CysC and GM-CSF Participate in Shaping the Malignancy Phenotype of Melanoma Cells in the Brain

Introduction: Melanoma brain metastasis (MBM) is common in patients with malignant melanoma. We have shown that in vitro stroke-like conditions as well as treatment with melanoma-conditioned medium (MCM) increased Cystatin C (CysC) and Granulocyte-macrophages colony-stimulating factor (GM-CSF) secretion by brain microenvironmental cells. The present study is aimed to elucidate the role of CysC and GM-CSF in MBM progression.

Methods: Infection with virions containing shCysC and shGM-CSF was used to downregulate the expression of these genes. Wound healing and transmigration assays were performed to determine the effect of CysC and GM-CSF on cell migration or BEC penetration. NGS mice were subjected to ischemic stroke. MBM were generated in mice 7 days after stroke Non-stroked mice were inoculated similarly. Control group was neither stroked nor inoculated. IC inoculation of melanoma cells into nude mice and human-specific RT-qPCR were performed to determine GM-CSF effect on MBM formation.

Results and Discussion: Microglia-conditioned medium (MGCM) upregulated CysC secretion from melanoma cells. Reciprocally, MCM upregulated CysC secretion from microglia. Whereas CysC enhanced melanoma migration and penetration through a layer of brain endothelial cells (BEC), it inhibited the migration of microglia cells toward melanoma cells. CysC promoted the formation of melanoma 3D structures. IHC revealed increased expression levels of CysC in the brains of mice bearing xenografted human MBM compared to the brains of control mice. Increased expression levels of CysC were detected in the regenerating brains of mice after stroke. Post-stroke brains with MBM showed an even stronger expression of CysC. Put together these results demonstrated that CysC may promote but also inhibit MBM formation. MCM also stimulated GM-CSF secretion from BEC and astrocytes. GM-CSF was found to enhance melanoma transendothelial migration, effecting only the BEC and not melanoma cells. MGCM inhibited GM-CSF secretion from melanoma cells. TNF-α and IL-1A, secreted by microglia in its pro-inflammatory (anti-tumor) state, enhanced GM-CSF secretion by melanoma. Upregulation of GM-CSF secretion by melanoma cells was inhibited when melanoma cells were treated with TNF-α or IL-1 in combination with MGCM, indicating that microglia-derived factors either inhibit or enhance GM-CSF secretion by melanoma. All these results demonstrae the complexity of melanoma-microglia interactions. IC inoculation of melanoma cells knocked down to GM-CSF resulted in an increase in MBM formation compared to control cells, showing that despite increasing BEC penetration, GM-CSF is an anti-metastatic factor in the context of MBM.

Conclusion: Cells and molecules in the brain microenvironment exert yin yang functions either supporting or inhibiting tumor progression. The overall balance between the two determines the metastatic fate.