Influence of head and neck cancer exosomes on macrophage polarization

Background: Tumor cells within the tumor microenvironment (TME) release exosomes that influence macrophage phenotypes, either pro-tumorigenic or anti-tumorigenic. This mechanism, especially in head and neck squamous cell carcinoma (HNSCC), remains poorly understood. This study investigates the role of HNSCC exosomes in macrophage polarization.

Methodology: Exosomes were isolated from HPV16-positive (93VU147T, UDSCC2) and HPV-negative (OCT1) HNSCC cell lines. These exosomes were characterized for their potential to modulate macrophage polarization. Uptake of PKH-26 labeled exosomes by macrophages was monitored via confocal microscopy. Changes in macrophage polarization were assessed using quantitative real-time PCR and immunoblotting. Exosomal transcripts and proteome cargo was examined for polarization associated mediators.

Results: HPV-negative exosomes showed higher uptake by THP1 resting macrophages (M0). Exosomes from HPV-positive cells induced a mixed macrophage phenotype (M1 and M2), whereas HPV-negative exosomes favored M1 polarization. Immunoblotting analysis revealed that this polarization was driven by the activation of transcription factors STAT1, NF-κB, and AP1. Transcriptomic analysis of HNSCC exosomes revealed reads for AP1 (c-Jun, c-Fos, FosB, Fra1, Fra2) and NF-κB (p50/105, p52/100, RelA, RelB, c-Rel), along with their known upstream mediators MEK1--7, JNK1-3, JAK1-3, TYK2, IKKα, and IKKβ. Splice variants of macrophage polarization markers, including iNOS and TGFβ, were also identified, though none of the exosomal proteome component corresponded to these factors.

Conclusion: HPV-negative exosomes are efficiently internalized by macrophages, promoting M1 polarization likely via modulation of STAT1, NF-κB, and AP1 signaling. These findings provide novel insights into role of tumor exosomes in modulation of macrophage-mediated TME dynamics in HNSCC.