Beschreibung
Therapeutic options for metastatic malignant melanoma are severely limited in both veterinary and human medicine, particularly for patients in stage IV with a poor prognosis.
This study aimed to address these limitations by conducting multiple hepatic passages of the murine melanoma cell line WT31, which harbors a mutation in human NRAS. Through intravenous injection, a melanoma cell line called WT31P5IV was generated, which metastasizes about two thirds (P=0.0077) less to the lungs and generates about twice as many (P=0.0531) liver metastases. The ratio of lung to liver metastases also shows a reduction (P=0.0005) in WT31_P5IV, which means that there are fewer lung metastases per liver metastasis than in WT31. H&E staining was performed to further characterize this phenotype. No morphological difference in size, necrosis and growth pattern could be detected in either the lung or liver metastases. Further investigations were carried out using immunohistochemistry. Ki67, a marker for proliferation, showed a signal reduced by half (P=0.0171) in the lung metastases of WT31_P5IV compared to lung metastases of WT31. This indicates a reduced division activity of the passaged melanoma cells in the lung. In order to analyze the influence of hepatic passaging of WT31 on the tumorigenic factors of WT31_P5IV, immunohistochemical studies were also performed in the liver metastases. Staining of Ki67 (proliferation marker) and cleaved caspase-3 (marker for apoptotic cells) showed no difference between the liver metastases of WT31 and WT31_P5IV. Furthermore, the analysis of the vascular pattern in the liver metastases of WT31 and WT31_P5IV also showed no significant difference. In the metastases of both cell lines, mainly markers of continuous endothelium are expressed (Endomucin, CD31). Thus, liver metastases of WT31 and WT31_P5IV show the same morphological structure and no difference in vascularization (P=0.3776), proliferation (P=0.6282) and necrosis (P=0.1163). To understand the altered metastatic behavior of WT31P5IV, comprehensive molecular genetic characterization was performed on both cell lines.
Remarkably, the vascular route taken by the cells during the passaging process caused significant modifications in tumor-intrinsic properties. A comparative analysis of the gene expression of WT31 and WT31_P5IV in the HALLMARK gene sets showed changes in genes involved in cell metabolism, such as oxidative phosphorylation and glycolysis, and in signaling pathways of MYC targets, mitotic spindle, DNA repair and PI3K/AKT/mTor.
The Seahorse Analyzer was used to measure mitochondrial ATP production and ATP produced by glycolysis in both cell lines. No quantitative difference was detectable (mito-ATP production rate: P=0.4145 and glyco-ATP production rate: P=0.1908).
Igfpb3, as a strongly downregulated gene in WT31_P5IV (P<0.000.1), interacts negatively with AKT signaling pathway and was analyzed in more detail. Western blot analyses of phosphorylated AKT (pAKT) and total AKT (AKT) showed an increase in the ratio of pAKT to AKT by about one third (P=0.0291), confirming activation of the signaling pathway.
The gene expression profile of the passaged cell line WT31P5IV underwent changes, primarily affecting genes associated with the retention and adhesion of tumor cells to the pulmonary vascular bed. This alteration was validated in vivo by a retention experiment comparing WT31 and WT31P5IV. Notably, the initial attachment of WT31P5IV to the lung endothelium was significantly reduced (P=0.0016) compared to WT31 and because of that, melanoma cells were more prone to reach the liver via the bloodstream. This phenomenon of repeated colonization of tumor cells in specific organs may also happen in patients who have undergone multiple treatments and should be taken into account in future considerations.
