Abstract 119: Enteric glial cells promote chemoresistance in ATM-expressing cancer stem cells

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Abstract In colon cancer, the subset of cells fueling the clonal growth of the tumor have been shown to also possess enhanced chemoresistance abilities. These cells, known as cancer stem cells (CSC), are controlled by molecules and cells within the tumor microenvironment. In the latter reside enteric glial cells (EGC) that heavily regulate intestinal epithelial barrier function in a healthy colon. However, EGC influence on colon carcinogenesis remains poorly understood. Recent work from our lab shows that EGC promote CSC-driven tumorigenesis via the release of prostaglandin E2. Here, we investigate the impact of EGC on CSC chemoresistance. CD24Hi-CD44Hi CSC were FACS-isolated from human colon cancer primary tumors or cell lines and 3D-grown in the presence of murine or human EGC seeded on Transwell filters in vitro in the presence of the chemotherapeutic drug 5-Fluorouracil (5-FU) or injected subcutaneously with(out) EGC in vivo in 5-FU treated-immunodeficient mice. EGC impact was assessed on 5-FU-induced restriction of tumor formation and growth in vitro and in vivo. EGC-conditioned medium (CM) impact on 5-FU-induced apoptosis in CSC was measured by flow cytometry. EGC impact on expression of 48 chemoresistance-related genes in CSC was assessed by high throughput RT-qPCR. Impact of inhibition of ATM activity and ATM activation by the Mre11-Rad50-Nbs1 (MRN) complex in CSC was tested using 0.1µM KU-55933 and 12µM mirin. To understand the impact of 5-FU on EGC, levels of senescence markers p16, p19, p21 and H3K9me3 and β-gal activity were assessed. Proteomic analyses of EGC-CM were conducted by mass spectrometry. EGC promoted growth of CSC-derived tumors in the presence of 5-FU in vivo. EGC increased the number and size of tumor-organoids grown from CSC isolated from cell lines (HT29, HCT116, HCT15) and primary tumors in the presence of 5-FU in vitro. EGC-CM reduced apoptosis in 5-FU-treated CSC. Out of all genes tested, only ATM mRNA was significantly enriched in 5-FU-treated CSC cultured with EGC vs. alone. Co-culture studies using CSC derived from the ATM deficient SW1222 cell line or using the ATM inhibitor KU-55933 reduced EGC pro-chemoresistance effects. Furthermore, inhibition of ATM activation by the upstream DNA damage sensor MRN complex using mirin abolished EGC protective effects on CSC resistance to 5-FU. 5-FU-treated EGC showed increased levels of senescence markers and protein secretion, indicative of a senescence-associated secretory phenotype (SASP). Moreover, CM from 5-FU-treated EGC further reduced 5-FU-induced apoptosis in CSC as compared to CM from untreated EGC. Mass spectrometric analyses revealed that IGFBP7, a putative ATM activator, was >100 times more abundant in the CM of 5-FU-treated EGC vs. untreated EGC. Our data strongly indicate that EGC promote CSC chemoresistance via increased ATM signaling. Future studies will investigate the implication of IGFBP7 and downstream targets of ATM involved in DNA repair. Citation Format: Gregory Bacola, Simon Vales, Alice Prigent, Kelsie A. Dougherty, Deanna M. Peperno, Shaian Lashani, Bradley A. Wieland, Melissa Touvron, Lisa Oliver, François M. Vallette, Michel Neunlist, Laurianne Van Landeghem. Enteric glial cells promote chemoresistance in ATM-expressing cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 119.