Introduction: Angiogenesis is essential for tumour development. Tumour blood vessels may be modulated by immunotherapies that polarise macrophages towards a pro-inflammatory, anti-angiogenic phenotype resulting in tumour regression.
Aim: To examine the effects interleukin-2 (IL-2) +/- CD40L or CD40 antibody (Ab) and TLR7/8 agonist-activated macrophages exert on endothelial cells and tumour cells.
Methods: Human and murine macrophages, mesothelioma cell lines, and human umbilical vein endothelial cells (HUVEC) were first treated directly with IL-2 +/- agonist CD40Ab (murine) or CD40L (human) +/- a TLR7/8 agonist and their viability assessed with MTT assays. Macrophage polarisation was confirmed by assessing changes in cytokine production and phenotype using flow cytometry. The effect of IL-2/CD40 or TLR7/8-activated macrophages on endothelial cells was assessed by treating HUVECS with macrophage-derived conditioned media and analysing changes in activation and tight junction proteins using flow cytometry. A multicellular tumour spheroid model involving macrophages, tumour and endothelial cells was established to reflect the tumour microenvironment and test the effect of IL-2/CD40 or TLR7/8 agonism on the three cell types.
Outcomes: A TLR7/8 agonist induced apoptosis in tumour and HUVEC cells, and macrophage proliferation. IL-2/CD40 was not toxic to any cell type, and induced macrophage proliferation and polarisation to pro-inflammatory cells. Preliminary data show that polarised macrophages did not affect HUVEC viability and induced morphological and phenotypical changes. The spheroid model shows promise as the three cells types integrate to form spheroids.
Conclusions: TLR7/8 agonism induced HUVEC and tumour cell death, and activated macrophages. Macrophages polarised by IL-2/CD40 induced endothelial cell activation.