Title Establishment and characterization of a new cell line derivedfrom human colorectal laterally spreading tumor

AIM: To study the molecular mechanism of laterally spreading tumor (LST), a cell line [Laterally Spreading Tumor-Rectum 1 (LST-R1)] was derived and the characteristics of this cell line were investigated. METHODS: A new cell line (LST-R1) originated from laterally spreading tumor was established. Properties of the cell line were characterized using scanning and transmission electron microscopy, immunohistochemistry method, cytogenetic analysis and nude mice xenograft experiments. In vitro invasion assay, cDNA microarray and Western blott ing were used to compare the difference between the LST-R1 and other colorectal cancer cell lines derived from prudent colon cancer. RESULTS: Our study demonstrated that both epithelial special antigen (ESA) and cytokeratin-20 (CK20) were expressed in LST-R1. The cells presented microvilli and tight junction with large nuclei. The karyotypic analysis showed hyperdiploid features with structural chromosome aberrations. The in vivo tumorigenicity was also demonstrated in nude mice xenograft experiments. The invasion assay suggested this cell line has a higher invasive ability. cDNA microarray and Western blotting show the loss of the expression of E-cadherin in LST-R1 cells. Online Submissions: wjg.wjgnet.com World J Gastroenterol 2008 February 28; 14(8): 1204-1211 www.wjgnet.com World Journal of Gastroenterology ISSN 1007-9327 wjg@wjgnet.com © 2008 WJG. All rights reserved. Moreover, our previous study has also shown that the incidence of LST is not low in China. Flat appearance makes it difficult to detect it and previous study has shown that laterally spreading tumors have a higher malignant potential than conventional adenomas. To study the distinctive biological behavior and the underlying biological mechanisms responsible for LST cancer progression will certainly help improving the detection rate of LST and eventually lead to the decreasing morbidity of colon cancer. Invasion is an important step in the intricate process leading to the formation of cancer and has close relationship with the adhesion ability of the cancer cells. To investigate the higher malignant tendency of colorectal superficial tumor, we established a stable cell line derived from LST and assessed its invasion ability by in vitro invasion assay. Here we show that the invasion ability of LST is higher than SW480 and lovo cells, which is originated from prudent colon cancer. To elucidate the reason for this difference, we have developed a cDNA microarray, representing 18 000 cDNA clusters to profile the gene expression patterns in Laterally Spreading Tumor-Rectum 1 (LST-R1), SW480, lovo cell lines and found that many genes associated with adhesion showed a different expression profile. Our data suggest that LSTR1cells have some distinct characteristics comparing with SW480 and lovo cells. Further investigations on the cells should enhance our understanding on the distinct biology of LST. MATERIALS AND METHODS Tumor origin Laterally spreading tumor cells were derived from a rectal LST of 59-year-old Chinese woman. Magnifying endoscope showed a flat granular tumor with nodus (about 70 mm × 60 mm) in rectum, 3 cm far from anus. Examination of the biopsy specimen revealed that it has the characteristic morphology of a villous adenoma accompanied by moderate sever atypical hyperplasia (Figure 1B). Tissue and cell culture Tissue specimens were obtained by endoscopic partial mucosal resection (EPMR), and were transferred to a transport medium containing five folds penicillin, streptomycin (Invitrogen, Carlsbad, CA) and amphotercin B. The specimens were vibrated for about ten minutes to get rid of filth and washed five times with transport medium. They were then trimmed to remove fat and connective tissue, minced into pieces in a sterile culture dish, and subsequently plated in a 25 cm flask. Cells were incubated in a 37 incubator with 5% CO2. RPMI1640 medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA)was added to the cells four hours later and media was changed every 2-3 d. Clonal growth was observed after 27 d of culture, and colonies were identified and subcultured. After 2 passages, the cells grew rapidly and were polymorphic with a few fibroblasts. To purify the cells, they were inoculated in 96-well plates and were cultured separately to get rid of Wang XY et al. LST-R1 derived from human colorectal laterally spreading tumor 1205 www.wjgnet.com fibroblasts. Finally, the polymorphic cells (more than 90%) were designated as LST-R1. HCT116, lovo, SW480 and colo205 colon tumor cell lines were commercially obtained from the American Type Culture Collection and maintained in RPMI 1640 medium supplemented with 100 U/mL penicillin,100 μg/mL streptomycin, 10% fetal bovine serum in 75 cm tissue culture flasks at 37 in a 5% CO2 environment. Confirmation of epithelial origin Cells were grown on chamber slides and stained for CK20 and ESA expression with a specific antibody (Beijing ZhongShan Biology technology Ltd, Beijing, China). Scanning and transmission electron microscopy LST cells grown on slides were trypsinized and centrifuged. After fixed in 2.5% glutaraldehyde and postfixed in 1% osmium, they were dehydrated with acetone. The sample was divided into two parts: one half was added with acetas, dried on critical point of CO2 and observed under scanning electron microscope, another half was embedded in paraffin and cut into thin pieces with a thickness of about 700 mm, stained with uranyl acetate and citrate, and observed under transmission electron microscope. Metaphase chromosome preparation and spectral karyotyping (SKY) Metaphase cells were obtained by treatment of the cultured cells with Colcemid (Gibco, Grand Island, NY, USA) at a final concentration of 0.03 μg/mL for 3 h. The harvested cells were treated with 0.8% sodium citrate for 15 min at 37 and fixed in 3:1 methanol/acetic acid. Metaphase chromosome spreading was performed as previously described. The slide with metaphase cells was aged at room temperature (R.T.) for 5-7 s prior to SKY. The slide was treated with DNase-free RNase solution (0.1 g/L) at 37 for 1 h, and briefly rinsed in distilled water. After washing in 2 × SSC for 10 min at R.T, the slide was treated with proteinase K (0.5 μg/mL) solution for 10 min at 37 and washed in 2 × SSC at R.T. for 10 min. The slide was then fixed in 1% paraformaldehyde and washed in 2 × SSC at R.T. for 10 min each, followed by dehydration in 70%, 85%, and 95% ethanol at R.T. for 2 min. The slide was placed in 70% formamide/2 × SSC at 70 for 4 min and dehydrated in 70%, 85%, and 95% ethanol for 2 min Figure 1 Endoscope and the histopathology of LST. A: LST under endoscope which grows laterally in the rectum, 3 cm far from the anus and has a diameter of 60 mm × 70 mm; B: The histopathology of the tissue: a villous adenoma accompanied by moderate severe atypical hyperplasia. A B each. Other protocols including SKY probe (Applied Spectral Imaging, Migdal Ha’Emek, Israel) denaturation, hybridization and detection were done according to the recommendations of SKY probe manufacturer. SKY image capturing and karyotyping were performed using the SkyVision Imaging System equipped with a Zeiss Axioplan 2 fluorescence microscope. Recommendations were followed in karyotyping descriptions. In vitro and in vivo growth properties of the cells To determine the population doubling time and mitotic index of LST-R1 cells, the cells were suspended at a concentration of 1 × 10 per mL and 100 μL suspension was cultured in each well of 96 well plates. The cell growth was monitored after 12 h, 24 h, 36 h and 48 h by 3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT, the final concentration = 0.5 g/L) assay. The value indicated the mean of three independent experiments. In v i vo tumorigenici ty studies were perfor med using 5-wk-old athymic nude mice obtained from the Animal Center of the First Military Medical University (Guangzhou, China). LST-R1 cells at passage 51 were trypsinized, washed twice with serum-free RPIM1640 medium, and resuspended in serum-free RPMI-1640 (1 × 10 per mL). A final volume of 0.2 mL was injected per site on the back of 8 athymic nude mice. Tumor growth was measured weekly using calipers. The tumor area was calculated according to the following formula: tumor area = π × (width/2 × length/2). When the tumors grew to a certain extent (about 6 mm × 4 mm), tissue was removed and fixed in neutral buffered formalin for histological examination. Some cells from the xenografts were re-established in vitro to compare their morphology with that of the original cell line. In vitro invasion assay The invasion assay was performed by using 24-well BD BioCoatTM MatrigelTM Invasion Chamber with 8-μm polycarbonated filters (Becton Dickinson, Bedford, MA), as described by Albini et al. 1 × 10 cells were seeded on Matrigel invasion chamber plates and cultured in routine medium. Cells were incubated for 22 h at 37 in a humidified incubator with 5% CO2. Nonmigratory cells on the upper surface of the filter were removed by wiping with a cotton swab. Invasive cells that penetrated through pores and migrated to the underside of the membrane were stained with Giemsa solution after fixation with 4% formaldehyde in PBS. The cell number was counted under microscopic vision. Screening of differentially expressed genes Total RNA was extracted using standard Trizol RNA isolation protocol (Life Technologies, Inc., Grand Island, NY). The poly (A) mRNA was isolated from the total RNA using a poly (dT) resin (Qiagen, Hilden, Germany). Nylon membrane-based cDNA microarrays were prepared by using Spotter (BioRobotics, Cambridge, U.K.) containing clones from the present work and other tissue resources and the internal controls. The procedures for probe preparation, hybridization, washing, scanning, and signal intensity normalization of the spots were performed also using a previo