Cancer cell-derived extracellular vesicles drive pre-metastatic niche formation of lymph node via IFNGR1/JAK1/STAT1-activated-PD-L1 expression on FRCs in head and neck cancer.

Objective: The aim of this study is to evaluate the role of FRCs regulated by cancer cell-derived extracellular vesicles (CEVs) played in pre-metastatic niche (PMN) formation of lymph node (LN).

Materials And Methods: The FRCs in sixty fresh cervical LNs from 20 patients were evaluated by flow cytometric analysis. Cells in LN with or without metastasis were analyzed by single-cell RNA sequencing (scRNA-seq). CEVs were isolated from the culture supernatant of primarily cultured cancer cells and cocultured with FRCs. Mass Spectrometry was used to identify LN metastasis related protein in CEVs. The activation of IFNGR1/JAK1/STAT1-activated-PD-L1 pathway in FRCs was detected by western blotting. FRCs were co-cultured with CD8+ T lymphocytes to confirm the cytotoxicity assay of FRCs.

Results: The proportion of fibroblastic reticular cells (FRCs) was significantly higher in micro-metastatic LN in head and neck squamous cell carcinoma patients (HNSCC, p < 0.05) and scRNA-seq analysis further showed a high focus of extracellular vesicles-related pathway on FRCs in LN with metastasis (p < 0.05). Interferon gamma receptor 1 (IFNGR1) in CEVs can be engulfed by FRCs and promote PD-L1 expression on FRCs via JAK1-STAT1 pathway, resulting in an increased CD8 T cell exhaustion.

Conclusion: IFNGR1, originated from cancer cell-derived extracellular vesicles, promote PD-L1 expression on FRCs and subsequent CD8+ T cell exhaustion via JAK1-STAT1 activation, which facilitate pre-metastatic niche formation and tumor metastasis in sentinel lymph node in HNSCC.