Abstract
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 (Mφ) 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 Mφs from an M1- (CD206loCD64hi) to M2-like (CD206hiCD64lo) phenotype. Lipopolysaccharide (LPS) and IFNγ treatment reversed M2-like Mφ polarisation, indicating the plasticity of Mφs. Importantly, antigen-specific CD8+ T cell responses were reduced in the presence of tumour explant-conditioned Mφs, but not spheroid-conditioned Mφs, 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 Mφ 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 Mφ-induced immunosuppression.
Highlights
Solid cancers comprise malignant cells, and additional cancer-associated stromal and hematopoietic cells
Macrophages are the most abundant tumour-infiltrating immune cells and exist following the recruitment of blood monocytes or macrophages to the tumour tissue, where they differentiate into tumour-associated macrophages (TAMs) [1]
The addition of CD14+ PBMCs to homotypic H522 spheroids resulted in spheroid expansion, whilst their addition to homotypic AG02603 fibroblasts (AGFB) or heterotypic H522/AGFB
Summary
Solid cancers comprise malignant cells, and additional cancer-associated stromal and hematopoietic cells. Macrophages are the most abundant tumour-infiltrating immune cells and exist following the recruitment of blood monocytes or macrophages to the tumour tissue, where they differentiate into tumour-associated macrophages (TAMs) [1]. TAMs can promote or suppress anti-tumour immune responses, as discussed later. M1-like macrophages are activated by Th1 cytokines, including IFNγ and TNFα, or Toll-like receptor ligands, namely lipopolysaccharide (LPS) [4], and have been shown to promote cytotoxic CD8+ T cell responses through production of pro-inflammatory cytokines (IL-6, IFNγ, TNFα, and inducible nitric oxide (iNOS)) [5]. M2-like macrophage activation is induced by Th2 cytokines (IL-4, 4.0/)
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