Cytotoxic T Lymphocytes Efficiently Recognize Human Colon Cancer Stem-Like Cells
2011; Elsevier BV; Volume: 178; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2011.01.004
ISSN1525-2191
AutoresSatoko Inoda, Yoshihiko Hirohashi, Toshihiko Torigoe, Rena Morita, Akari Takahashi, Hiroko Asanuma, Munehide Nakatsugawa, Satoshi Nishizawa, Yasuaki Tamura, Tetsuhiro Tsuruma, Takeshi Terui, Tôru Kondo, Kunihiko Ishitani, Tadashi Hasegawa, Koichi Hirata, Noriyuki Sato,
Tópico(s)Cancer Research and Treatments
ResumoCancer stem-like cells (CSCs) and tumor-initiating cells (TICs) are a small population of cancer cells that share three properties: tumor initiating ability, self-renewal, and differentiation. These properties suggest that CSCs/TICs are essential for tumor maintenance, recurrence, and distant metastasis. Here, we show that cytotoxic T lymphocytes (CTLs) specific for the tumor-associated antigen CEP55 can efficiently recognize colon CSCs/TICs both in vitro and in vivo. Using Hoechst 33342 dye staining, we isolated CSCs/TICs as side population (SP) cells from colon cancer cell lines SW480, HT29, and HCT15. The SP cells expressed high levels of the stem cell markers SOX2, POU5F1, LGR5, and ALDH1A1 and showed resistance to chemotherapeutic agents such as irinotecan or etoposide.To evaluate the susceptibility of SP cells to CTLs, we used CTL clone 41, which is specific for the CEP55-derived antigenic peptide Cep55/c10orf3_193 (10) (VYVKGLLAKI). The SP cells expressed HLA class I and CEP55 at the same level as the main population cells. The SP cells were susceptible to CTL clone 41 at the same level as main population cells. Furthermore, adoptive transfer of CTL clone 41 inhibited tumor growth of SW480 SP cells in vivo. These observations suggest that Cep55/c10orf3_193(10) peptide-based cancer vaccine therapy or adoptive cell transfer of the CTL clone is a possible approach for targeting chemotherapy-resistant colon CSCs/TICs. Cancer stem-like cells (CSCs) and tumor-initiating cells (TICs) are a small population of cancer cells that share three properties: tumor initiating ability, self-renewal, and differentiation. These properties suggest that CSCs/TICs are essential for tumor maintenance, recurrence, and distant metastasis. Here, we show that cytotoxic T lymphocytes (CTLs) specific for the tumor-associated antigen CEP55 can efficiently recognize colon CSCs/TICs both in vitro and in vivo. Using Hoechst 33342 dye staining, we isolated CSCs/TICs as side population (SP) cells from colon cancer cell lines SW480, HT29, and HCT15. The SP cells expressed high levels of the stem cell markers SOX2, POU5F1, LGR5, and ALDH1A1 and showed resistance to chemotherapeutic agents such as irinotecan or etoposide.To evaluate the susceptibility of SP cells to CTLs, we used CTL clone 41, which is specific for the CEP55-derived antigenic peptide Cep55/c10orf3_193 (10) (VYVKGLLAKI). The SP cells expressed HLA class I and CEP55 at the same level as the main population cells. The SP cells were susceptible to CTL clone 41 at the same level as main population cells. Furthermore, adoptive transfer of CTL clone 41 inhibited tumor growth of SW480 SP cells in vivo. These observations suggest that Cep55/c10orf3_193(10) peptide-based cancer vaccine therapy or adoptive cell transfer of the CTL clone is a possible approach for targeting chemotherapy-resistant colon CSCs/TICs. Colon cancer is one of the most common malignancies worldwide. With recent progress in treatment, the prognosis has improved to some extent. In advanced disease, however, the prognosis remains unfavorable, because of recurrence, distant metastasis, and resistance to treatment. Thus, novel treatment modalities are needed. Cancers contain morphologically heterogeneous populations. This fact has led to the cancer stem cell theory,1Papailiou J. Bramis K.J. Gazouli M. Theodoropoulos G. Stem cells in colon cancer A new era in cancer theory begins.Int J Colorectal Dis. 2011; 26: 1-11Crossref PubMed Scopus (51) Google Scholar the idea that cancers are composed of several types of cells, and that only a small population of cancer cells that can regenerate cancer tissues, much as normal tissue can be regenerated only by a small population of stem-like cells. Recently, cancer stem-like cells and tumor-initiating cells (CSCs/TICs) have been isolated from various types of malignancies, including colon cancer.2Clarke M.F. Dick J.E. Dirks P.B. Eaves C.J. Jamieson C.H. Jones D.L. Visvader J. Weissman I.L. Wahl G.M. Cancer stem cells—perspectives on current status and future directions: AACR workshop on cancer stem cells.Cancer Res. 2006; 66: 9339-9344Crossref PubMed Scopus (2503) Google Scholar, 3Dalerba P. Dylla S.J. Park I.K. Liu R. Wang X. Cho R.W. Hoey T. Gurney A. Huang E.H. Simeone D.M. Shelton A.A. Parmiani G. Castelli C. Clarke M.F. Phenotypic characterization of human colorectal cancer stem cells.Proc Natl Acad Sci USA. 2007; 104: 10158-10163Crossref PubMed Scopus (1767) Google Scholar, 4O'Brien C.A. Pollett A. Gallinger S. Dick J.E. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice.Nature. 2007; 445: 106-110Crossref PubMed Scopus (3453) Google Scholar, 5Ricci-Vitiani L. Lombardi D.G. Pilozzi E. Biffoni M. Todaro M. Peschle C. De Maria R. Identification and expansion of human colon-cancer-initiating cells.Nature. 2007; 445: 111-115Crossref PubMed Scopus (3400) Google Scholar, 6Huang E.H. Hynes M.J. Zhang T. Ginestier C. Dontu G. Appelman H. Fields J.Z. Wicha M.S. Boman B.M. Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis.Cancer Res. 2009; 69: 3382-3389Crossref PubMed Scopus (841) Google Scholar In colon cancer, CSCs/TICs can reinitiate tumors that resemble mother colon cancer tissues morphologically when transplanted into immunodeficient mice.3Dalerba P. Dylla S.J. Park I.K. Liu R. Wang X. Cho R.W. Hoey T. Gurney A. Huang E.H. Simeone D.M. Shelton A.A. Parmiani G. Castelli C. Clarke M.F. Phenotypic characterization of human colorectal cancer stem cells.Proc Natl Acad Sci USA. 2007; 104: 10158-10163Crossref PubMed Scopus (1767) Google Scholar Furthermore, these CSCs/TICs have higher tumorigenic potential than do non-CSCs/TICs. Previous reports have shown that CSCs/TICs are resistant to a variety of treatments, including chemotherapy and radiotherapy, with varied mechanisms of resistance, including high expression of drug transporters, relative cell cycle quiescence, high levels of DNA repair machinery, and resistance to apoptosis.7Dean M. Fojo T. Bates S. Tumour stem cells and drug resistance.Nat Rev Cancer. 2005; 5: 275-284Crossref PubMed Scopus (3066) Google Scholar These reports3Dalerba P. Dylla S.J. Park I.K. Liu R. Wang X. Cho R.W. Hoey T. Gurney A. Huang E.H. Simeone D.M. Shelton A.A. Parmiani G. Castelli C. Clarke M.F. Phenotypic characterization of human colorectal cancer stem cells.Proc Natl Acad Sci USA. 2007; 104: 10158-10163Crossref PubMed Scopus (1767) Google Scholar, 4O'Brien C.A. Pollett A. Gallinger S. Dick J.E. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice.Nature. 2007; 445: 106-110Crossref PubMed Scopus (3453) Google Scholar, 5Ricci-Vitiani L. Lombardi D.G. Pilozzi E. Biffoni M. Todaro M. Peschle C. De Maria R. Identification and expansion of human colon-cancer-initiating cells.Nature. 2007; 445: 111-115Crossref PubMed Scopus (3400) Google Scholar, 6Huang E.H. Hynes M.J. Zhang T. Ginestier C. Dontu G. Appelman H. Fields J.Z. Wicha M.S. Boman B.M. Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis.Cancer Res. 2009; 69: 3382-3389Crossref PubMed Scopus (841) Google Scholar support the hypothesis that malignant cancers comprise heterogeneous populations that organize in a hierarchical differentiation model. The CSCs/TICs are located at the top of this hierarchy, and targeting CSCs/TICs is essential to achieve efficient effects for treatment of malignant diseases. Recently, some trials targeting CSCs/TICs have been reported for hematopoietic malignancies.8Low J.A. de Sauvage F.J. Clinical experience with Hedgehog pathway inhibitors.J Clin Oncol. 2010; 28: 5321-5326Crossref PubMed Scopus (168) Google Scholar Hedgehog signaling is essential for maintenance of myeloid leukemia stem cells, and inhibition of hedgehog signaling by cyclopamine is effective for imatinib-resistant myeloid leukemia.9Zhao C. Chen A. Jamieson C.H. Fereshteh M. Abrahamsson A. Blum J. Kwon H.Y. Kim J. Chute J.P. Rizzieri D. Munchhof M. VanArsdale T. Beachy P.A. Reya T. Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia.Nature. 2009; 458 ([Erratum appeared in Nature 2009, 460:652]): 776-779Crossref PubMed Scopus (748) Google Scholar To date, however, no such CSC/TIC targeting approach has been reported for colon cancer. In the present study, we evaluated the efficiency of CTL-based immunotherapy targeting colon CSCs/TICs. Using Hoechst 33342 dye, we isolated colon CSCs/TICs as side population (SP) cells from six colon cancer cell lines. The SP cells derived from SW480, HT29, and HCT15 showed higher tumorigenicity than did main population (MP) cells. On the other hand, SP cells from KM12LM, Lovo, and Colo320 did not show any increase in tumorigenicity, compared with MP cells. This suggests that SW480, HT29, and HCT15 SP cells (but not KM12LM, Lovo, and Colo320 SP cells) were enriched with CSCs/TICs. In RT-PCR analysis the SW480, HT29, and HCT15 SP cells showed a stem cell-like gene expression signature, including SOX2, POU5F1, LGR5, and ALDH1A1. Furthermore, these SP cells also showed resistance to chemotherapeutic agents, including irinotecan and etoposide. These observations support the idea that these SP cells had stem cell-like features. To assess the immunogenicity of SP cells, we evaluated the expression of HLA class I and of CEP55, which is a tumor-rejection antigen of breast and colon cancer.10Inoda S. Hirohashi Y. Torigoe T. Nakatsugawa M. Kiriyama K. Nakazawa E. Harada K. Takasu H. Tamura Y. Kamiguchi K. Asanuma H. Tsuruma T. Terui T. Ishitani K. Ohmura T. Wang Q. Greene M.I. Hasegawa T. Hirata K. Sato N. Cep55/c10orf3, a tumor antigen derived from a centrosome residing protein in breast carcinoma.J Immunother. 2009; 32: 474-485Crossref PubMed Scopus (78) Google Scholar, 11Inoda S. Morita R. Hirohashi Y. Torigoe T. Asanuma H. Nakazawa E. Nakatsugawa M. Tamura Y. Kamiguchi K. Tsuruma T. Terui T. Ishitani K. Hashino S. Wang Q. Greene M.I. Hasegawa T. Hirata K. Asaka M. Sato N. The feasibility of Cep55/c10orf3 derived peptide vaccine therapy for colorectal carcinoma.Exp Mol Pathol. 2011; 90: 55-60Crossref PubMed Scopus (38) Google Scholar The SP cells expressed HLA class I (and also HLA-A24) at the same level as MP cells. The SP cells also expressed CEP55 messenger RNA (mRNA) at the same level as MP cells in RT-PCR. To confirm the susceptibility of SP cells to cytotoxic T lymphocytes (CTLs), we used CTL clone 41, which recognizes CEP55 in an HLA-A24-restricted manner.10Inoda S. Hirohashi Y. Torigoe T. Nakatsugawa M. Kiriyama K. Nakazawa E. Harada K. Takasu H. Tamura Y. Kamiguchi K. Asanuma H. Tsuruma T. Terui T. Ishitani K. Ohmura T. Wang Q. Greene M.I. Hasegawa T. Hirata K. Sato N. Cep55/c10orf3, a tumor antigen derived from a centrosome residing protein in breast carcinoma.J Immunother. 2009; 32: 474-485Crossref PubMed Scopus (78) Google Scholar CTL clone 41 killed SW480, HT29, and HCT15 SP cells at the same level as it killed MP cells and presorted cells. These observations suggest that colon CSCs/TICs are also sensitive to CTLs, as non-CSC/TIC populations are. Furthermore, adoptive transfer of CTL clone 41 inhibited the tumor growth of SW480 SP cells in immunodeficient mice. These observations suggest that CTL-based colon cancer immunotherapy is efficient for colon CSCs/TICs. To our knowledge, the present study provides the first direct evidence that colon CSCs/TICs are susceptible to CTLs and thus opens possibilities for future applications in immunotherapy using CSC/TIC-specific vaccines. Colon adenocarcinoma cell lines SW480 (HLA-A*0201/2402), HCT15 (HLA-A*0201/2402), HT29 (HLA-A1/24), Lovo, and Colo320 were kind gifts of Dr. K. Imai (Sapporo, Japan), and KM12LM was a kind gift of Dr. K. Itoh (Kurume, Japan). All cell lines except K562 were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). K562 was cultured in RPMI-1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum. HCT15-B2M, a stable transfectant of HCT15 cells with B2M (β2 microglobulin) cDNA, was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 10 μg/mL puromycin (Sigma-Aldrich).11Inoda S. Morita R. Hirohashi Y. Torigoe T. Asanuma H. Nakazawa E. Nakatsugawa M. Tamura Y. Kamiguchi K. Tsuruma T. Terui T. Ishitani K. Hashino S. Wang Q. Greene M.I. Hasegawa T. Hirata K. Asaka M. Sato N. The feasibility of Cep55/c10orf3 derived peptide vaccine therapy for colorectal carcinoma.Exp Mol Pathol. 2011; 90: 55-60Crossref PubMed Scopus (38) Google Scholar Side population analysis was performed as described previously, with some modifications.12Goodell M.A. Brose K. Paradis G. Conner A.S. Mulligan R.C. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.J Exp Med. 1996; 183: 1797-1806Crossref PubMed Scopus (2484) Google Scholar Trypsinized cultured cells were washed with PBS and were resuspended at 37°C in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. After 10 minutes preincubation, the cells were labeled with Hoechst 33342 dye (Lonza, Walkersville, MD) for 90 minutes at concentrations of 3.75 μg/mL for Colo320, 5 μg/mL for SW480 and Lovo, 7.5 μg/mL for HT29 and KM12LM, and 10 μg/mL for HCT15, with or without verapamil (Sigma-Aldrich), which is an inhibitor of ABC transporters, at concentrations of 50 μmol/L for SW480, HCT15, and Colo320, 75 μmol/L for Lovo, and 100 μmol/L for HT29. Cells were counterstained with 1 μg/mL propidium iodide to label dead cells. Next, 1 × 106 viable cells were analyzed and sorted using a BD FACSAria II fluorescence-activated cell sorting system (BD Biosciences, Franklin Lakes, NJ). The Hoechst dye was excited at 355 nm, and its fluorescence was measured at two wavelengths using optical filters 405 DF20 [450/20 nm band-pass filter O (Hoechst Blue)] and 635LP [635 nm long-pass edge filter (Hoechst Red)]. Propidium iodide labeling was measured through a 630/BP30 filter for discrimination of dead cells. The SP cells, MP cells, and presorted cells from colon cancer cell lines were mixed 1:1 by volume with Matrigel (BD Biosciences) and were injected subcutaneously into the backs of female 4- to 8-week-old nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Tumor size in cubic millimeters was assessed weekly with calipers and was calculated as Tumor Size = (Longest Diameter × Shortest Diameter2)/2. RT-PCR analysis was performed as described previously.10Inoda S. Hirohashi Y. Torigoe T. Nakatsugawa M. Kiriyama K. Nakazawa E. Harada K. Takasu H. Tamura Y. Kamiguchi K. Asanuma H. Tsuruma T. Terui T. Ishitani K. Ohmura T. Wang Q. Greene M.I. Hasegawa T. Hirata K. Sato N. Cep55/c10orf3, a tumor antigen derived from a centrosome residing protein in breast carcinoma.J Immunother. 2009; 32: 474-485Crossref PubMed Scopus (78) Google Scholar Total RNAs were isolated from both SP cells and MP cells using an RNeasy mini kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from 2 μg of total RNA by reverse transcription using SuperScript III reverse transcriptase (Invitrogen). The PCR amplification was performed in 20 μL of PCR mixture containing 1 μL of cDNA mixture, 0.5 μL of Taq DNA polymerase (Qiagen) and 4 pmol of primers. The PCR mixture was initially incubated at 98°C for 2 minutes, followed by 30 cycles of denaturation at 98°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The following primer pairs were used for RT-PCR analysis (forward and reverse, respectively): 5′-CATGATGGAGACGGAGCTGA-3′ and 5′-ACCCCGCTCGCCATGCTATT-3′ for SOX2, with an expected PCR product size of 410 bp; 5′-TGGAGAAGGAGAAGCTGGAGCAAAA-3′ and 5′-GGCAGATGGTCGTTTGGCTGAATA-3′ for POU5F1, with an expected PCR product size of 163 bp; 5′-CTCTTCCTCAAACCGTCTGC-3′ and 5′-GATCGGAGGCTAAGCAACTG-3′ for LGR5, with an expected PCR product size of 181 bp; 5′-TGTTAGCTGATGCCGACTTG-3′ and 5′-TTCTTAGCCCGCTCAACACT-3′ for ALDH1A1, with an expected PCR product size of 154 bp; 5′-TGAGTTTGCCATCACAGAGC-3′ and 5′-TTGCTTGCTGGTGCATTAAC-3′ for CEP55, with an expected PCR product size of 521 bp; and 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), with an expected product size of 452 bp. GAPDH was used as an internal control. Quantitative real-time PCR was performed using an ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. Primers and probes were designed by the manufacturer (TaqMan gene expression assays; Applied Biosystems). Thermal cycling was performed using 40 cycles of 95°C for 15 seconds followed by 60°C for 1 minute. Each experiment was done in triplicate, with normalization to the GAPDH gene as an internal control. Cells were incubated with mouse monoclonal antibodies at saturation concentration for 30 minutes on ice, washed with PBS, and stained with a polyclonal goat anti-mouse antibody coupled with fluorescein isothiocyanate for 30 minutes. Samples were analyzed using a BD FACSCalibur flow cytometry system (Becton Dickinson, Mountain View, CA). Anti-pan HLA class I (W6/32) and anti-HLA-A24 monoclonal antibodies (C7709A2.6 hybridoma, a kind gift from Dr. P.G. Coulie, Brussels, Belgium) were prepared from hybridomas. We isolated SP and MP cells of SW480 and HCT15 and seeded them into 96-well culture plates at 1 × 104 cells per well for each population of cells. The cells in both populations were treated with etoposide (1 and 5 μg/mL) or irinotecan (40 and 400 μg/mL for SW480, 10 and 100 μg/mL for HCT15). After 72 hours of exposure to the chemotherapeutic agents, viability of the cells was determined using the SOD assay kit WST-1, which was performed according to the manufacturer's protocol (Dojindo Molecular Technologies, Kumamoto, Japan; Rockville, MD). We had previously established CTL clone 41, which recognizes an HLA-A24 restricted antigenic peptide (VYVKGLLAKI) termed Cep55/c10orf3_193(10), from an HLA-A24-positive breast cancer patient's peripheral blood mononuclear cells.8Low J.A. de Sauvage F.J. Clinical experience with Hedgehog pathway inhibitors.J Clin Oncol. 2010; 28: 5321-5326Crossref PubMed Scopus (168) Google Scholar The lytic activity of CTL clone 41 for SP cells, MP cells, and presorted cells was evaluated by 51Cr release assay. Briefly, SP cells, MP cells and presorted cells were labeled with 100 μCi of 51Cr for 1 hour at 37°C, washed four times with PBS, and resuspended in AIM-V medium (Invitrogen). The 51Cr-labeled target cells (2000 cells/well) were then incubated with various numbers of effector cells for 6 hours at 37°C in 96-well culture plates. Radioactivity of the culture supernatant was measured with a gamma counter. The percentage of cytotoxicity was calculated as follows: % Specific Lysis = (Experimental Release − Spontaneous Release) × 100/(Maximum Release − Spontaneous Release). Target cells were treated with 100 units/mL interferon-γ for 48 hours before the assay. SW480 SP cells were mixed with CTL clone 41 at a ratio of 1 SP cell to 10 CTL cells. The resulting mixture (200 μL with 1 × 106 CTL clone 41 and 1 × 105 SP cells) was injected subcutaneously into the backs of NOD/SCID mice. A control group of five mice was injected with SP cells alone. Tumor size was assessed weekly. NOD/SCID mice were inoculated subcutaneously on the back with 1 × 103 SW480 SP cells. Three weeks later, when the tumor started to be palpable, 5 × 104 Cep55/c10orf3_193(10)-specific CTL clone cells or PBS was injected intravenously. The same adoptive transfer procedure was performed 4 weeks after inoculation with SP cells. Tumor size was assessed weekly. In the xenograft model, survival studies using chemotherapeutic agents, cytotoxicity assay, Winn assay, and adoptive transfer model, the data were analyzed using the Mann-Whitney U-test, with P < 0.05 conferring statistical significance. Several methods to isolate colon cancer CSCs/TICs has been reported, including cell surface markers such as CD44 or PROM1 (CD133), SP cells, and the Aldefluor assay.3Dalerba P. Dylla S.J. Park I.K. Liu R. Wang X. Cho R.W. Hoey T. Gurney A. Huang E.H. Simeone D.M. Shelton A.A. Parmiani G. Castelli C. Clarke M.F. Phenotypic characterization of human colorectal cancer stem cells.Proc Natl Acad Sci USA. 2007; 104: 10158-10163Crossref PubMed Scopus (1767) Google Scholar, 4O'Brien C.A. Pollett A. Gallinger S. Dick J.E. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice.Nature. 2007; 445: 106-110Crossref PubMed Scopus (3453) Google Scholar, 5Ricci-Vitiani L. Lombardi D.G. Pilozzi E. Biffoni M. Todaro M. Peschle C. De Maria R. Identification and expansion of human colon-cancer-initiating cells.Nature. 2007; 445: 111-115Crossref PubMed Scopus (3400) Google Scholar, 6Huang E.H. Hynes M.J. Zhang T. Ginestier C. Dontu G. Appelman H. Fields J.Z. Wicha M.S. Boman B.M. Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis.Cancer Res. 2009; 69: 3382-3389Crossref PubMed Scopus (841) Google Scholar, 13Haraguchi N. Utsunomiya T. Inoue H. Tanaka F. Mimori K. Barnard G.F. Mori M. Characterization of a side population of cancer cells from human gastrointestinal system.Stem Cells. 2006; 24: 506-513Crossref PubMed Scopus (515) Google Scholar In the present study, we isolated colon CSCs/TICs using SP cell analysis. Several colon cancer cell lines were dyed with Hoechst 33342 and then analyzed with a BD FACSAria II flow cytometer as described under Materials and Methods. Side population cells could be detected in all six colon cancer cell lines analyzed (ie, SW480, HT29, HCT15, Colo320, Lovo, and KM12LM) (Figure 1, A and B). The frequency of SP cells ranged from 3.3% for SW480 to 11.1% for HCT15 cells. All these SP cells were specifically inhibited by verapamil, as has been shown previously,14Murase M. Kano M. Tsukahara T. Takahashi A. Torigoe T. Kawaguchi S. Kimura S. Wada T. Uchihashi Y. Kondo T. Yamashita T. Sato N. Side population cells have the characteristics of cancer stem-like cells/cancer-initiating cells in bone sarcomas.Br J Cancer. 2009; 101: 1425-1432Crossref PubMed Scopus (116) Google Scholar suggesting that these SP cells were specific for ABC transporter expression. Because previous studies showed that some colon cancer SP cells were not enriched with a CSC/TIC population,15Burkert J. Otto W.R. Wright N.A. Side populations of gastrointestinal cancers are not enriched in stem cells.J Pathol. 2008; 214: 564-573Crossref PubMed Scopus (105) Google Scholar it was essential to confirm the presence of CSCs/TICs in SP cells for further analysis. We inoculated these SP cells subcutaneously into the back of immunodeficient NOD/SCID mice using serial dilution. The SP cells derived from SW480, HCT15, and HT29 showed higher tumor initiating ability, compared with MP cells (Table 1). Furthermore, SW480, HT29, and HCT15 SP cells showed faster tumor growth, compared with MP cells (Figure 1, C and D), suggesting the presence of CSCs/TICs in these SP cells. In contrast, the SP cells derived from Colo320, Lovo, and KM12LM did not show any difference in tumorigenicity or tumor growth, compared with MP cells. We therefore restricted further analysis to the SW480, HT29, and HCT15 SP cells as colon cancer CSCs/TICs.Table 1Tumor Initiating Ability of Colon Cancer SP CellsCell line (% SP cells)Tumor initiating ability⁎Tumor initiating ability is expressed as the ratio of tumor-initiation to injection.1 × 104†The tumor initiation abilities were evaluated at day 42 after injection of the indicated number of cells.1 × 103†The tumor initiation abilities were evaluated at day 42 after injection of the indicated number of cells.1 × 102†The tumor initiation abilities were evaluated at day 42 after injection of the indicated number of cells.SW480 (3.3) SP cells4/44/64/4 MP cells2/43/50/4HT29 (10.4) SP cells3/32/33/3 MP cells3/30/30/3HCT15 (11.1) SP cells3/33/43/3 MP cells1/31/40/3Colo320 (10.9) SP cells2/21/21/2 MP cells2/22/21/2Lovo (9.3) SP cells0/11/10/1 MP cells1/10/10/1KM12LM (9.1) SP cells1/22/21/1 MP cells1/22/21/1MP, main population; SP, side population. Tumor initiating ability is expressed as the ratio of tumor-initiation to injection.† The tumor initiation abilities were evaluated at day 42 after injection of the indicated number of cells. Open table in a new tab MP, main population; SP, side population. To examine the molecular properties of SP cells, we performed RT-PCR analysis. SOX2 and POU5F1 are representative markers for embryonal stem cells and CSCs/TICs.16Tysnes B.B. Tumor-initiating and -propagating cells: cells that we would like to identify and control.Neoplasia. 2010; 12: 506-515Abstract Full Text PDF PubMed Scopus (77) Google Scholar The SP cells derived from SW480, HT29, and HCT15 showed higher expression of both SOX2 and POU5F1, compared with MP cells (Figure 2A). ALDH1A1, a colon CSC/TIC marker,6Huang E.H. Hynes M.J. Zhang T. Ginestier C. Dontu G. Appelman H. Fields J.Z. Wicha M.S. Boman B.M. Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis.Cancer Res. 2009; 69: 3382-3389Crossref PubMed Scopus (841) Google Scholar was expressed at a higher level in SP cells of HCT15 than in MP cells, but SP cells of SW480 and HT29 did not show any difference in comparison with MP cells. SW480 and HT29 SP cells also showed higher expression of LGR5, which is known as a normal colon stem cell marker.17Vermeulen L. Todaro M. de Sousa Mello F. Sprick M.R. Kemper K. Perez Alea M. Richel D.J. Stassi G. Medema J.P. Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity.Proc Natl Acad Sci USA. 2008; 105: 13427-13432Crossref PubMed Scopus (602) Google Scholar To confirm the expression of stem cell markers, we also performed real-time PCR. The SW480 SP cells expressed 90 times higher SOX2, 7 times higher POU5F1, 153 times higher LGR5, and 6.1 times higher ALDH1A1, compared with MP cells (Figure 2B). These findings indicate that these SP cells had molecular properties similar to those of embryonal stem cells. Although SP cells derived from liver cancer cell line HuH7 have showed resistance to chemotherapy,13Haraguchi N. Utsunomiya T. Inoue H. Tanaka F. Mimori K. Barnard G.F. Mori M. Characterization of a side population of cancer cells from human gastrointestinal system.Stem Cells. 2006; 24: 506-513Crossref PubMed Scopus (515) Google Scholar we know of no conclusive previous studies of such resistance in colon SP cells. We performed a cell survival study of colon cancer SP cells using the chemotherapeutic agents irinotecan and etoposide. The SW480 and HCT15 SP cells were more resistant to both irinotecan and etoposide than were MP cells (Figure 3, A and B). This finding is consistent with findings for CSCs/TICs derived from other organs.22Ho M.M. Ng A.V. Lam S. Hung J.Y. Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells.Cancer Res. 2007; 67: 4827-4833Crossref PubMed Scopus (853) Google Scholar, 24Wang J. Guo L.P. Chen L.Z. Zeng Y.X. Lu S.H. Identification of cancer stem cell-like side population cells in human nasopharyngeal carcinoma cell line.Cancer Res. 2007; 67: 3716-3724Crossref PubMed Scopus (349) Google Scholar Because CTLs recognize tumor-associated antigen (TAA)-derived antigenic peptides presented by HLA class I molecules, expression of HLA class I molecules is essential for activation of CTLs. Several types of malignancies have been reported to lose the expression of HLA class I molecules through various mechanisms and so escape CTL attack.18Campoli M. Ferrone S. HLA antigen changes in malignant cells: epigenetic mechanisms and biologic significance.Oncogene. 2008; 27: 5869-5885Crossref PubMed Scopus (294) Google Scholar We therefore evaluated the expression of HLA class I molecules and TAA. We assessed the differences of HLA class I and HLA-A24 expression between SP cells and MP cells by flow cytometry. Because ELISA study has revealed that HCT15 cells lack B2M because of gene mutations of B2M,19Bicknell D.C. Rowan A. Bodmer W.F. Beta 2-microglobulin gene mutations: a study of established colorectal cell lines and fresh tumors.Proc Natl Acad Sci USA. 1994; 91: 4751-4755Crossref PubMed Scopus (133) Google Scholar we transduced wild-type B2M cDNA into HCT15 cells and so established HCT15-B2M cells. The SW480, HT29, and HCT15-B2M SP cells showed HLA class I and HLA-A24 expression at the same level as MP cells (Figure 4, A and B). Furthermore, we assessed the expression of one of the colon cancer TAAs, CEP55, by both RT-PCR and real-time PCR (Figure 2, A and B). Both SP cells and MP cells derived from SW480, HT29, and HCT15-B2M expressed CEP55 mRNA at the same level. These data raised the possibility that SP cells are also sensitive to CTLs specific for the CEP55-derived antigenic peptide. Because both SP cells and MP cells expressed CEP55 mRNA at the same level, this appeared to be an ideal target for comparing the susceptibilities of SP cells and MP cells to CTLs. We had previously established CTL clone 41, which is specific for the cancer-related, antigen-derived, HLA-A24-restricted peptide Cep55/c10orf3_193(10).10Inoda S. Hirohashi Y. Torigoe T. Nakatsugawa M. Kiriyama K. Nakazawa E. Harada K. Takasu H. Tamura Y. Kamiguchi K. Asanuma H. Tsuruma T. Terui T. Ishitani K. Ohmura T
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