Cancer-induced nerve injury promotes resistance to anti-PD-1 therapy [scRNA-seq II]

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: Six-week-old male and female littermate control (Trpv1wt::Diptheria-Toxinfl/wt) mice were intradermally injected in the left paw with 2×105 B16F10 cells or non-tumorigenic keratinocytes (MPEK-BL6). Seven days later, tumor-infiltrating neurons (or neurons in MPEK-injected skin) were retrogradely labeled by an acute intra-tumor injection of AAV9-CAG-mCherry-WPRE-SV40p (10 µL, 5.5×109). Fourteen days post-inoculation, mice were euthanized, and the L3–L5 dorsal root ganglia (DRG) were harvested and digested. Most CD45+ cells were depleted using the MagniSort Mouse CD45 Depletion Kit (Invitrogen, #8804-6864). The remaining neurons were resuspended in FACS buffer (PBS with 2% fetal calf serum and EDTA) and stained with Viability Dye eFluor 780 (eBioscience, 65-0865-14) at 4 °C for 15 minutes. Subsequently, nuclei were stained with SYTO 40 (10 µM, Thermofisher, #S11351) for 5 minutes at room temperature to distinguish cells from axonal debris. Cells were then purified by FACS on a BD FACSAria IIu cell sorter and subjected to single-cell barcoding using the Chromium Next GEM Single Cell 3' GEM, Library, and Gene Expression v3.1 system (10x Genomics). Libraries were sequenced on an Illumina NovaSeq X platform, generating 120 million total reads. Reads were aligned to the mouse reference genome using Cell Ranger (10x Genomics) and analyzed with Seurat. Low-RNA-content and dead cells were excluded (nFeature_RNA > 100, percent.mt < 25%). AAV9 expression was not detected and therefore subsequent analysis was performed using principal component analysis which identified 19 clusters, and which were assigned cell types using known markers and the Tabula Muris reference atlas. Clusters were grouped into major cell types for comparing gene expression under different conditions. Four biological replicates per group were analyzed to compare B16F10-injected mice and keratinocyte-injected mice. To increase statistical robustness, a pseudobulk approach was used by averaging expression for each cell type in each sample, followed by differential expression analysis with DESeq2. Gene set enrichment analysis (GSEA) was then used to evaluate gene ontology gene sets.