Abstract 6015: A newly established neuroendocrine carcinoma cell line from a human ascending colon tumor shows high proliferation and CD133 expression in spherical cancer stem cell-like formation
Authors: Shinji Seiichi , Sasaki Norihiko , Yamada Takeshi , Koizumi Michihiro , Ohta Ryo , Matsuda Akihisa , Takahashi Goro , Hotta Masahiro , Iwai Takuma , Hara Keisuke , Takeda Kohki , Ueda Koji , Kuriyama Sho , Ishiwata Toshiyuki , Yoshida Hiroshi 
Published: September 2020

Abstract
BACKGROUND: The current incidence of neuroendocrine tumor (NET) is 0.6 cases per 100,000 people, although it has increased 50-fold over the last 35 years and is becoming increasingly clinically important. Neuroendocrine carcinoma (NEC) is a type of malignant NET associated with poor prognoses. However, the molecular basis and clinical characteristics of NEC have not yet been fully investigated due to a lack of patient biopsies to derive cell lines and established cell lines. Here, we report a newly established and characterized NEC cell line (SS-2) derived from a human ascending colon tumor. Furthermore, we investigated the ability of SS-2 cells to form spherical cancer stem cells (CSCs) and express CSC markers.
MATERIALS AND METHODS: The SS-2 cell line was derived from a tumor resection of the ascending colon from 59-year old Japanese woman with stage four NEC. The Ki-67 labeling index of the resected tumor was &gt;20% and, thus, the tumor was diagnosed as NEC. The characteristics of SS-2 cells transplanted subcutaneously into nude mice was determined. siRNA suppression of the neuroendocrine marker, INSM-1 was detected in SS-2 cells by western blotting. Additionally, SS-2 cells were seeded onto ultra-low attachment plates to detect sphere-formation and the expression of CSC markers, with confirmation by FACS analysis. Additionally, we compared the effect of commonly-used anti-colorectal chemotherapeutics on SS-2 cells and conventional colorectal cancer cells (Caco-2) using cell viability assay. Statistical analysis was done using Student's t test with P-values &lt; 0.05 considered statistically significant.
RESULTS: Characteristics of the resected tumor were retained in the SS-2 cells and in the xenograft in nude mice. INSM-1 was detected in SS-2 cell lysates and in nuclei of established SS-2 cells. siRNA targeting INSM-1 decreased INSM-1 mRNA levels but did not affect levels of chromogranin A and synaptophysin mRNA. SS-2 cells formed spheres when seeded onto ultra-low attachment plates that expressed higher levels of CD133 compared to SS-2 cells cultured under adherent conditions, which was also confirmed upon FACS analysis. After the addition of oxaliplatin and 5-FU, cell viabilities were higher in SS-2 cells than Caco-2 cells.
CONCLUSION: We established a novel NEC cell line, derived from a human ascending colon tumor, which we transplanted into nude mice. We determined that the CSC marker, CD133 is highly expressed in SS-2 cells, and hence posit that combination therapy of chemotherapeutics together with anti-CSC therapy may have increased efficacy against NEC.
Citation Format: Seiichi Shinji, Norihiko Sasaki, Takeshi Yamada, Michihiro Koizumi, Ryo Ohta, Akihisa Matsuda, Goro Takahashi, Masahiro Hotta, Takuma Iwai, Keisuke Hara, Kohki Takeda, Koji Ueda, Sho Kuriyama, Toshiyuki Ishiwata, Hiroshi Yoshida. A newly established neuroendocrine carcinoma cell line from a human ascending colon tumor shows high proliferation and CD133 expression in spherical cancer stem cell-like formation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6015.