc2). Although this response was even stronger in WT31, both cell lines showed a solid treatment response. Necrosis or pseudocystic degeneration is not seen when subcutaneous B16 melanoma is treated with Sorafenib [56]. This indicates that the outcome of Sorafenib treatment and anti-angiogenesis depends on the organ-specific microenvironment. In this context lower oxygen levels in the liver may be a contributing factor. Like anti-angiogenic treatment in desmoplastic CRC [57] a small rim of viable tumor cells was still present after treatment of our models. This indicates that a combinatorial approach is likely necessary to achieve complete remission of hepatic metastasis. Due to the subtle effect of chemotherapeutics alone and the missing improvement of therapy response to Sorafenib when combined with Dacarbazine [58], a combination of standard of care ICI or targeted therapy with anti-angiogenic drugs seems appealing for hepatic metastasis. This approach is particularly promising as a breakthrough in the therapy of hepatocellular carcinoma was achieved by the combination of atezolizumab and bevacizumab replacing the former gold standard Sorafenib [59].

Altogether, molecular and phenotypic diversity of murine melanoma determine liver metastatic propensity involving cell migration and angiogenesis as major processes. Metastatic vascularization correlates with metastatic size. Interestingly, both highly and poorly vascularized hepatic lesions responded to Sorafenib indicating a broad efficacy for Sorafenib in this specific context. Therefore, anti-angiogenic therapy is an appealing approach to treat liver metastasis in advanced melanoma patients.

Heterogeneity of hepatic metastasis of cutaneous melanoma was studied using a murine orthotopic model with five genetically different cell lines. Efficacies and phenotypic features of liver colonization of these melanoma cell lines were comprehensively compared. Migration and angiogenesis were among the differentially regulated functions identified by comparative RNA-seq analysis. Overall, molecular and phenotypic heterogeneity of hepatic melanoma metastasis revealed angiogenesis as a targetable determinant of hepatic colonization paving the way for organ-specific anti angiogenic treatment.