Abstract
Cancer stem cells (CSCs) have been proposed to underlie the initiation and maintenance of tumor growth and the development of chemoresistance in solid tumors. The identification and role of these important cells in pancreatic cancer remains controversial. Here, we isolate side population (SP) cells from the highly aggressive and metastatic human pancreatic cancer cell line L3.6pl and evaluate their potential role as models for CSCs. SP cells were isolated following Hoechst 33342 staining of L3.6pl cells. SP, non-SP, and unsorted L3.6pl cells were orthotopically xenografted into the pancreas of nude mice and tumor growth observed. RNA was analyzed by whole genome array and pathway mapping was performed. Drug resistant variants of L3.6pl were developed and examined for SP proportions and evaluated for surface expression of known CSC markers. A distinct SP with the ability to self-renew and differentiate into non-SP cells was isolated from L3.6pl (0.9 % ± 0.22). SP cells showed highly tumorigenic and metastatic characteristics after orthotopic injection. Transcriptomic analysis identified modulation of gene networks linked to tumorigenesis, differentiation, and metastasization in SP cells relative to non-SP cells. Wnt, NOTCH, and EGFR signaling pathways associated with tumor stem cells were altered in SP cells. When cultured with increasing concentrations of gemcitabine, the proportion of SP cells, ABCG2+, and CD24+ cells were significantly enriched, whereas 5-fluorouracil (5-FU) treatment lowered the percentage of SP cells. SP cells were distinct from cells positive for previously postulated pancreatic CSC markers. The Hoechst-induced side population in L3.6pl cells comprises a subset of tumor cells displaying aggressive growth and metastasization, increased gemcitabine-, but not 5-FU resistance. The cells may act as a partial model for CSC biology.
Similar content being viewed by others
References
Pardal R, Clarke MF, Morrison SJ (2003) Applying the principles of stem-cell biology to cancer. Nat Rev Cancer 3:895–902
Nowell PC (1976) The clonal evolution of tumor cell populations. Science 194:23–8
Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414:105–11
Donnenberg VS, Donnenberg AD (2005) Multiple drug resistance in cancer revisited: the cancer stem cell hypothesis. J Clin Pharmacol 45:872–7
Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB et al (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444:756–60
McDonald SA, Graham TA, Schier S, Wright NA, Alison MR (2009) Stem cells and solid cancers. Virchows Arch 455:1–13
Bonnet D, Dick JE (1997) Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3:730–7
Dick JE (2005) Acute myeloid leukemia stem cells. Ann N Y Acad Sci 1044:1–5
Bhagwandin VJ, Shay JW (2009) Pancreatic cancer stem cells: fact or fiction? Biochim Biophys Acta 1792:248–59
Sergeant G, Vankelecom H, Gremeaux L, Topal B (2009) Role of cancer stem cells in pancreatic ductal adenocarcinoma. Nat Rev Clin Oncol 6:580–6
Hermann PC, Bhaskar S, Cioffi M, Heeschen C (2010) Cancer stem cells in solid tumors. Semin Cancer Biol 20:77–84
Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V et al (2007) Identification of pancreatic cancer stem cells. Cancer Res 67:1030–7
Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M et al (2007) Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 1:313–23
Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC (1996) Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183:1797–806
Bruns CJ, Harbison MT, Kuniyasu H, Eue I, Fidler IJ (1999) In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice. Neoplasia 1:50–62
Krebs S, Fischaleck M, Blum H (2009) A simple and loss-free method to remove TRIzol contaminations from minute RNA samples. Anal Biochem 387:136–8
Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S et al (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5:R80
Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP (2003) Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res 31:e15
Smyth GK (2004) Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol;3:Article3
Moll AG, Lindenmeyer MT, Kretzler M, Nelson PJ, Zimmer R, Cohen CD (2009) Transcript-specific expression profiles derived from sequence-based analysis of standard microarrays. PLoS ONE 4:e4702
Notohamiprodjo S, Djafarzadeh R, Rieth N, Hofstetter M, Jaeckel C, Nelson PJ (2012) Cell surface engineering of renal cell carcinoma with glycosylphosphatidylinositol-anchored TIMP-1 blocks TGF- beta 1 activation and reduces regulatory ID gene expression. Biol Chem 393:1463–70
Ebert B, Kisiela M, Wsol V, Maser E (2011) Proteasome inhibitors MG-132 and bortezomib induce AKR1C1, AKR1C3, AKR1B1, and AKR1B10 in human colon cancer cell lines SW-480 and HT-29. Chem Biol Interact 191:239–49
Huntly BJ, Gilliland DG (2005) Cancer biology: summing up cancer stem cells. Nature 435:1169–70
Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J et al (1994) A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 367:645–8
Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C et al (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445:111–5
Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T et al (2004) Identification of human brain tumour initiating cells. Nature 432:396–401
Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100:3983–8
Challen GA, Little MH (2006) A side order of stem cells: the SP phenotype. Stem Cells 24:3–12
Wulf GG, Wang RY, Kuehnle I, Weidner D, Marini F, Brenner MK et al (2001) A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia. Blood 98:1166–73
Chiba T, Kita K, Zheng YW, Yokosuka O, Saisho H, Iwama A et al (2006) Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology 44:240–51
Dou J, Wen P, Hu W, Li Y, Wu Y, Liu C et al (2009) Identifying tumor stem-like cells in mouse melanoma cell lines by analyzing the characteristics of side population cells. Cell Biol Int 33:807–15
Harris MA, Yang H, Low BE, Mukherjee J, Guha A, Bronson RT et al (2008) Cancer stem cells are enriched in the side population cells in a mouse model of glioma. Cancer Res 68:10051–9
Zhao Y, Bao Q, Schwarz B, Zhao L, Mysliwietz J, Ellwart J, et al (2014) Stem cell like side populations in esophageal cancer: a source of chemotherapy resistance and metastases. Stem Cells Dev 23:180--92
Ho MM, Ng AV, Lam S, Hung JY (2007) Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res 67:4827–33
Haraguchi N, Utsunomiya T, Inoue H, Tanaka F, Mimori K, Barnard GF et al (2006) Characterization of a side population of cancer cells from human gastrointestinal system. Stem Cells 24:506–13
Kabashima A, Higuchi H, Takaishi H, Matsuzaki Y, Suzuki S, Izumiya M et al (2009) Side population of pancreatic cancer cells predominates in TGF-beta-mediated epithelial to mesenchymal transition and invasion. Int J Cancer 124:2771–9
Zhou J, Wang CY, Liu T, Wu B, Zhou F, Xiong JX et al (2008) Persistence of side population cells with high drug efflux capacity in pancreatic cancer. World J Gastroenterol 14:925–30
Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ et al (2001) The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7:1028–34
Doyle LA, Ross DD (2003) Multidrug resistance mediated by the breast cancer resistance protein BCRP (ABCG2). Oncogene 22:7340–58
Alt R, Wilhelm F, Pelz-Ackermann O, Egger D, Niederwieser D, Cross M (2009) ABCG2 expression is correlated neither to side population nor to hematopoietic progenitor function in human umbilical cord blood. Exp Hematol 37:294–301
Conroy T, Desseigne F, Ychou M, Bouche O, Guimbaud R, Becouarn Y et al (2011) FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med 364:1817–25
Shmelkov SV, Butler JM, Hooper AT, Hormigo A, Kushner J, Milde T et al (2008) CD133 expression is not restricted to stem cells, and both CD133+ and CD133− metastatic colon cancer cells initiate tumors. J Clin Invest 118:2111–20
Dittfeld C, Dietrich A, Peickert S, Hering S, Baumann M, Grade M et al (2010) CD133 expression is not selective for tumor-initiating or radioresistant cell populations in the CRC cell line HCT-116. Radiother Oncol 94:375–83
Barski OA, Tipparaju SM, Bhatnagar A (2008) The aldo-keto reductase superfamily and its role in drug metabolism and detoxification. Drug Metab Rev 40:553–624
Diez-Dacal B, Gayarre J, Gharbi S, Timms JF, Coderch C, Gago F, et al (2011) Identification of Aldo-keto reductase AKR1B10 as a selective target for modification and inhibition by prostaglandin A1: implications for anti-tumoral activity. Cancer Res 71:4161--71
Fukumoto S, Yamauchi N, Moriguchi H, Hippo Y, Watanabe A, Shibahara J et al (2005) Overexpression of the aldo-keto reductase family protein AKR1B10 is highly correlated with smokers’ non-small cell lung carcinomas. Clin Cancer Res 11:1776–85
Yan R, Zu X, Ma J, Liu Z, Adeyanju M, Cao D (2007) Aldo-keto reductase family 1 B10 gene silencing results in growth inhibition of colorectal cancer cells: implication for cancer intervention. Int J Cancer 121:2301–6
Sasajima Y, Tanaka H, Miyake S, Yuasa Y (2005) A novel EID family member, EID-3, inhibits differentiation and forms a homodimer or heterodimer with EID-2. Biochem Biophys Res Commun 333:969–75
Koopmann J, Buckhaults P, Brown DA, Zahurak ML, Sato N, Fukushima N et al (2004) Serum macrophage inhibitory cytokine 1 as a marker of pancreatic and other periampullary cancers. Clin Cancer Res 10:2386–92
Zimmerman AL, Wu S (2011) MicroRNAs, cancer and cancer stem cells. Cancer Lett 300:10–9
Zhu S, Wu H, Wu F, Nie D, Sheng S, Mo YY (2008) MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res 18:350–9
Miller TE, Ghoshal K, Ramaswamy B, Roy S, Datta J, Shapiro CL et al (2008) MicroRNA-221/222 confers tamoxifen resistance in breast cancer by targeting p27Kip1. J Biol Chem 283:29897–903
Park JK, Lee EJ, Esau C, Schmittgen TD (2009) Antisense inhibition of microRNA-21 or -221 arrests cell cycle, induces apoptosis, and sensitizes the effects of gemcitabine in pancreatic adenocarcinoma. Pancreas 38:e190–9
Hu T, Li C (2010) Convergence between Wnt-beta-catenin and EGFR signaling in cancer. Mol Cancer 9:236
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Additional information
This research was supported by the FöFoLe Research Program (no. 570/548/636) of the University of Munich, Munich, Germany and the SPP1190/2 “Tumor vessel interface” (BR 1614/8-2) of the German Research Society (DFG).
All animal experiments were performed in accordance with institutional and governmental guidelines and approval obtained from the ethics commission of the State of Bavaria (no. 55.2-1-54-2531-19-08).
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 696 kb)
Rights and permissions
About this article
Cite this article
Niess, H., Camaj, P., Renner, A. et al. Side population cells of pancreatic cancer show characteristics of cancer stem cells responsible for resistance and metastasis. Targ Oncol 10, 215–227 (2015). https://doi.org/10.1007/s11523-014-0323-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11523-014-0323-z