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: 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
Journal: Cancer Research (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 &amp;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 &amp;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.