Article gene and protein expression is altered by ascorbate availability in murine macrophages cultured under tumour-like conditions

Abstract

Tumour-associated macrophages (TAMs) are ubiquitously present in tumours and com-monly associated with poor prognosis. In immune cells, ascorbate affects epigenetic regulation, differentiation and phenotype via its co-factor activity for the 2-oxoglutarate dependent dioxygenase enzymes. Here, we determined the effect of ascorbate on TAM development in response to tumour microenvironmental cues. Naïve murine bone marrow monocytes were cultured with Lewis Lung Carcinoma conditioned media (LLCM) or macrophage colony-stimulating factor (MCSF) to encour-age the development into tumour-associated macrophages. Cells were stimulated with hypoxia (1% O2 ), with or without ascorbate (500 µM) supplementation. Cells and media were harvested for gene, cell surface marker and protein analyses. LLCM supported bone marrow monocyte growth with >90% of cells staining CD11b+ F4/80+, indicative of monocytes/macrophages. LLCM-grown cells showed increased expression of M2-like and TAM genes compared to MCSF-grown cells, which further increased with hypoxia. In LLCM-grown cells, ascorbate supplementation was associated with increased F4/80 cell surface expression, and altered gene expression and protein secretion. Our study shows that ascorbate modifies monocyte phenotype when grown under tumour mi-croenvironmental conditions, but this was not clearly associated with either a pro-or anti-tumour phenotype, and reflects a complex and nuanced response of macrophages to ascorbate. Overall, ascorbate supplementation clearly has molecular consequences for TAMs, but functional and clinical consequences remain unknown.