Autophagy Promotes Growth of High Tumor Mutational Burden Tumors by Inhibiting a T Cell Immune Response [RNA-seq]

Autophagy degrades and recycles intracellular components to sustain metabolism and survival during starvation. Tumor cells upregulate and require autophagy to support their metabolism and enhance their proliferation and malignancy, and host autophagy also promotes tumor growth by providing essential tumor nutrients such as arginine in the circulation or alanine in the local tumor microenvironment. In addition to its metabolic role, autophagy regulates immune cell homeostasis and function, and suppresses inflammation, which may also play a role in cancer. Although host autophagy does not promote a T cell anti-tumor immune response in tumors with low tumor mutational burden (TMB), whether this was the case in tumors with high TMB was not known. Here we show that in contrast to low TMB tumors, host-specific deletion of the essential autophagy gene Atg7 induces a pro-inflammatory cytokine response and limits the growth of high TMB tumors, which is rescued by T-cell depletion. Expression of immune-related genes is increased in tumors from Atg7Δ/Δ hosts in a T-cell dependent manner. Tumors from Atg7Δ/Δ hosts also have decreased T regulatory cells (Tregs), and depletion of Tregs phenocopies the reduced tumor growth observed in Atg7Δ/Δ hosts. Moreover, loss of Stimulator of interferon genes (Sting) or IFNg restores growth of high TMB tumors on Atg7Δ/Δ hosts. Finally, similar to whole-body loss of autophagy, specific loss of autophagy in the liver is sufficient to limit tumor growth. Thus, autophagy, especially in the liver, promotes tumor immune tolerance by limiting STING, T cells, and IFNg, which enables tumor growth. We have designated this: Hepatic Autophagy Immune Tolerance (HAIT). Autophagy thereby promotes tumor growth through both metabolic and immune mechanisms depending on mutational load, and autophagy inhibition is an effective means to promote an anti-tumor T-cell response in high TMB tumors. Resected mouse tumors from Atg7+/+ and Atg7∆/∆ hosts were placed in complete media supplemented with 10% FBS and kept on ice. Dissociation of the tumor tissue was carried out using a commercially-available mouse tumor dissociation kit and the Miltenyi GentleMACS dissocation system (Miltenyi Biotec, Cologne, Germany). Isolated cells were filtered through a 40mm mesh strainer and PBMC’s were isolated by density-dependent centrifugation. Cells were washed complete media (10% FBS), then counted and assessed for viability using a Countess II cell counter (Invitrogen). Cells were then suspended to 1x10^6 per ml in complete media (10% FBS) for single cell emulsion preparation. scRNA-seq libraries were prepared according to 10X Genomics specifications (10X Genomics, Pleasanton, CA). Independent cell suspensions were loaded for droplet-encapsulation by the Chromium Controller (10X Genomics). Single-cell cDNA synthesis, amplification, and sequencing libraries were generated using the Single cell 5’ Reagent Kit following the manufacturer’s instructions. The libraries were shipped on dry ice to Novogene (Sacramento, CA), and sequenced in a single lane of an Illumina NovaSeq 6000 (Settings: Read1: 26, i7: 8, Read2: 91, for 200M bp paired-end reads per sample). mRNA profiling of 8 mouse tumors (4 with Atg7+/+ genotype and 4 with Atg7∆/∆ genotype) using 10x Chromium Novaseq 6000 instruments.