Investigating the Relationship Between Ascorbate and the Differentiation of Monocytes in an In-Vitro Melanoma Model

Abstract

Melanoma, a tumour of the skin, has exceptionally high mortality rates, claiming approximately 350 people every year within New Zealand. This makes melanoma the fourth most common cause of cancer-related deaths within the country, indicating a need for better treatments to improve patient outcomes. Anti-PD1 immunotherapy (e.g., Pembrolizumab) targets and stimulates suppressed cytotoxic T cells to eliminate the cancer. However, long term efficacy remains low and immune-related adverse events (irAEs) remain frequent.

Previous research in our laboratory using a murine model showed that ascorbate supplementation modifies tumour-associated Macrophages (TAM) into a population of macrophages that have neither definitive inflammatory (M1-like) or anti-inflammatory (M2-like) phenotypes. These changes in gene and protein expression could exert either pro- or anti-tumour effects and potentially mediate phenotypical change that affects anti-tumour immunity, as well as the chronic inflammatory cascade induced by immunotherapy that underlies many irAEs. Interestingly, ascorbate supplementation can enhance activity of epigenetic modifiers within immune cells, including Ten-eleven Translocation 2 (TET2), which has been suggested to simultaneously support and limit cancer progression. The aim of this study was to establish an in-vitro model to study the effects of melanoma cells on monocytes/macrophage phenotype and function, and to answer the following questions: 1) Can ascorbate supplementation alter TAM phenotype in a human model and 2) Does ascorbate supplementation alter TET2 activity in these macrophages.

To address these questions, relevant markers were measured after monocytes were cultured in melanoma cell line NZM40-derived condition media (20%) for 7 days, with or without ascorbate supplementation (100μM). Quantitative PCR was used to determine relative gene expression of TAM related genes. Immunocytochemistry and high-throughput imaging were used to identify and quantify M1- and M2-affiliated phenotypic cell surface markers, HLADR and CD206, respectively. Mass Spectrometry was used to assay the epigenetic marker 5-hydroxymethylcytosine, indicative of TET2 activity. These experiments demonstrated that NZM40-derived condition media supported monocyte growth, but did not stimulate gene expression and surface marker presence expected of M2-like TAMs. Rather, it generated Macrophage populations that were of mixed M1- and M2-like phenotype. When compared to NZM40-derived condition media alone, ascorbate supplementation enhanced TET2 activity (as evidenced by the increase DNA demethylation) and increased the presence of the M1 surface marker, HLADR, but did not induce significant changes in gene expression.

This study provided some evidence to support the concept that ascorbate supplementation alters TET2 activity and TAM phenotype. However, this study also highlighted the limitations of the in vitro model used, and future studies would aim to develop a co-culture model for bi-directional communication to study TAM phenotype and function in melanoma.