Cancer-induced nerve injury promotes resistance to anti-PD-1 therapy [RNA-Seq - cell line]
Peri-neural invasion (PNI) is a well-established poor prognostic factor in multiple cancer types. However, the mechanisms driving the PNI's detrimental clinical effect remain elusive. Here, we provide clinical and mechanistic insights into PNI and cancer-induced injury of tumor-associated nerves (TANs) and their role in resistance to anti-PD-1 therapy. Our work demonstrates that poor response to anti-PD-1 therapy in cutaneous squamous cell carcinoma (cSCC), melanoma, and gastric cancer is associated with PNI and TANs injury. Ultrastructural electron microscopy analysis reveals that direct contact between cancer cells and nerve fibers leads to cancer-induced nerve injury (CINI) via myelin degradation. Injured neurons respond by autonomously initiating an interleukin (IL)-6 and interferon (IFN) type I inflammatory response. This inflammatory response alters the immune activity in the peri-neural niche in melanoma, cSCC, and pancreatic adenocarcinoma, leading to an immuno-suppressive activity aimed at nerve healing and regeneration. As the tumor grows, the CINI burden increases, the inflammatory signal within the niche becomes chronic, and eventually skews the general immune tone within the tumor microenvironment to a suppressive and exhaustive state. The CINI-driven anti-PD-1 resistance can be reversed by targeting multiple steps in the CINI signaling process: denervating the tumor, conditional knockout of the transcription factor mediating the injury signal within neurons (cKO-Atf3), knockout of the IFN-a receptor signaling (Ifnar1-/-), or by combining anti-PD-1 and anti-IL-6-receptor blockade. Our findings demonstrate the direct immuno-regulatory roles of TANs and their therapeutic potential. Overall design: Human neurons were co-cultured with the human cutaneous squamous cell carcinoma IC8 cell line (obtained from the laboratory of Drs. Irene Leigh and Catherine Harwood, UK 23), the human oral squamous cell carcinoma PCI-13 cell line (obtained from the laboratory of Dr. Jeffrey Myers, MD Anderson Cancer Center, USA), or with the human epidermal keratinocyte HEK cells (ATCC, PCS-200-011). Sensory or motor neurons were cultured to assess the impact of nerves' degenerative/regenerative status on immunotherapy in vitro.Neurons were plated on laminin-coated (Life Technologies, Cat# 23017015) round glass coverslips set in 12-well plates. Two hours after plating, the wells were flooded with 1 mL of warm (37°C) Ham's F12 culture medium (Sigma-Aldrich) supplemented with 10% EV-depleted fetal bovine serum (Gibco, Thermo Fisher Scientific) and 1% penicillin/streptomycin and incubated at 37°C in 5% CO2 and 95% air. The carcinoma cells were plated (1:10 neuron: cancer cells ratio), and cultures were grown in RPMI 1640 medium containing 10% EV-depleted fetal bovine serum in 37°C and 5% CO2 incubation conditions, for 72 hours, with or without anti-PD1 antibody (Cemiplimab, 1000ug/ml,  or RMP1-14, 100ug/ml, InVivoMAb Antibodies, BioXCell, for human and murine, respectively), before neuron sorting and RNA extraction.