Cytosolic DNA structures produced by mismatch-repair deficiency coordinate anti-tumor immunity in colorectal cancer

Patients with the microsatellite instable (MSI) subtype of colorectal cancer (CRC) have better prognosis and immunotherapy response than patients with the chromosomally instable (CIN) subtype due to improved cytotoxic T cell responses. This is in part due to high production of the chemokines CXCL10 and CCL5 from constitutive activation of the cytosolic DNA (cyDNA) sensor cGAS/STING by specific features of MSI cyDNA that lead to more effective cGAS/STING pathway activation. Here, we investigate MSI and CIN cyDNA structure and show that MSI cyDNA is enriched in G-quadruplexes which improve cGAS/STING and CD8+ T cell activation. We also show that micronuclei are less effective at inducing anti-tumor immunity and instead increase Treg activation and IL10 production. Overall, these data highlight the role of specific cyDNA structures in anti-tumor immunity and provide knowledge for improved design of therapeutic DNA-based cGAS/STING agonists to improve the prognosis of poorly immunogenic tumors like CIN CRCs. Overall design: On day 1, C57BL/6 mice were injected orthotopically into the descending colon wall with 5x10E5 MSI or CIN MC38 cells as performed previously using a flexible needle (Hamilton) inserted through the working channel of a Wolfe endoscope and visualized using the ColoView Imaging System. Fresh bone marrow derived dendritic cells (BMDCs) were resuspended in RPMI at 2x10E6 cells/mL for stimulation using 2ug MSI or CIN free cyDNA or micronuclei and 0.125uL/mL Lipofectamine 2000 per 2x10E6 cells for 30 minutes at 37°C as per manufacturer instructions. Following 2 PBS washes, 2x10E6 BMDCs were injected intraperitoneally into the tumor bearing mice on days 1, 6, and 12. On day 15, tumors were collected and pulverized using glass beads (Sigma Aldrich) and a Mini Beadbeater before RNA isolation using TRIzol (Invitrogen). Equal RNA quantities from each of the 3 biological replicates were then combined for bulk RNA sequencing.