ORIGINAL ARTICLE pISSN 2288-6575 • eISSN 2288-6796 http://dx.doi.org/10.4174/astr.2016.90.4.183 Annals of Surgical Treatment and Research Characterization of sphere-forming HCT116 clones by whole RNA sequencing Eunkyung Chung*, Inkyung Oh1,*, Kil Yeon Lee1 R&D Center, L&K Biomed Co. Ltd., Seoul, 1Department of Surgery, College of Medicine, Kyung Hee University, Seoul, Korea Purpose: To determine CD133+ cells defined as cancer stem cells (CSCs) in colon cancer, we examined whether CD133+ clones in HCT116 demonstrate known features of CSCs like sphere-forming ability, chemodrug-resistance, and metastatic potential. Methods: Magnetic cell isolation and cell separation demonstrated that <1% of HCT116 cells expressed CD133, with the remaining cells being CD133- clones. In colon cancer cells, radioresistance is also considered a CSC characteristic. We performed clonogenic assay using 0–4 Gy g-irradiation. Results: Interestingly, there were no differences between HCT116 parental and HCT116 CD133+ clones when the cells comprised 0.5% of the total cells, and CD133- clone demonstrated radiosensitive changes compared with parental and CD133+ clones. Comparing gene expression profiles between sphere-forming and nonforming culture conditions of HCT116 subclones by whole RNA sequencing failed to obtain specific genes expressed in CD133+ clones. Conclusion: Despite no differences of gene expression profiles in monolayer attached culture conditions of each clone, sphere-forming conditions of whole HCT116 subclones, parental, CD133+, and CD133- increased 1,761 coding genes and downregulated 1,384 genes related to CSCs self-renewal and survival. Thus, spheroid cultures of HCT116 cells could be useful to expand colorectal CSCs rather than clonal expansion depending on CD133 expressions. [Ann Surg Treat Res 2016;90(4):183-193] Key Words: Neoplastic stem cell, Colon neoploasms, RNA sequence analysis, HCT116 cells INTRODUCTION sticity, capability of metastasis, chemotherapy resistance, and can continually adapt to changing microenvironments [5,6]. Cancer stem cells (CSCs) are tumor cells that have the CSCs were initially considered a population with well-defined principal properties of self-renewal, clonal tumor initiation phenotypic and molecular features. However, accumulating capacity, and clonal long-term repopulation potential. CSCs evidence suggests instead that CSCs are a dynamic population are a distinct cellular subpopulation in colon cancer that continuously shaped by a convergence of genetic, epigenetic, is essential for tumor maintenance [1,2]. Human colorectal and microenvironmental factors [1]. CSCs were first isolated on the basis of CD133 expression and CSCs reside in niches that are anatomically distinct regions were demonstrated to induce tumors in mice that resembled within the tumor microenvironment, which are cellular and the original malignancy [3,4]. CSCs have some unexpected noncellular elements surrounding tumors [7]. Interactions Reviewed properties, including a high degree of heterogeneity and pla- between malignant and nontransformed cells create the January February March April May Received December 11, 2015, Reviewed February 5, 2016, *Eunkyung Chung and Inkyung Oh contributed equally to this study as co-first June Accepted February 11, 2016 authors. July Corresponding Author: Kil Yeon Lee Copyright ⓒ 2016, the Korean Surgical Society August Department of Surgery, College of Medicine, Kyung Hee University, 26 cc Annals of Surgical Treatment and Research is an Open Access Journal. All September Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Korea articles are distributed under the terms of the Creative Commons Attribution Non- October Tel: +82-2-958-8261, Fax: +82-2-969-9366 Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which November E-mail: [email protected] permits unrestricted non-commercial use, distribution, and reproduction in any December medium, provided the original work is properly cited. Annals of Surgical Treatment and Research 183 Annals of Surgical Treatment and Research 2016;90(4):183-193 tumor microenvironment [8]. Nonmalignant cells of the tumor Radiosensitivity assay microenvironment including immune cells, tumor vasculatures The association between the expression of monolayer and and lymphatics, as well as fibroblasts, pericytes, and adipocytes sphere formed cells and radiation sencitivity was evaluated have dynamic and often tumor-promoting functions at all by sorting and collecting CD133+ and CD133- clones. The cells stages of carcinogenesis [9,10]. CD133+ colon cancer cells are were pre-plated in a 6-cm diameter culture dish. The following reportedly more interactive with the tumor microenvironment day the cells were exposed to externally applied 0-4 Gray (Gy) than are CD133- cells [11]. HCT116 cell populations are g-irradiation. After 10 days the cells were washed in phosphate- composed mainly of stem-like cancer cells, as demonstrated by buffered saline and fixed with methanol: acetic acid (3:1) for 10 their colonosphere forming capability and CD133 expression minutes. The colonies were stained with 5% trypan blue for 10 [12,13]. Long-term cultured self-renewing CD133+CD44+ cells minutes, rinsed in water, and counted. enriched in the CD133+CD44 (high) subset, which express the epithelial-to-mesenchymal transition marker, are more invasive RNA sequencing in vitro and are solely responsible for liver metastasis in vivo [14]. Transcriptome libraries were prepared following Illumina’s However, it has not yet been defined whether CD133+ clones of TruSeq RNA kit protocol, using 1–2 μg of total RNA. Poly(A)+ HCT116 cells have distinct molecular signatures compared with RNA was isolated using AMPure XP beads (Beckman Coulter, CD133- clones in terms of known CSCs properties like sphere Brea, CA, USA) and fragmented with the Ambion Fragmentation forming ability, angiogenesis and vascular niche formation, Reagents kit (Ambion, Austin, TX, USA). cDNA synthesis, end- chemoresistance, radioresistance, and invasive migration for repair, A-base addition, and ligation of the Illumina indexed metastasis. adapters were performed according to Illumina’s protocol. Libra- In the present study, we performed whole RNA sequencing ries were size-selected for 250–300 bp cDNA fragments on a 3% in CD133+ and CD133- clones both in attached monolayer Nusieve 3:1 (Lonza) agarose gel, recovered using QIAEX II gel cultures and colonosphere forming condition to identify CSC- extraction reagents (Qiagen, Hilden, Germany), and polymerase specific gene expression profiles. Moreover, we also intended chain reaction (PCR)-amplified using Phusion DNA polymerase to establish in vitro three-dimensional (3D) tumor model (New England Biolabs, Ipswich, MA, USA) for 14 PCR cycles. consisting of CSCs by applying tumor-sphere formation The amplified libraries were purified using AMPure XP beads. depending on CD133 expression. Library quality was measured on an Agilent 2100 Bioanalyzer for product size and concentration. Paired-end libraries were METHODS sequenced with the Illumina HiSeq 2500 (2×100 nucleotide read length). Reads that passed the chastity filter of Illumina Monolayer and tumor spheres cultures BaseCall software were used for subsequent analysis. The HCT116 human colorectal cancer cell line was obtained from the American Type Culture Collection (Manassas, VA, RESULTS USA) Cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum HCT116 cells display colonosphere forming capa- o and antibiotics at 37 C and 5% CO2. city and radioresistance regardless of CD133 ex- To obtain sphere cultures, monolayer cells were enzymatically pression and manually dissociated into a single cell suspension using In order to identify whether CD133 expression in HCT116 Trypsin-EDTA (0.125%) followed by passage through a 25-gauge cancer cells is a prerequisite to define CSCs, CD133+ cells were needle. Cells were seeded at 5,000 cells/mL into nonadherent isolated using MACS. Fewer than 1% of HCT116 cells expressed plates coated with 1.2% poly-(2-hydroxyethylmethacrylate)/95% CD133 with the remainder being CD133-. Whole subclones of ethanol (Sigma-Aldrich, St Louis, MO, USA). Stem cell medium HCT116 separated by the expression of CD133 formed irregular consisted in DMEM/F12 supplemented with B27 (Gibco-Life shaped colonospheres in culture (Fig. 1A). In colon cancer cells, Technologies, Carlsbad, CA, USA) and MEGM SingleQuots radioresistance is also considered a CSC characteristic [15,16]. (human epidermal growth factor, insulin, hydrocortisone and Appropriately we performed a clonogenic assay using 0–4 Gy GA-100; Lonza, Basel, Switzerland). Tumor sphere cultured g-irradiation. Interestingly, there were no differences between (TSC) cells were disaggregated by incubation with the StemPro HCT116 parental and CD133- clones when cells comprised 0.6% accutase Cell Dissociation Reagent (Gibco-Life Technologies) of the total cells (Fig. 1B). Only CD133+ clones demonstrated until a single cell suspension was obtained. TSC cells were radioresistant changes at 1 Gy g-irradiation compared with subcultured every 5 to 7 days for up to 5 generations. other HCT116 clones. It suggests that whole clones of HCT116 have some CSC properties determined by colonosphere forming capacity, while there is a difference in CD133+ clones 184 Eunkyung Chung, et al: Characterization of sphere-forming HCT116 clones by whole RNA sequencing A CD133+ CD133 HCT116 HCT116 HCT116 B CD133+ HAT116 (0.6%) 1.0 Fig.