Macrophage Plasticity And Function In The Lung Tumour Microenvironment Revealed In 3d Heterotypic Spheroid And Explant Models

In non-small cell lung cancer (NSCLC), stroma-resident and tumour-infiltrating macrophages may facilitate an immunosuppressive tumour microenvironment (TME) and hamper immunotherapeutic responses. Analysis of tumour-associated macrophage (TAM) plasticity in NSCLC is largely lacking. We established a novel, multi-marker, dual analysis approach for assessing monocyte-derived macrophage (M phi) polarisation and M1/M2 phenotypic plasticity. We developed a flow cytometry-based, two-marker analysis (CD64 and CD206) of CD14(+) cells. The phenotype and immune function of in vitro-induced TAMs was studied in a heterotypic spheroid and tumour-derived explant model of NSCLC. Heterotypic spheroids and NSCLC explants skewed M phi s from an M1- (CD206(lo)CD64(hi)) to M2-like (CD206(hi)CD64(lo)) phenotype. Lipopolysaccharide (LPS) and IFN gamma treatment reversed M2-like M phi polarisation, indicating the plasticity of M phi s. Importantly, antigen-specific CD8(+) T cell responses were reduced in the presence of tumour explant-conditioned M phi s, but not spheroid-conditioned M phi s, suggesting explants are likely a more relevant model of the immune TME than cell line-derived spheroids. Our data indicates the importance of multi-marker, functional analyses within M phi subsets and the advantages of the ex vivo NSCLC explant model in immunomodulation studies. We highlight the plasticity of the M1/M2 phenotype using the explant model and provide a tool for studying therapeutic interventions designed to reprogram M2-like M phi-induced immunosuppression.