Abstract P022: Investigating the role of the tumor microenvironment in the rectal cancer response to preclinical radio-immunotherapy combinations
Authors: Melissourgou-Syka Lydia , Gillespie Michael A. , Hillson Lily V.S. , Gilroy Kathryn , Stevenson Katrina , Susanti Susanti , Lannagan Tamsin R.M. , Mulholland Eoghan , Dunne Philip D. , Edwards Joanne , O'Cathail Sean M. , Steele Colin W. , Sansom Owen J. , Roxburgh Campbell S.D. 
Published: January 2025

Abstract
Background: Locally-advanced rectal cancer (LARC) is traditionally treated with neoadjuvant chemoradiotherapy before surgery, however, response heterogeneity necessitates the development of novel treatment combinations. Immune checkpoint blockade (ICB) with or without radiotherapy (RT) elicits clinical benefit in various solid tumors, yet ICB’s effectiveness so far is limited to colorectal tumors with microsatellite instability and the role of the tumor microenvironment (TME) is unclear. We present a novel orthotopic immunocompetent mouse model of LARC, where combinations of RT and ICB are tested to elucidate the role of TME in the response to treatment. Methods: Autochthonous murine tumor-derived organoids expressing common driver mutations (Apcfl/fl; KrasG12D/+; p53fl/fl; Tgfbr1fl/fl: ‘AKPT’) were orthotopically injected into syngeneic C57Bl/6 immunocompetent mice. Tumor response to RT-ICB was studied on timepoint and aging cohorts of mice with tumors growing in different TME settings: (a) in the rectal submucosa of wildtype mice, (b) subcutaneously in wildtype mice, and (c) in the rectal submucosa of mice lacking the iCCR locus (CCR1, CCR2, CCR3, and CCR5: ‘iCCRKO ’), which inhibits inflammatory monocyte recruitment into the TME. Radiation was delivered as 15Gy in 3# (Mon-Wed-Fri) using a Small Animal Radiation Research Platform on day 13 following tumor engraftment for the orthotopic models and on day 7 for the subcutaneous. PD-1 inhibition (PD-1i) was administered intraperitoneally (10mg/kg twice weekly) 3 days pre-RT until endpoint. Mice in the timepoint experiments were sampled 5 days following RT, while aging mice were sampled at clinical endpoint or 100 days, whichever came first. Results: RT and PD-1i offered significant survival extension and tumor control in the orthotopic wildtype setting (a). Transcriptomic characterization at 5 days revealed that untreated and irradiated tumors harbored a stroma-rich microenvironment, low in immune cells, which PD-1i partly reversed. This phenotypic ‘switch’ was also evident in an aging cohort, suggesting that RT + PD-1i have long-lasting effects on the stroma. In the subcutaneous setting (b), RT significantly increased tumor control but PD-1 blockade offered little additional advantage. Interestingly, subcutaneous AKPT tumors had significantly more CD8+ cells at baseline than orthotopic ones, highlighting the inherent immune permissiveness of the subcutaneous TME. Finally, tumors growing in the iCCRKO setting (c) were more radio-sensitive, leading to 3 instances of tumor clearance: one in the RT group and two in the RT + PD-1i. Conclusion: Our findings demonstrate the synergistic potential of RT and PD-1i to sensitize an inherently radio-resistant orthotopic mouse model of LARC. Tumor location and inflammatory monocytes emerged as key regulators of the immune response to RT-ICB, which could have important implications for LARC modeling. Importantly, we highlight that TME components can facilitate treatment resistance and give rise to targets that can be therapeutically exploited.
Citation Format: Lydia Melissourgou-Syka, Michael A. Gillespie, Lily V.S. Hillson, Kathryn Gilroy, Katrina Stevenson, Susanti Susanti, Tamsin R.M. Lannagan, Eoghan Mulholland, Philip D. Dunne, Joanne Edwards, Sean M. O'Cathail, Colin W. Steele, Owen J. Sansom, Campbell S.D. Roxburgh.Investigating the role of the tumor microenvironment in the rectal cancer response to preclinical radio-immunotherapy combinations.[abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P022