Characterising the surfaceome of hypoxic myeloid cells infiltrating the tumour microenvironment.

Tumour-associated macrophages (TAMs) are one of the most abundant populations in the tumour microenvironment and can contribute to cancer progression. The behaviour of macrophages is influenced by environmental stimuli, including hypoxia (oxygen deprivation), to further promote therapy resistance and cancer relapse. Whilst there is an interest in targeting TAMs to improve anti-tumour efforts, further study is needed to identify markers for hypoxic TAMs before developing therapies. An in silico surfaceome dataset (Bausch-Fluck et al.) revealed over 100 genes encoding cell surface molecules were upregulated in human monocytes treated with dimethyloxalylglycine (DMOG), a hypoxia mimetic. To identify candidate TAM markers, genes were then filtered based on their known expression profile and a literature review was conducted to identify 9 genes of interest to explore further. Monocytes are recruited to the tumour microenvironment where they differentiate into TAMs. Of these 9, TNFRSF1B, TREM1, HCAR3, and PLAUR expression was increased in hypoxic human monocyte-derived macrophages (MDMs). The product of these genes was then assessed by flow cytometry in differentially treated MDMs. Cell surface CD87 (encoded by PLAUR), TREM1, and TNFR2 (TNFRSF1B) was upregulated in the presence of hypoxia and also in MDMs skewed with pro-inflammatory cytokines, representing relevant hypoxia-sensitive markers to investigate further. To investigate the expression of these markers in more clinically relevant models, their expression was assessed on TAM-like macrophages generated using cancer cell line conditioned media (CM). CM from cell lines that may constitutively express hypoxia-related proteins considerably upregulated the candidate markers at normoxia. To elucidate the factors regulating their expression, cytokines, chemokines, and exosomes released by cancer cells will be assessed by ELISA, PCR, and ultracentrifugation. These markers will be further assessed by immunohistochemistry in primary human tumour samples. Following subsequent validation, monoclonal antibodies capable of modulating the receptor will be sourced or generated to explore their ability to modulate macrophage behaviour.