Abstract 77: Recapitulating metastatic colorectal cancer in somatic mutation models for investigating the tumor immune microenvironment in minimal residual disease
Authors: Mohamed Alaa M. , Soeung Melinda , Alshenaifi Jumanah , Fowlkes Natalie , Manyam Ganiraju , Davis Jennifer , Anderson Amanda , Norton Will , Deem Angela K. , Gao Sisi , Khanduri Isha , Bistow Christopher , Carbone Federica , Napolitano Stefania , Huang Justin , Maru Dipen M. , Menter David G. , Draetta Giulio F. , Genovese Giannicola , Kopetz Scott 
Published: April 2023

Abstract
Colorectal cancer (CRC) is the second leading cause of cancer deaths worldwide. One major factor contributing to the high mortality rate of CRC is the presence of microscopic minimal residual disease (MRD) that remains radiographically undetectable and persist regardless of therapeutic interventions. In fact, many CRC patients who have received therapy with curative intent will experience recurrence years later due to MRD, which can progress to recurrent disease that results in clinical morbidity. While immune modulation is crucial for mediating progression and metastasis of primary CRC tumors, how micrometastases suppress or evade antitumor immunity and/or maintain a state of dormancy before growing into macrometastases remains poorly understood. Filling this knowledge gap requires establishing and utilizing models capable of accurately recapitulating the immune phenotype of human microsatellite stable (MSS) metastatic CRC. To this end, we generated genetically engineered, syngeneic mouse colonic organoids using CRE recombinase technology to recapitulate MSS metastatic CRC models with APC and TP53 mutations. After establishing hematologic experimental metastases, histopathological examination confirmed that those present in the liver bore a close resemblance to human colorectal adenocarcinoma liver metastases. We then leveraged our model to test the hypothesis that the activation of suppressive immune cell populations is key for enabling the establishment and progression of MRD. We performed high-plex immunofluorescent co-localization analysis to delineate the immune compartments of micro- and macrometastases. Our results showed that, overall, leukocytes (CD45+ cells) decreased as metastatic tumor foci in the liver progressed. Specifically, cytotoxic CD8+ T cell densities were markedly reduced as tumor metastases progressed to macroscopic disease. Furthermore, populations of CD4+FOXP3+ (T-reg) cells increased as tumors progressed, with an increase in the ratio of CD4+FOXP3+ to CD8+ cells during tumor progression. Additionally, M2 macrophage density (IBA-1+, CD163+) increased as the liver metastases progressed in size. Overall, our findings support our hypothesis, and initiate the first step for identifying suppressive immune infiltrates that may be exploited for therapeutic targeting. Importantly, our somatic mutation-based modeling strategy enables a highly precise recapitulation of the mutational drivers and heterogeneity that occurs in CRC patients as well as provides a valuable resource for research aimed at elucidating the immunobiology of MRD.
Citation Format: Alaa M. Mohamed, Melinda Soeung, Jumanah Alshenaifi, Natalie Fowlkes, Ganiraju Manyam, Jennifer Davis, Amanda Anderson, Will Norton, Angela K. Deem, Sisi Gao, Isha Khanduri, Christopher Bistow, Federica Carbone, Stefania Napolitano, Justin Huang, Dipen M. Maru, David G. Menter, Giulio F. Draetta, Giannicola Genovese, Scott Kopetz. Recapitulating metastatic colorectal cancer in somatic mutation models for investigating the tumor immune microenvironment in minimal residual disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 77.