Abstract 5026: Alcohol enriches therapy-resistant cancer cells with high autophagy, CD44 expression and tumor initiating capability
Authors: Tanaka Koji , Kijima Takashi , Chandramouleeswaran Prasanna Modayur , Whelan Kelly A , Kasagi Yuta , Shimonosono Masataka , Furukawa Haruna , Hara Takeo , Makino Tomoki , Yamasaki Makoto , Klein-Szanto Andres J , Natsugoe Shoji , Mori Masaki , Doki Yuichiro , Nakagawa Hiroshi 
Published: August 2018

Abstract
Introduction: Esophageal squamous cell carcinoma (ESCC) is the deadliest of all human cancers with alcohol (EtOH) as a major risk factor. Malignant properties of ESCC have been attributed to ESCC cells with high CD44 expression (CD44H) and autophagy; however, the pathogenic role of EtOH in ESCC remains elusive
Methods: With a goal of translation in personalized medicine, we analyzed therapy-naïve ESCC tumor biopsies from patients and ESCC cell lines in three-dimensional (3D) organoids for functional characterization of ESCC cells following exposure to 5-fluorouracil (5FU) or EtOH ex vivo. Tumor growth was assessed in EtOH-fed mice carrying xenograft tumors with two independent ESCC cell lines in the presence or absence of 4-methylpyrazole (4MP), an alcohol dehydrogenase inhibitor. 3D organoids and tumors were analyzed by flow cytometry and immunostaining for CD44, Ki67, p53 and autophagic vesicle (AV) content.
Results: ESCC patient biopsies contained CD44H cells with increased AV content. ESCC 3D organoids were grown successfully from 11 out of 16 tumors (68.8%) and passaged with recapitulation of the histopathology, proliferation, p53 and CD44 expression and autophagy present in the original in situ tumors. Successful organoid formation was significantly associated with poor chemoradiation therapy response (progressive and stable diseases, n=10 vs. partial response, n=6). In 3D organoids surviving 5FU treatment, CD44H cells with high autophagic activity was found to be enriched. Pharmacological autophagy flux inhibition by chloroquine augmented 5FU-mediated cytotoxicity in 3D ESCC organoids. 5FU-resistant CD44H cells were more capable of forming 3D organoids compared to bulk populations of ESCC cells. EtOH promoted expansion of CD44H cells with significantly increased proliferation and organoid formation rate upon serial passages, suggesting the increased self-renewal of CD44H cells. In xenograft tumors, alcohol drinking not only promoted tumor growth but increased the intratumoral CD44H cell content which was antagonized by 4MP. Moreover, pharmacological autophagy flux impairment depleted CD44H cells in xenograft tumors.
Conclusions: The novel single cell-based 3D ESCC organoid system may serve as a highly efficient platform to explore the role of alcohol and other environmental factors as well as cancer therapeutics and therapy resistance mechanisms in conjunction with morphological and functional assays with implications for translation in personalized medicine.
Citation Format: Koji Tanaka, Takashi Kijima, Prasanna Modayur Chandramouleeswaran, Kelly A Whelan, Yuta Kasagi, Masataka Shimonosono, Haruna Furukawa, Takeo Hara, Tomoki Makino, Makoto Yamasaki, Andres J Klein-Szanto, Shoji Natsugoe, Masaki Mori, Yuichiro Doki, Hiroshi Nakagawa. Alcohol enriches therapy-resistant cancer cells with high autophagy, CD44 expression and tumor initiating capability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5026.