Identification of a unique hepatocellular carcinoma line, Li-7, with CD13(+) cancer stem cells hierarchy and population change upon its differentiation during culture and effects of sorafenib

BMC Cancer

Takeshi Yamada²  Masato Abei¹  Inaho Danjoh³  Ryoko Shirota²  Taro Yamashita⁴  Ichinosuke Hyodo¹  Yukio Nakamura² 

[1] Division of Gastroenterology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8575, Ibaraki, Japan

[2] Cell Engineering Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba 305-0074, Ibaraki, Japan

[3] Functional Evaluation of Genomic Polymorphisms, Tohoku Medical Megabank Organization, Sendai, Japan

[4] Department of Gastroenterology, Kanazawa University Hospital, 13-1, Takara-machi, Kanazawa 920-8641, Ishikawa, Japan

关键词: Population change;    Sorafenib;    CD166;    CD13;    Hepatocellular carcinoma;    Cancer stem cell;   

Others  :  1171700
DOI  :  10.1186/s12885-015-1297-7

 received in 2014-10-05, accepted in 2015-03-31,  发布年份 2015

【 摘 要 】

Backgrounds

Cancer stem cell (CSC) research has highlighted the necessity of developing drugs targeting CSCs. We investigated a hepatocellular carcinoma (HCC) cell line that not only has CSC hierarchy but also shows phenotypic changes (population changes) upon differentiation of CSC during culture and can be used for screening drugs targeting CSC.

Methods

Based on a hypothesis that the CSC proportion should decrease upon its differentiation into progenitors (population change), we tested HCC cell lines (HuH-7, Li-7, PLC/PRF/5, HLF, HLE) before and after 2 months culture for several markers (CD13, EpCAM, CD133, CD44, CD90, CD24, CD166). Tumorigenicity was tested using nude mice. To evaluate the CSC hierarchy, we investigated reconstructivity, proliferation, ALDH activity, spheroid formation, chemosensitivity and microarray analysis of the cell populations sorted by FACS.

Results

Only Li-7 cells showed a population change during culture: the proportion of CD13 positive cells decreased, while that of CD166 positive cells increased. The high tumorigenicity of the Li-7 was lost after the population change. CD13(+)/CD166(−) cells showed slow growth and reconstructed the bulk Li-7 populations composed of CD13(+)/CD166(−), CD13(−)/CD166(−) and CD13(−)/CD166(+) fractions, whereas CD13(−)/CD166(+) cells showed rapid growth but could not reproduce any other population. CD13(+)/CD166(−) cells showed high ALDH activity, spheroid forming ability and resistance to 5-fluorouracil. Microarray analysis demonstrated higher expression of stemness-related genes in CD166(−) than CD166(+) fraction. These results indicated a hierarchy in Li-7 cells, in which CD13(+)/CD166(−) and CD13(−)/CD166(+) cells serve as slow growing CSCs and rapid growing progenitors, respectively. Sorafenib selectively targeted the CD166(−) fraction, including CD13(+) CSCs, which exhibited higher mRNA expression for FGF3 and FGF4, candidate biomarkers for sorafenib. 5-fluorouracil followed by sorafenib inhibited the growth of bulk Li-7 cells more effectively than the reverse sequence or either alone.

Conclusions

We identified a unique HCC line, Li-7, which not only shows heterogeneity for a CD13(+) CSC hierarchy, but also undergoes a “population change” upon CSC differentiation. Sorafenib targeted the CSC in vitro, supporting the use of this model for screening drugs targeting the CSC. This type of “heterogeneous, unstable” cell line may prove more useful in the CSC era than conventional “homogeneous, stable” cell lines.

【 授权许可】

   
2015 Yamada et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Sugihara E, Saya H: Complexity of cancer stem cells. Int J Cancer 2013, 132(6):1249-59.
• [2]Nagano H, Ishii H, Marubashi S, Haraguchi N, Eguchi H, Doki Y, et al.: Novel therapeutic target for cancer stem cells in hepatocellular carcinoma. J Hepatobil Pancreat Sci 2012, 19(6):600-5.
• [3]Touil Y, Igoudjil W, Corvaisier M, Dessein AF, Vandomme J, Monte D, et al.: Colon cancer cells escape 5FU chemotherapy-induced cell death by entering stemness and quiescence associated with the c-Yes/YAP axis. Clin Cancer Res. 2014, 20(4):837-46.
• [4]Schepers AG, Snippert HJ, Stange DE, van den Born M, van Es JH, van de Wetering M, et al.: Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas. Science 2012, 337(6095):730-5.
• [5]Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM: Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010, 127(12):2893-917.
• [6]Abei M: Clinical staging and treatment selection for hepatocellular carcinoma: Overview of the current status and perspectives for the future. In Therapy for hepatocellular carcinoma: etiology and treatment. Edited by Ohkohchi N. Nova Science Publishers Inc, New York; 2014:61-100.
• [7]Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al.: Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008, 359(4):378-90.
• [8]Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, Kim JS, et al.: Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol 2009, 10(1):25-34.
• [9]Chiba T, Kita K, Zheng YW, Yokosuka O, Saisho H, Iwama A, et al.: Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology 2006, 44(1):240-51.
• [10]Haraguchi N, Utsunomiya T, Inoue H, Tanaka F, Mimori K, Barnard GF, et al.: Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells 2006, 24(3):506-13.
• [11]Suetsugu A, Nagaki M, Aoki H, Motohashi T, Kunisada T, Moriwaki H: Characterization of CD133+ hepatocellular carcinoma cells as cancer stem/progenitor cells. Biochem Biophys Res Commun 2006, 351(4):820-4.
• [12]Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO, et al.: Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology 2007, 132(7):2542-56.
• [13]Yang ZF, Ho DW, Ng MN, Lau CK, Yu WC, Ngai P, et al.: Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell 2008, 13(2):153-66.
• [14]Yamashita T, Ji J, Budhu A, Forgues M, Yang W, Wang HY, et al.: EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology 2009, 136(3):1012-24.
• [15]Zhu Z, Hao X, Yan M, Yao M, Ge C, Gu J, et al.: Cancer stem/progenitor cells are highly enriched in CD133 + CD44+ population in hepatocellular carcinoma. Int J Cancer. 2010, 126(9):2067-78.
• [16]Lee TK, Castilho A, Cheung VC, Tang KH, Ma S, Ng IO: CD24(+) liver tumor-initiating cells drive self-renewal and tumor initiation through STAT3-mediated NANOG regulation. Cell Stem Cell 2011, 9(1):50-63.
• [17]Ma S, Chan KW, Lee TK, Tang KH, Wo JY, Zheng BJ, et al.: Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations. Mol Cancer Res. 2008, 6(7):1146-53.
• [18]Haraguchi N, Ishii H, Mimori K, Tanaka F, Ohkuma M, Kim HM, et al.: CD13 is a therapeutic target in human liver cancer stem cells. J Clin Invest 2010, 120(9):3326-39.
• [19]Muramatsu S, Tanaka S, Mogushi K, Adikrisna R, Aihara A, Ban D, et al.: Visualization of stem cell features in human hepatocellular carcinoma reveals in vivo significance of tumor-host interaction and clinical course. Hepatology 2013, 58(1):218-28.
• [20]Masters JR: Cell-line authentication: End the scandal of false cell lines. Nature 2012, 492(7428):186.
• [21]Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J: Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res 1982, 42(9):3858-63.
• [22]Hirohashi S, Shimosato Y, Kameya T, Koide T, Mukojima T, Taguchi Y, et al.: Production of alpha-fetoprotein and normal serum proteins by xenotransplanted human hepatomas in relation to their growth and morphology. Cancer Res 1979, 39(5):1819-28.
• [23]Alexander JJ, Bey EM, Geddes EW, Lecatsas G: Establishment of a continuously growing cell line from primary carcinoma of the liver. South African Med J 1976, 50(54):2124-8.
• [24]Dor I, Namba M, Sato J: Establishment and some biological characteristics of human hepatoma cell lines. Gan 1975, 66(4):385-92.
• [25]Sugiyama H, Onuki K, Ishige K, Baba N, Ueda T, Matsuda S, et al.: Potent in vitro and in vivo antitumor activity of sorafenib against human intrahepatic cholangiocarcinoma cells. J Gastroenterol 2011, 46(6):779-89.
• [26]Fujiwara D, Kato K, Nohara S, Iwanuma Y, Kajiyama Y: The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines. Biochem Biophys Res Commun 2013, 434(4):773-8.
• [27]Arao T, Ueshima K, Matsumoto K, Nagai T, Kimura H, Hagiwara S, et al.: FGF3/FGF4 amplification and multiple lung metastases in responders to sorafenib in hepatocellular carcinoma. Hepatology 2013, 57(4):1407-15.
• [28]Lopez-Terrada D, Cheung SW, Finegold MJ, Knowles BB: Hep G2 is a hepatoblastoma-derived cell line. Hum Pathol 2009, 40(10):1512-5.
• [29]Yin C, Lin Y, Zhang X, Chen YX, Zeng X, Yue HY, et al.: Differentiation therapy of hepatocellular carcinoma in mice with recombinant adenovirus carrying hepatocyte nuclear factor-4alpha gene. Hepatology 2008, 48(5):1528-39.
• [30]Wang F, He L, Dai WQ, Xu YP, Wu D, Lin CL, et al.: Salinomycin inhibits proliferation and induces apoptosis of human hepatocellular carcinoma cells in vitro and in vivo. PLoS One 2012, 7(12):e50638.
• [31]Meacham CE, Morrison SJ: Tumour heterogeneity and cancer cell plasticity. Nature 2013, 501(7467):328-37.
• [32]Borovski T, De Sousa EMF, Vermeulen L, Medema JP: Cancer stem cell niche: the place to be. Cancer Res 2011, 71(3):634-9.
• [33]Martin-Padura I, Marighetti P, Agliano A, Colombo F, Larzabal L, Redrado M, et al.: Residual dormant cancer stem-cell foci are responsible for tumor relapse after antiangiogenic metronomic therapy in hepatocellular carcinoma xenografts. Lab Invest. 2012, 92(7):952-66.
• [34]Muraro MG, Mele V, Daster S, Han J, Heberer M, Cesare Spagnoli G, et al.: CD133+, CD166 + CD44+, and CD24 + CD44+ phenotypes fail to reliably identify cell populations with cancer stem cell functional features in established human colorectal cancer cell lines. Cell Transl Med 2012, 1(8):592-603.
• [35]Singh AK, Arya RK, Maheshwari S, Singh A, Meena S, Pandey P, et al.: Tumor heterogeneity and cancer stem cell paradigm: Updates in concept, controversies and clinical relevance. Int J Cancer 2015, 136(9):1991-2000.
• [36]Magee JA, Piskounova E, Morrison SJ: Cancer stem cells: impact, heterogeneity, and uncertainty. Cancer Cell 2012, 21(3):283-96.
• [37]Nguyen LV, Vanner R, Dirks P, Eaves CJ: Cancer stem cells: an evolving concept. Nat Rev Cancer 2012, 12(2):133-43.
• [38]Xin HW, Ambe CM, Hari DM, Wiegand GW, Miller TC, Chen JQ, et al.: Label-retaining liver cancer cells are relatively resistant to sorafenib. Gut 2013, 62(12):1777-86.
• [39]Shan J, Shen J, Liu L, Xia F, Xu C, Duan G, et al.: Nanog regulates self-renewal of cancer stem cells through the insulin-like growth factor pathway in human hepatocellular carcinoma. Hepatology 2012, 56(3):1004-14.
• [40]Abdullah LN, Chow EK: Mechanisms of chemoresistance in cancer stem cells. Clin Transl Med. 2013, 2(1):3. BioMed Central Full Text
• [41]Chen X, Lingala S, Khoobyari S, Nolta J, Zern MA, Wu J: Epithelial mesenchymal transition and hedgehog signaling activation are associated with chemoresistance and invasion of hepatoma subpopulations. J Hepatol 2011, 55(4):838-45.
• [42]Gedaly R, Galuppo R, Musgrave Y, Angulo P, Hundley J, Shah M, et al.: PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation. J Surg Res 2013, 185(1):225-30.
• [43]Goel HL, Mercurio AM: VEGF targets the tumour cell. Nat Rev Cancer 2013, 13(12):871-82.
• [44]Katoh M, Katoh M: Cross-talk of WNT and FGF signaling pathways at GSK3beta to regulate beta-catenin and SNAIL signaling cascades. Cancer Biol Ther 2006, 5(9):1059-64.
• [45]Deng L, Ren Z, Jia Q, Wu W, Shen H, Wang Y: Schedule-dependent antitumor effects of 5-fluorouracil combined with sorafenib in hepatocellular carcinoma. BMC Cancer 2013, 13:363. BioMed Central Full Text
• [46]Wild AT, Gandhi N, Chettiar ST, Aziz K, Gajula RP, Williams RD, et al.: Concurrent versus sequential sorafenib therapy in combination with radiation for hepatocellular carcinoma. PLoS One 2013, 8(6):e65726.
• [47]Yamashita T, Forgues M, Wang W, Kim JW, Ye Q, Jia H, et al.: EpCAM and alpha-fetoprotein expression defines novel prognostic subtypes of hepatocellular carcinoma. Cancer Res 2008, 68(5):1451-61.
• [48]Fan ST, Yang ZF, Ho DW, Ng MN, Yu WC, Wong J: Prediction of posthepatectomy recurrence of hepatocellular carcinoma by circulating cancer stem cells: a prospective study. Ann Surg 2011, 254(4):569-76.
• [49]Liu S, Li N, Yu X, Xiao X, Cheng K, Hu J, et al.: Expression of intercellular adhesion molecule 1 by hepatocellular carcinoma stem cells and circulating tumor cells. Gastroenterology 2013, 144(5):1031-41. e1010