Live immunogenic melanoma cells generated by etoposide treatment upregulate B2M and lose tumor-forming potential
Cytotoxic chemotherapy has been the primary cancer treatment modality for decades, however, chemotherapy drugs do not selectively target cancer cells. These drugs are toxic to all proliferating cells including immune cells that constitute the host-defense system against pathogens and cancer cells. In an effort to develop non-toxic combinations of chemotherapy and immunotherapies for cancer, we previously reported in Sriram et al. 2021 that etoposide-treated B16 melanoma cells expressing ovalbumin, when co-cultured in vitro with dendritic cells and T-cells, enhance T-cell activation. The live fraction of melanoma cells after etoposide treatment was critical to promote T-cell activation as dead cells alone or in combination with cell-free supernatants were not effective. In this study, we show that the live fraction of etoposide-treated B16-Ova cells, which gain the ability to stimulate T-cells, are growth-arrested, but remain viable past the co-incubation period with dendritic cells and T-cells post etoposide washout. Furthermore, using a soft-agar colony-formation assay as a proxy for tumor-forming potential, we show that these live etoposide-treated B16-Ova cells have drastically reduced ability to form colonies displaying a near-complete loss of anchorage-independent growth. We also show that etoposide treatment upregulates the production of Beta-2-microglobulin (B2M), a subunit of Class I MHC. Intriguingly, we observed using immunofluorescence that the live etoposide-treated B16-Ova cells show enhanced intracellular perinuclear B2M accumulation but B2M surface staining, while detectable, is not enhanced by etoposide treatment. Furthermore, while the live etoposide-treated B16-Ova cells express Class I MHC protein, there is only a marginal drug treatment-induced increase observed in the expression levels by western blotting. Our results suggest that T-cell activation by live etoposide-treated B16-Ova cells is likely not driven by drug treatment-induced enhancement in cell surface levels of Class I MHC and point to other mechanisms.