Oropharyngeal Cancer-derived Extracellular Vesicles Elicit a Pro-inflammatory Macrophage Phenotype

Objectives: Human papillomavirus positive (HPV+) oropharyngeal cancer (OPC) has an improved prognosis over HPV negative (HPV-) OPC, possibly due to signalling differences in the tumour microenvironment. We hypothesised that extracellular vesicles (EVs) derived from HPV+ and HPV- OPC cell lines differentially regulate polarisation of tumour-associated macrophages. We aimed to isolate and characterise these EVs and to determine macrophage phenotype after treatment with conditioned medium or EVs.
Methods: EVs were isolated from the conditioned medium of HPV+ (SCC2 and SCC90) and HPV- OPC (SCC72 and SCC89) cell lines by differential centrifugation and size exclusion chromatography to produce large and small EV populations. EVs were characterised by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blotting. Monocyte-derived macrophages (MDM) were generated from monocytes isolated from human blood. MDM were polarised to M0/M1/M2 phenotypes using cytokines (n=3), or treated with conditioned medium (n=4) or purified EVs (n=3) and the resulting MDM phenotypes analysed using qPCR. ELISA was used to quantify MDM-derived cytokines released upon stimulation.
Results: NTA and TEM confirmed the isolation of large (~400 nm) and small EVs (~140 nm) that were positive for common EV markers (CD63 and CD9) by western blotting. Cytokine polarisation produced M0/M1/M2 baseline MDM phenotypes. Treatment with HPV+ or HPV- OPC conditioned medium and EVs caused phenotypic changes to MDM. Treatment of MDM with large EVs caused a significant (p<0.01) increase in the expression of the M1 inflammatory marker CD80. In addition, a significant increase in CXCL8 secretion from small EV-treated MDM was detected (p<0.05).
Conclusions: Stimulation of MDM with EVs from HPV+ and HPV- OPC cell lines altered MDM phenotype marker expression. These data significantly contribute to our understanding of the role of EVs in modulating the tumour microenvironment.