Cancer stem cells in human gastrointestinal cancers

Human Cell 2006; 19: 24–29 doi: 10.1111/j.1749-0774.2005.00004.x

© 2006 The Authors24 Journal compilation © 2006 Japan Human Cell Society

Blackwell Publishing Ltd

FEATURE: GENE DIAGNOSIS – TREATMENT OF CARCINOMA

Cancer stem cells in human gastrointestinal cancers

Naotsugu HARAGUCHI, Hiroshi INOUE, Fumiaki TANAKA, Koshi MIMORI, Tohru UTSUNOMIYA, Atsushi SASAKI and Masaki MORIDepartment of Surgery, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu 874-0838, Japan

Abstract

The concept of cancer stem cell has developed in leukemia. Recently, it has expanded to includesolid tumors such as brain or breast tumors. However, the descriptions are not recognized inhuman gastrointestinal cancers. We used flow cytometry and the DNA-binding dye (Hoechst33342) to isolate side population (SP) cells from various human gastrointestinal system cancercell lines. The SP cell fraction is considered to contain abundant stem cells. Fifteen of 16 cancercell lines from the gastrointestinal system contained 0.3–2.2% SP cells. We studied the char-acteristics of the SP cells in hepatic or colon cancer cell lines. The results demonstrated that cancersof the gastrointestinal system do contain SP cells that show some characters of so-called stemcells. In this paper, we report our study results with a review of the literature.

Key words: chemoresistance, flow cytometry, microarray, side population, stem cell.

INTRODUCTIONThe cancer stem cell (CSC) hypothesis suggests thatneoplastic clones are maintained exclusively by a smallsubpopulation of cells that give rise to phenotypicallydiverse cancer cells.1–3 To date, the possible existence ofCSCs has been demonstrated in leukemia and some solidtumors,4–6 including breast cancer and brain tumors.7–12

Cancers of the gastrointestinal system are a leadingcause of death, and are much more prevalent worldwidecompared with breast cancer and brain tumors. In Japan,cancers of gastrointestinal system sites, including theesophagus, stomach, colorectum, liver and pancreas arelisted among the top 10 causes of cancer death. Althoughpatients with gastrointestinal cancers are numerous, the

existence of CSC in these cancers has not been clarified.Clinical experience suggests that most tumors mayregrow even if the original tumors have been eliminatedeffectively with chemotherapy or radiation. This may bedue to the existence of CSCs that show strong resistanceto therapy. So to make significant progress in cancer treat-ment, especially in cancers of the gastrointestinal system,it is necessary to characterize CSC.

In this paper we review the literature and report ourexperiences in identifying cancer side population (SP)cells in 15 of 16 human gastrointestinal system cancer celllines. Moreover, we evaluate the capacity for self-renewal,multilineage potential and resistance to chemotherapeuticagents in the SP cells of representative hepatoma andcolorectal cell lines, HuH7 and SW480.

IDENTIFICATION OF SIDE POPULATION CELLS

Stem cells identified from solid tumors usually expressorgan-specific markers. In the case of breast cancers,a cell surface marker profile CD44+CD24–/low Lin– wasreported.7 The cells isolated from central nervous sys-tem tumors express cell surface antigen CD133, which

Correspondence: Dr Masaki Mori, Department of Surgery, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu 874-0838, Japan. Email: [email protected]

This report was presented at the 23rd Annual Meeting of theJapan Human Cell Society held on 25–27 August 2005 inTsukuba, Japan.

Received 4 November 2005; accepted 14 November 2005.

Cancer stem cells in gastrointestinal cancers

© 2006 The AuthorsJournal compilation © 2006 Japan Human Cell Society 25

is known as a marker of hematopoietic stem cells.12

However, distinct regulated molecules characterizingCSCs are largely unknown. The drug transport propertyconferred by the ABC transporter is an important markerin the isolation and analysis in hematopoietic stem cellsand termed “side population” (SP) cells. The SP fractionis a useful tool for stem cell studies, especially whenspecific cell surface markers are unknown.

Human gastrointestinal system cancer cell linesincluded esophageal (TE1, TE2 and TE13), gastric(NUGC3, MKN1, MKN7 and MKN28), colorectal (WiDr,SW480, HSC15 and CCK81), pancreas (PK9 andPK45H) and liver (HuH7, Hep3B and HepG2) cell lines.The culture condition was described elsewhere. To deter-mine whether a selection of cancer cell lines contain SPcells, we stained them with Hoechst 33342,13 which isactively extruded by verapamil-sensitive ABC transporters.

Representative results analyzed by flow cytometry areshown (Fig. 1). Cancer cell lines from the esophagus con-tained 0.3–1.4% SP cells; stomach 0.6–2.2% SP cells;colorectal 0.3–0.5% SP cells; liver 0–1.8% SP cells; andpancreas 0.3–1.3% SP cells. In each case, by definition,the number of SP cells was decreased greatly by treatmentwith verapamil (Fig. 1). We could not detect SP cells inonly a single liver cancer cell line HepG2. Thus, most ofthe gastrointestinal system cancer cell lines do contain asmall component of cancer SP cells.

IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES BETWEEN SP AND NON-SP CELLSUsing a Human Whole Genome Oligo Microarray of41 000 clones, we tried to identify differentially expressed

Figure 1 Analysis of side population (SP) cells in gastrointestinal cancers. Representative SP cell isolation from esophageal, gastricand colon cancer cell lines. Cell lines of esophageal (TE13), gastric (MKN28) and colon (SW480) were labeled with Hoechst 33342and analyzed by flow cytometry. Results when cells are treated with 50 µM verapamil during the labeling procedure. The SP cells,which disappear in the presence of verapamil (lower panel), are outlined and shown as a percentage of the total cell population.

N. Haraguchi et al.


genes between SP and non-SP lineages of HuH7, and theresults are summarized in Table 1. The top up-regulatedelement was CEACAM6, a glycosylphosphatidylinositol-linked immunoglobulin super-family member that isover-expressed in a variety of gastrointestinal cancers.Over-expression of CEACAM6 promotes cell survivalunder anchorage-independent conditions, and protectscells from apoptosis.14 Furthermore, CEACAM6 over-expression may serve as a determinant of gemcitabinechemoresistance.15 The expression of AREG, which hasrecently been associated with resistance to gefitinib,16

was also up-regulated. In addition, the expression of ABCtransporters, such as BCRP1 and multidrug resistance1(MDR1) were up-regulated in the HuH7 SP cells. Genesassociated with a “transporter,” including ABC trans-porters, were frequently up-regulated. The expression of

GATA6 was increased in HuH7 SP cells. This GATA6 isreported to be associated with embryonic developmentand hepatocytic differentiation,17–19 and is thought tointeract with BMP pathways.20 Several elements, such aspim-1, that have been linked to the Akt/PI3kinasepathway were also up-regulated and the findingwould be interesting from the view-point of the HuH7propagation.21 We noted increased expression of“structural proteins,” such as VIM, KRT14 and KRT19.Over expression of VIM plays an important role inhuman liver cancer metastasis.22 In addition, human liverprogenitor cells express VIM,23 KRT14 and KRT19.23,24

Therefore, these molecules could be distinctive markersfor liver cancer SP cells. Down-regulated genes in SP cellscould equally be very important but are not furtherdiscussed here.

Table 1 Results of our attempt to identify differentially expressed genes between SP and non-SP lineages of HuH7 using a HumanWhole Genome Oligo Microarray of 41 000 clones

Gene name Systematic name Description

Up-regulated genesCEACAM6 NM_002483 Homo sapiens carcinoembryonic antigen-related cell adhesion

molecule 6 (CEACAM6), mRNAACE2 NM_021804 Homo sapiens angiotensin I converting enzyme

(peptidyl-dipeptidase A) 2 (ACE2), mRNAAREG NM_001657 Homo sapiens amphiregulin (schwannoma-derived growth factor)

(AREG), mRNAPIGR NM_002644 Homo sapiens polymeric immunoglobulin receptor (PIGR), mRNASLC3A1 NM_000341 Homo sapiens solute carrier family 3, member 1 (SLC3A1), mRNAPLAC8 NM_016619 Homo sapiens placenta-specific 8 (PLAC8), mRNAHEPH NM_014799 Homo sapiens hephaestin (HEPH), transcript variant 2, mRNALGALS2 NM_006498 Homo sapiens lectin, galactoside-binding, soluble, 2 (galectin 2)

(LGALS2), mRNAAK074229 AK074229 Homo sapiens cDNA FLJ23649 fis, clone COL06395, highly

similar to Oryctolagus cuniculus lacrimal lipase mRNA PLAC8 NM_016619 Homo sapiens placenta-specific 8 (PLAC8), mRNA

Down-regulated genesAHSG NM_001622 Homo sapiens alpha-2-HS-glycoprotein (AHSG), mRNAFLJ10156 NM_019013 Homo sapiens hypothetical protein FLJ10156 (FLJ10156), mRNAENST00000308206 ENST00000308206 UnknownAK021738 AK021738 Homo sapiens cDNA FLJ11676 fis, clone HEMBA1004752, highly

similar to Homo sapiens mRNA for LAK-4pI_958237 I_958237 Lysyl oxidase, copper-dependent enzyme that oxidizes lysine residues

to alpha-aminoadipic-delta-semialdehyde to initiate cross-linking of collagens and elastin, regulates HRAS, deficiency is associated with cutis laxa and over expression with fibrosis

H2BFS NM_017445 Homo sapiens H2B histone family, member S (H2BFS), mRNAPUS1 NM_025215 Homo sapiens pseudouridylate synthase 1 (PUS1), mRNAENST00000330311 ENST00000330311 UnknownFLJ32009 NM_152718 Homo sapiens hypothetical protein FLJ32009 (FLJ32009), mRNAHIST1H2BB NM_021062 Homo sapiens histone 1, H2bb (HIST1H2BB), mRNA



CHARACTERIZATION OF SIDE POPULATION CELLS

Self-renewal ability of side population cells

To examine the cellular characteristics of HuH7 SP cells,we examined which factors maintained HuH7 SP cells.We determined that culture medium supplemented withleukocyte inhibitory factor could effectively expandthe HuH7 SP cells. To compare the self-renewal abilityof SP cells, we maintained them in this supplementedmedium for 2 weeks, and then again sorted by flowcytometry. We found that the cultures initiated with SPcells contained both SP and non-SP cells, whereas thosewith non-SP cells generated only non-SP cells (Fig. 2).The percentage of SP cells was increased when analyzed

after 2 weeks culture of the SP cells compared to that ofthe pre-isolated HuH7 cell line. In addition, when thesorting and 2-week culture was repeated four times, thepercentage of SP cells was greatly increased comparedwith that of the 1st sort and 2-week culture. Comparableresults were obtained for colorectal cancer cell line SW480.

Stem cells show properties of self-renewal and pluri-potential differentiation. The cancer stem cell sharesmany properties with normal stem cells. In centralnervous system tumors, stem cells with a capacity forself-renewal and pluripotential development have beenisolated.12 Our study demonstrated that, at least in HuH7and SW480, a tumor hierarchy exists in which SP cellscan generate both SP and non-SP cells; this is in accord-ance with previous observations that the SP fractioncan divide asymmetrically and display a capacity forself-renewal.8,9,12

Figure 2 Side population (SP) cells display a capacity for self-renewal. Self-renewal capacity of HuH7 SP cells. The HuH7 cell linecontains 0.9% SP cells (left). After the isolation of both the HuH7 SP and non-SP fractions we cultured them in media supplementedwith LIF for 2 weeks and then they were re-analyzed by flow cytometry. In these culture conditions, the SP cells expanded sufficientlyand maintained the SP subpopulation (center) whereas no SP cells were found in the fraction initiated from non-SP cells (right).

N. Haraguchi et al.


Bipotential differentiation of side population cellsWe confirmed the expression of ALBU, KRT19, KRT14 andCD133 immunocytochemically in both SP and non-SPcells of HuH7. Corresponding to the observed increasedgene-expression in the microarray analysis, staining forKRT19 (cholangiocyte marker) and KRT14 (thought to bea liver stem cell marker) were strongly positive in SP cellsand negative in non-SP cells. Conversely, ALBU (maturehepatocyte marker) expression was weak in SP cells andstrong in non-SP cells. The staining for CD133 (hemato-poietic and neural stem cell marker) was strongly positivein SP cells and negative in non-SP cells. In addition, SPcells were morphologically quite distinguishable (smalland round in shape) from non-SP cells (fibroblast-like).

Chemoresistance of SP cells

Various types of ATP-binding cassette (ABC) transportershave been shown to contribute to drug resistance in manycancers by pumping drugs out of cells. Interestingly,some ABC transporters, such as breast cancer-resistanceprotein1 (BCRP1), are expressed by many kinds of stemcells,4,9,25 and this was confirmed in our study ofhepatoma or colon cancer as mentioned above. HuH7 SPcells express families of ABC transporters such as MDR1and BCRP1. We examined the sensitivity of HuH7 SP andnon-SP cells to Doxorubicin, which is well known asan ABC transporter dependent anticancer drug.25 Theviability of the SP cells was markedly higher comparedto that of the non-SP cells. HuH7 SP cells also expresshigh levels of CEACAM6 and AREG. The expression ofCEACAM6 is associated with resistance to gemcitabineand AREG to gefitinib.15,16 To determine, whether SPcells resist ABC transporter independent anticancer drugsmore than do non-SP cells, we tested 5-FU and gemci-tabine. In the case of 5-FU treatment, the viability of SPcells was again significantly higher than that of non-SPcells, and similarly for Gemcitabine. Even a small popu-lation of CSCs is potentially important because they maybe responsible for recurrence after cancer treatments,such as chemotherapy, even when most of the cancer cellsappear to be killed.

CONCLUSION

It is generally accepted that normal stem cells showproperties that provide for a long lifespan such as relativequiescence, resistance to drugs through the expression ofABC transporters and anti-apoptosis. The cancer SP cells

may also be relatively quiescent compared with non-SPcells, while still playing important roles to maintaincancer foci, that is to differentiate and proliferate afterchemotherapy and radiotherapy using their self-renewaland chemo-resistance capacities. As the specific markersof normal stem cells have not been clarified, the char-acteristics of normal stem cells have not been fully studied.To understand the characteristics of CSCs, it is necessaryto clarify the normal stem cells. This study is ongoing inour laboratory. If specific surface markers of normal stemcells and/or CSC are discovered, it may permit isolationof this important population of cells, and cancer therapyand science will be revolutionized.

ACKNOWLEDGMENTS

This study was supported by CREST of Japan, Scienceand Technology Agency; a Grant-in-Aid for ScientificResearch (S) (17109013) from the Japan Society for thePromotion of Science; a Grant-in-Aid for ScientificResearch on Priority Areas (17015032) from the Ministryof Education, Culture, Sports, Science and Technology ofJapan; a Grant for The Third Term Comprehensive Ten-year Strategy for Cancer Control, and a Grant for CancerResearch from the Ministry of Health, Labor and Welfareof Japan.

We thank Miss T. Shimooka, Miss H. Yasunami, MissM. Oda and Miss M. Kasagi for their excellent technicalassistance.

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Cancer stem cells in human gastrointestinal cancers