the antigen asb4 on cancer stem cells serves as a target...

Research Article

The Antigen ASB4 on Cancer Stem CellsServes as a Target for CTL Immunotherapy ofColorectal CancerSho Miyamoto1,2, Vitaly Kochin1, Takayuki Kanaseki1, Ayumi Hongo1, Serina Tokita1,Yasuhiro Kikuchi1, Akari Takaya1, Yoshihiko Hirohashi1, Tomohide Tsukahara1,Takeshi Terui3, Kunihiko Ishitani3, Fumitake Hata4, Ichiro Takemasa5, Akihiro Miyazaki2,Hiroyoshi Hiratsuka2, Noriyuki Sato1, and Toshihiko Torigoe1

Abstract

Colorectal cancer consists of a small number of cancer stemcells (CSC) and many non-CSCs. Although rare in number, CSCsare a target for cancer therapy, because they survive conventionalchemo- and radiotherapies and perpetuate tumor formation invivo. In this study, we conducted an HLA ligandome analysis tosurvey HLA-A24 peptides displayed by CSCs and non-CSCs ofcolorectal cancer. The analysis identified an antigen, ASB4, whichwas processed and presented by a CSC subset but not by non-CSCs. The ASB4 gene was expressed in CSCs of colorectal cancer,but not in cells that had differentiated into non-CSCs. BecauseASB4 was not expressed by normal tissues, its peptide epitopeelicited CD8þ cytotoxic T-cell (CTL) responses, which lysed CSCs

of colorectal cancer and left non-CSCs intact. Therefore, ASB4 is atumor-associated antigen that can elicit CTL responses specific toCSCs and can discriminate between two cellular subsets of colo-rectal cancer. Adoptively transferred CTLs specific for the CSCantigen ASB4 could infiltrate implanted colorectal cancer celltumors and effectively prevented tumor growth in a mousemodel. As the cancer cells implanted in these mice containedvery few CSCs, the elimination of a CSC subset could be thecondition necessary and sufficient to control tumor formation invivo. These results suggest that CTL-based immunotherapiesagainst colorectal CSCs might be useful for preventing relapses.Cancer Immunol Res; 6(3); 358–69. �2018 AACR.

IntroductionSolid tumors are heterogeneous, consisting of a variety of

cell types. One of these cell types, the cancer stem cells (CSC),or cancer-initiating cells, comprise a small subset of tumorcells and are responsible for tumorigenesis (1–3). The exis-tence of CSCs was first reported in a hematologic tumor, andthereafter in a variety of solid tumors, including colon, breast,head and neck, uterine, brain, pancreas, and prostate cancers(4–10). Intratumoral heterogeneity and the presence of CSCsubsets have been established in primary solid-cancer tissues(11). CSCs are resistant to conventional chemotherapy andradiotherapy, most likely due to their quiescent status. CSCsmay also have mechanisms to activate drug transporters orDNA-damage checkpoint responses (12–14). Leukemic stemcells are resistant to a targeted agent, imatinib (15). With these

attributes, CSCs may contribute to relapse or metastasis inclinical settings, increasing the demand for a therapy focusedon CSCs.

Our group and others have proposed that cytotoxic CD8þ

T lymphocytes (CTL) could be used for the immunotherapeutictargeting of CSCs. Indeed, CTL responses against CSCs havebeen demonstrated in the context of colon, kidney, cervical,brain, head and neck, and breast cancers (16–24). Thus, hostCTLs may target CSCs, thereby protecting against tumor growthin vivo. Meanwhile, it remains unclear whether CSCs are animportant therapeutic target. In some studies, CTLs respondingto CSC antigens also recognized or lysed non-CSC counterparts,which account for the majority of cells in solid cancers. Todisambiguate effects of CTLs on CSCs from effects on non-CSCs, we searched colorectal cancer cells for CSC-specificantigens that were naturally processed and elicited CTLresponses only to CSCs, leaving non-CSCs intact.

In this study, we took advantage of a pair of cell lines that arosefrom side-population (SP) and main-population (MP) cells ofcolorectal cancer, which provided us with a consistent source ofCSCs (25, 26).We thenmapped the HLA-A24 peptide landscapesof the CSCs and non-CSCs with HLA ligandome analysis usingmass spectrometry. The comparison between those two cellularsubsets revealed that many HLA-A24 peptides were expressed byboth cell types. A few peptides were identified only in CSCs. Wefurther analyzed one of these peptides: ankyrin repeat and SOCSbox protein 4 (ASB4). CSCs but not non-CSCs expressed the geneand the peptide. Here, we found that the CTLs responding to theASB4 antigen discriminate CSCs from non-CSCs of colorectal

1Department of Pathology, Sapporo Medical University, Sapporo, Japan.2Department of Oral Surgery, Sapporo Medical University, Sapporo, Japan.3Higashi-Sapporo Hospital, Sapporo, Japan. 4Sapporo Dohto Hospital, Sapporo,Japan. 5Department of Surgery, Sapporo Medical University, Sapporo, Japan.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Takayuki Kanaseki, Sapporo Medical University, Chuo-ku S1W17, Sapporo, Hokkaido 060-8556, Japan. Phone: 81-11-611-2111, ext. 25510;Fax: 81-11-643-2310; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-17-0518

�2018 American Association for Cancer Research.

CancerImmunologyResearch

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cancer and controlled colorectal cancer growth in vivo. These datamay suggest that control of CSCs is required to prevent colorectalcancer growth. The CSC-specific antigen ASB4 may be useful as atherapeutic target in immunotherapy against colorectal cancer.

Materials and MethodsCell lines

Cell lines were maintained in RPMI 1640 or DMEM supple-mented with 10% FBS and 1% antibiotics. The human cell linesused in this study were as follows: colon carcinoma (SW480,SW620, Colo205, HCT15, Colo320, HCT116, HT29, andCRC21), lung carcinoma (A549, SBC1, SBC5, and Lc817), kidneycarcinoma (Caki-1 and ACHN), liver carcinoma (HepG2), breastcarcinoma (MCF7 and MDA-MB-468), ovary carcinoma (ES2),prostate carcinoma (PC3), pancreas carcinoma (panc1), melano-ma1102-MEL, cervix carcinoma (HeLa), bladder carcinoma (UM-UC-3), osteosarcoma (U2OS), erythroleukemia (K562), and TAP-deficient T2 cell line stably expressing HLA-A�24:02 (T2-A24).SW480, SW620, Colo205, HCT15, Colo320, HCT116, HT29,A549, Caki-1, ACHN, HepG2, MCF7, MDA-MB-468, ES2, PC3,panc1, HeLa, UM-UC-3, U2OS, and K562 were purchased fromATCC. SBC1, SBC5, and Lc817 were purchased from the JapaneseCancer Research Resources Bank (JCRB,Osaka, Japan). 1102-MELwas a gift from Dr. F.M. Marincola (National Cancer Institute,Bethesda, MD). T2-A24 was a gift from Dr. K. Kuzushima (AichiCancer Center Research Institute). CRC21 was established in ourlab. The cell lines were immediately frozen in batches on arrival,and the culture period of each batchwas limited to amaximumof8 weeks. Because the HCT-15 line lacks b 2-microglobulin (b2m)expression, we used HCT-15 cells stably expressing b2m through-out the whole study.

AntibodiesHybridomas for anti-HLA-A24 (C7709A2, a gift from

Dr. P.G. Coulie, Ludwig Institute for Cancer Research, Brussels,Belgium), pan HLA-class I (W6/32, ATCC), and anti–HLA-DR(L243, ATCC) were cultured in Hybridoma-SFM (Gibco) supple-mented with 1% penicillin/streptomycin in CELLine bioreactorflasks (Corning). Produced mAbs were condensed and collectedthrough a semipermeable membrane during cell culture.

Side population assayThe original protocol has been previously described (46, 47).

Briefly, cells were labeled with 5 mg/mL Hoechst 33342 dye(Lonza) for 90 minutes in the presence or absence of 50 mmol/L verapamil (Sigma-Aldrich). Verapamil was used to inhibitABCG2 activity. Approximately 1 � 106 viable cells were thenanalyzed and sorted using a FACSAria II (BD Biosciences). TheHoechst dye was excited with the UV laser at 355 nm and itsfluorescence was measured using a 450/20 nm band-pass filter(Hoechst blue) and a 670 nm long-pass filter (Hoechst red). Deadcells were labeled with 1 mg/mL propidium iodide and excludedfrom the analysis.

Sphere formation assayCells were cultured in ultra-low attachment plates (Corning

Life Sciences) in serum-free Dulbecco's Modified Eagle Medium/F12 medium (Life Technologies) supplemented with 20 ng/mLhuman recombinant epidermal growth factor, 10 ng/mLhuman recombinant basic fibroblast growth factor (R&D

Systems), and 1% N2 supplement (Invitrogen). On day 7, wecollected spheres or counted the number of spheres of whichthe diameter was >100 mm.

HLA-A24 ligandome analysisThe peptides bound to HLA-A24 of SP-H and MP-A cells were

isolated and sequenced using mass spectrometry. We used twodifferent mass spectrometers, Q-Exactive Plus (Thermo FisherScientific) and 4800 Plus MALDI-TOF/TOF Analyzer (AB Sciex),and the detected peptides in three technical replicates each for SP-H andMP-Awere collectively analyzed. The precise workflow andprocedure have been previously described (27, 48). Cell lysateswere prepared from approximately 1.0 � 109 SP-H or MP-A cellswith lysate buffer containing 0.6% CHAPS (Dojindo MolecularTechnologies) and 1 � complete protease inhibitor (Roche) inPBS. The peptide–HLA-A24 complexes included in the sampleswere then isolated using affinity chromatography of an HLA-A24specific C7709A2 mAb coupled to cyanogen bromide-activatedSepharose 4B (GE Healthcare). The HLA-bound peptides werethen eluted with 0.2% TFA and purified using a 10 kDa ultra-centrifugal filter (Millipore), desalted using C18 ZipTip (Milli-pore), and condensed by vacuum centrifugation.

Samples were then loaded into a nano-flow UHPLC (Easy-nLC 1000 system, Thermo Fisher Scientific) online-coupled toan Orbitrap mass spectrometer equipped with a nanospray ionsource (Q-Exactive Plus, Thermo Fisher Scientific). We separat-ed the samples using a 75 mm � 20 cm capillary column with aparticle size of 3 mm (NTCC-360, Nikkyo Technos) by applyinga linear gradient ranging from 3% to 30% buffer B (100%acetonitrile and 0.1% formic acid) at a flow rate of 300 nL/minute for 80 minutes. In mass spectrometry analysis, surveyscan spectra were acquired at a resolution of 70,000 at 200 m/zwith a target value of 3e6 ions, ranging from 350 to 2,000 m/zwith charge states between 1þ and 4þ. We applied a data-dependent top 10 method, which generates high-energy colli-sion dissociation (HCD) fragments for the 10 most intenseprecursor ions per survey scan. MS/MS resolution was 17,500 at200 m/z with a target value of 1e5 ions.

For MS/MS data analysis, we used the Sequest HT (ThermoFisher Scientific) and Mascot ver 2.5 (Matrix Science) algorithmsembedded in the Proteome Discoverer 2.1 platform (ThermoFisher Scientific), and the peak lists were searched against theUniProt human databases. The tolerance of precursor ions andfragment ions were set to 10 ppm and 0.02 Da, respectively. Morethan 80% of the peptides including IV9 listed in SupplementaryTable S1 were detected with a false discovery rate (FDR) of 0.01;however, we applied a less stringent FDR of 0.05 as a threshold toavoid overlooking potential CSC antigens.

RT-PCR and quantitative PCRTotal RNA was isolated from cancer cell lines and cancer

tissues using an RNeasy Mini Kit (Qiagen) or an AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturer's instruc-tions. cDNA was synthesized from 2 mg of total RNA by reversetranscription with Superscript III and oligo(dT) primers (LifeTechnologies). cDNA from human fetal and adult tissueswas purchased from Clontech and Bio Chain. RT-PCR mixtureswere initially incubated at 94�C for 2 minutes, followed by35 cycles of denaturation at 94�C for 15 seconds, annealing at63�C for 30 seconds, and extension at 72�C for 30 seconds. Primerpairs were as follows: ASB4, 50-CTGTCTTGTTTGGCCATGTG-30

A Cancer Stem Cell Antigen for CTL Immunotherapy

www.aacrjournals.org Cancer Immunol Res; 6(3) March 2018 359




and 50-GCGTCTCCTCATCTTGGTTG-30 (product size 288 bp);G3PDH, 50-ACCACAGTCCATGCCATCAC-30 and50-TCCACCAC-CCTGTTGCTGTA-30 (product size 452 bp).

Gene expression was also quantified using a StepOne Real-Time PCR System (Applied Biosystems) with PowerUp SYBRGreen Master Mix (Thermo Fisher Scientific). An initial denatur-ation step of 95�C for 10 minutes was followed by 40 cycles ofdenaturation at 95�C for 15 seconds and annealing/extension at60�C for 60 seconds. Primer pairs for qPCRwere as follows: ASB4,50-CTGTCTTGTTTGGCCATGTG-30 and 50-GCGTCTCCTCATCT-TGGTTG-30; G3PDH, 50-GGATTTGGTCGTATTGGG-30 and 50-GGAAGATGGTGATGGGATT-30. Each sample was analyzed intriplicate and the threshold cycle values (Ct) of ASB4 werenormalized according to those of G3PDH.

Synthetic peptides and binding assaySynthetic peptides of the following purities were purchased

from Sigma Genosis [IV9 (IYPPQFHKV), 91.7%; HIVenv584-594

(RYLRDQQLL), 81.5%; GK12 (GYISPYFINTSK), 89.7%]. Pep-tides in a range of the indicated concentrations were pulsedonto T2-A24 cells, incubated for 3 hours at 27�C, and thenincubated for 2.5 hours at 37�C. Cells were incubated withC7709A2, followed by a secondary FITC-conjugated antibody,and then analyzed using flow cytometry. The difference inmean fluorescence intensity (DMFI) indicates the difference inMFI values between samples with and without the primaryantibody.

CTL induction and cloningWe used the established method for CTL induction (49).

Briefly, CD8þ cells were isolated from peripheral blood mono-nuclear cells (PBMC) of HLA-A�24:02-positive healthy donors orcolorectal cancer patients using anti-CD8 coupled to magneticmicrobeads (Miltenyi Biotec). Remaining CD8� cells were PHA-activated and used as PHA blasts. CD8þ cells were cultured inAIM-V medium (Life Technologies) containing 10% humanserum (kindly provided by Dr. Takamoto, Japanese Red CrossHokkaido Block Blood Center), 20 U/mL human IL2 (kindlyprovided by Takeda Pharmaceutical), and 10 ng/mL IL7 (R&DSystems) for 4 weeks. The CD8þ cells were repeatedly stimulatedwith autologous PHA-blasts pulsed with 20 mmol/L of the IV9peptide. To generate CD8þ T-cell clones, single cells binding bothanti-CD8 (Beckman Coulter) and an IV9-HLA-A24 tetramer(MBL, Japan), as well as the single cells positive for CD8 butnegative for binding to the tetramer,were isolated using FACSAriaII (BD Biosciences). They were expanded in AIM-V mediumcontaining 100 U/mL IL2, 1 mg/mL PHA, and X-ray–irradiatedPBMCs from healthy volunteers. The SM4 and control CTL cloneswere selected from the tetramer-positive and -negative clones,respectively.

ELISPOT IFNg assayTetramer-positive CTLs were added to ELISPOT plates coated

with antihuman IFNg (BD Biosciences) at 5.0 � 104 cells/mL perwell. T2-A24 or the indicated cancer lines at 5.0� 104 was addedto the corresponding wells. T2-A24 was preincubated at roomtemperature for 2 hours with 20 mmol/L of IV9 or irrelevant HIVpeptide. After incubation in a 5% CO2-incubator at 37�C for 24hours, the wells were incubated with a biotinylated antihumanIFNg antibody for 2 hours at room temperature, followed by theELISPOT Streptavidin-HRP antibody for 1 hour. Spots were

visualized using the ELISPOT AEC Substrate Set according to themanufacturer's instructions (BD Biosciences).

Biochemical analysis of naturally processed peptidesusing RP-HPLC

This analysis followed the original protocol withmodifications(50). Peptide extracts of 2 � 108 SP-H or MP-A were prepared byacid extraction using 10% formic acid in the presence of 2 mmol/Lof an irrelevant peptide followed by filtration using a <10k Dacutoff spin-column (Amicon). The samples were fractionatedusing RP-HPLC equipped a 2.1 � 250 mm C18 column with aparticle size of 2mm(ZORBAX300-SBC18, Agilent) by applying alinear gradient ranging from 23% to 45%buffer B for 45minutes.Each fraction was then dried using vacuum centrifugation over-night. Each fraction was incubated with 5� 104 SM4 and 5� 104

T2-A24. The IFNg produced by SM4 responding to its naturallyprocessed epitope was detected using the ELISPOT assay.

LDH cytotoxicity assayThe amount of lactate dehydrogenase (LDH) released from

lysed target cells was measured using an LDH cytotoxicity detec-tion kit according to themanufacturer's instructions (Takara Bio).Target cells (1.0 � 104) were co-incubated with the indicatednumbers (E/T ratio) of CTLs at 37�C for 6 hours. The percentage ofLDH released (cytotoxicity) was calculated as follows: % LDHrelease ¼ 100 � (experimental LDH release � spontaneous LDHrelease)/(maximal LDH release � spontaneous LDH release).LDH values from CTL alone and target cells treated with 2%Triton X-100 (Sigma-Aldrich) were used to estimate spontaneousand maximal LDH releases, respectively. In the HLA-blockingassay, target cells were preincubated for an hour with 100 mg/mLof anti-HLA-class I (W6/32), anti-HLA-2402 (C7709A2), or anti-HLA-DR (L243).

Mice and xenograft modelsNSG mice were purchased from The Jackson Laboratory. The

mice were maintained in the animal facility of Sapporo MedicalUniversity and all procedures were performed in accordance withthe institutional animal care guidelines. To evaluate the tumor-igenicity of SP-H and MP-A, NSG mice were subcutaneouslyinjected with 1.0 � 103 and 1.0 � 104 of SP-H or MP-A cells.In tumor-rejection models, 1.0 � 103 SW480 cells were subcu-taneously injected, followed by the adoptive intravenous transferof 5.0� 105 CTLs at the indicated time points. The major (x) andminor (y) axes of the tumors were routinely measured. Tumorvolume was calculated as follows: volume ¼ xy2/2. To assess CTLinfiltration into tumors, NSG mice were subcutaneously injectedwith 1.0 � 106 SW480 cells. Subsequently, the mice were intra-venously injected with 1.0� 105 and 2.0� 106 CTLs, on days 28and 29, respectively. The tumors and spleens were removed andfixed with 10% formalin on day 31. The paraffin-embeddedtissues were immunohistochemically stained with a human CD8antibody (DAKO). The numbers of CD8þ cells per HPF weremanually counted.

Patients and MethodsThe study was performed with approval of the Institutional

Review Board of Sapporo Medical University. Clinical samples ofpatients with colorectal cancer were included in this study, withinformed consent according to the guidelines of the Declarationof Helsinki. PBMCs were isolated using Lymphoprep (Nycomed)from whole blood samples of patients and healthy donors.

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Cancer Immunol Res; 6(3) March 2018 Cancer Immunology Research360




IV9-specific CD8þ T-cell precursor frequency of colorectalcancer patients

PBMCs from HLA-A24-positive colorectal cancer patients weredistributed over 24 wells per patient and cultured in AIM-Vmedium supplemented with 5% human serum and 50 U/mLIL2. PBMCswere stimulated with 20 mmol/L IV9 peptide on day 0and day 8, and then stained with anti-CD8, an IV9-HLA-A24tetramer, and an irrelevant HIV-HLA-A24 tetramer on day 15. Thefrequency of anti-IV9 CD8þ T-cell precursors was calculated asfollows: frequency ¼ the number of wells positive to IV9-HLA-A24/(24 � the initial number of CD8þ cells per well). Samplesshowing >0.02% positivity were counted as positive to the IV9-HLA-A24 tetramer.

ResultsA clone derived from human colorectal cancer cells shows CSCphenotypes

Various types of malignant tumors contain a CSC subset.However, the proportion of CSCs is low and unstable through

long-term in vitro culture, making their assessment challenging(28). To address this issue, we generated both CSC and non-CSClines from a single-cell clone of human colorectal cancer SW480cells (26). In that study, we isolated both side and main popu-lation cells using Hoechst 33342 dye staining, and demonstratedthat both CSC-like (SW480-SP-A, SP-B, and SP-H) and non-CSC-like (SW480-MP-A,MP-D, andMP-K) phenotypes were sustainedin culture. Although 1% to 2%of the parent SW480 cell lines wereside-population cells, a side-population assay demonstrated thatthe representative clone SP-H was 32.6% side-population cells,and the enriched side-population cells were maintained after 4months in vitro serum culture (Fig. 1A). Likewise, <0.1% of arepresentative clone of MP-A were side-population cells and thisclone never generated new side-population cells (Fig. 1B). Thenumber of spheres formed in nonserum culture was approxi-mately 10.6 times higher in SP-H than MP-A (Fig. 1C). Inaddition, SP-H formed significantly larger masses inmouse xeno-graft models in vivo (Fig. 1D–F). Tumor masses were palpable ondays 28 and 20 of 1.0 � 103 and 1.0 � 104 SP-H implantation,respectively, whereas MP-A never formed palpable tumors by day

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Figure 1.

The SP-H clone shows and maintains the CSC phenotypes in vitro and in vivo. A and B, Side-population assay of SP-H (A) and MP-A (B) clones. Top and bottompanels represent the density plots of flow cytometry stained with Hoechst Red and Blue on day 0 and 120 days after in vitro culture, respectively. The numbersinside the plots indicate the percentage of side-population cells surrounded by solid circles. Data are representatives of three independent experiments. C, Sphere-forming assay of SP-H and MP-A. The cells were cultured in 96-well ultra-low attachment plates without serum for 7 days. Spheres (diameter >100 mm) weremicroscopically detected and the bar chart summarizes the numbers of wells containing at least one sphere among 96wells. Data are shown asmeanþ SEM (n¼ 3)and P values were calculated using a two-tailed t test (� , P < 0.05). Photographs of SP-H and MP-A represent a sphere-positive and -negative wells, respectively.Magnification, � 100. D, A single NSG mouse subcutaneously implanted with 1.0 � 103 of SP-H (left) and MP-A (right) cells showing visible tumor formation(yellow circle) on the left side. Shown is a representative picture of 9 mice. E and F, Summaries of tumor formation in NSG mice implanted with 1.0 � 103 (E)and 1.0 � 104 (F) of SP-H and MP-A cells. The x-axis, days after implantation; the y-axis, tumor volumes. Data are shown as mean þ SD (n ¼ 9 in E, and n ¼ 14 in F)and P values were calculated using a two-tailed t test (� , P < 0.05 and �� , P < 0.01).






42. These data indicate that the tumor-initiating ability of SP-Hwas higher than that ofMP-A both in vitro and in vivo.We concludethat SP-H and MP-A clones represent CSC and non-CSC pheno-types. Both clones maintained stable phenotypes.

HLA-A24 ligandome analysis identified a CSC-specificpeptide, IV9

CSCs are long-lived in the host environment and harbortumor-initiating ability, hence are a target for immunotherapy(29). As antigen processing of CSCs is not fully understood, wedirectly surveyed the HLA-A24 peptide landscape of SP-H andMP-A (Fig. 2A). Briefly, 1 � 109 cells of SP-H or MP-A werelysed, and a mixture of peptide-HLA-A24 complexes wereimmunoprecipitated using an HLA-A24–specific antibody. Thebound peptides were then eluted and sequenced using massspectrometry coupled with nano-flow liquid chromatography.The analysis identified 178 sequences of nonredundant HLA-A24 peptides from SP-H and MP-A (Supplementary Table S1).The average length of the detected sequences was approximate-ly 9.1 amino acids (Fig. 2B), and both tyrosine (Y) at P2 andphenylalanine (F) or leucine (L) at PWwere conserved across thesequences (Fig. 2C). These peptide profiles ensured that ourligandome analysis identified HLA-A24 ligands from SP-H andMP-A (30).

Although most of the peptides were MP-A–specific (86 pep-tides) or shared between SP-H and MP-A (57 peptides), 35peptides were detected only in SP-H (Fig. 2D). Removal ofpeptides for which the source gene was expressed in normal organtissues left a 9-mer peptide (IYPPQFHKV, "IV9"; Fig. 2E). Thispeptide, IV9,was repeatedly isolated fromSP-H samples but never

detected in MP-A samples. Although both SP and MP clonesexpressed HLA-A24 on the cell surface, MP clones expressedmoreHLA-A24, potentially explaining a difference in the number ofisolated HLA-A24 peptides between SP and MP clones (Supple-mentary Fig. S1).

ASB4 is expressedby variety of cancers but notbynormal tissuesIV9 is encoded by both of two known isoforms of ASB4

(UniProt ID:Q9Y574), a potential component of the E3ubiquitinligase complex (Fig. 3A; ref. 31). TheASB4 genewas expressed by avariety of cancer cell lines, including lines derived from colorectalcancers (SW480, SW620, colo205, and HCT15), lung cancers(A549, SBC1, and SBC5), and a kidney cancer (Caki-1), as wellas a liver cancer (HepG2; Fig. 3B). Expression was higher inclinically dissected colorectal cancer tissues, greater than 5-foldhigher in 6 out of 21 cases (Fig. 3C). The ASB4 gene was littleexpressed by a panel of normal adult and fetal tissues as wellas cultured mesenchymal stem cells derived from bone marrow(Fig. 3C and Supplementary Fig. S2). Thus, expression of the ASB4gene was tumor specific in both cell lines and primary colorectalcancer tissues.

IV9 is a natural CD8þ T-cell epitope processed and presentedonly by SP-H

The synthetic IV9 peptide harboring Y at P2 and V at P9stabilizes expression of HLA-A24 on T2-A24 cells (Fig. 4A). Westimulated PBMCs derived from a healthy donor with the IV9peptide. From a cell population responding to an IV9-HLA-A24tetramer, we established the CD8þ T-cell clone line (SM4), 98.8%of which was positive for the tetramer (Fig. 4B). The SM4 line

Figure 2.

HLA-A24 ligandome analysis identifies a CSC-specific peptide, IV9. A, A workflow of HLA-A24 ligandome analysis using the C7709A2 mAb. The analysiscomprehensively sequenced HLA-A24 ligands of SP-H and MP-A on a large scale. The experiments were independently conducted two times for each cell type.B, Length distribution of detected peptides. The x-axis, numbers of amino acids (AA); the y-axis, numbers of peptides. C, Logo sequences of 9-mer HLA-A24 ligandsdetected in SP-H (top) and MP-A (bottom), showing that both Tyr (Y) at P2 and Phe, Leu, or Iso (F, L, or I) at P9 were strongly conserved across the sequences.D, Numbers of SP-H-specific (red, 35) and MP-A-specific (blue, 86) HLA-A24 ligands. Fifty-seven ligands were shared between SP-H and MP-A. E, Tandemmass (MS/MS) spectrum of IYPPQFHKV (IV9). Detected b- (or a-) and y-ions are indicated in red and blue, respectively.

Miyamoto et al.





responded to SW480 as well as to T2-A24 pulsed with 2 mmol/LIV9 peptides, producing IFNg (Fig. 4C). SM4 produced IFNg inresponse to SP-H and SW480, but not in response to MP-A cells.The SM4 line produced more IFNg against the MP-A cells expres-sing ASB4, demonstrating that ASB4 was the responsible antigenencoding the CTL epitope (Fig. 4D and E).

Next, we biochemically evaluated and quantified the naturalT-cell epitope produced in live SP-H and MP-A cells. Wefractionated cell extracts from 2 � 108 SP-H and MP-A usingRP-HPLC, then incubated these extracts with SM4 in the pres-ence of T2-A24 as antigen-presenting cells. We measured pro-duction of IFNg using an ELISPOT assay (Fig. 4F). We foundthat the SP-H extract contained the natural SM4 epitope atfraction #5, the same fraction as synthetic IV9 peptide. As thisassay used a consistent set of T cells and APCs across samples,only the amount of the IV9 peptide included in samplesinfluenced the number of IFNg spots. Therefore, we estimatedthe amount of IV9 in 2 � 108 SP-H at approximately 12 fmol ormore, because the number of positive spots at #5 was increasedby 1.28-fold (96/75) compared with that of the 10 fmolsynthetic peptide. MP-A extract did not contain IV9. Consid-ering that ASB4 gene overexpression restored SM4 responsesagainst MP-A, we assumed that the antigen-processing machin-ery of MP-A was capable of presenting the IV9 peptide. How-ever, loss of the ASB4 gene expression resulted in loss of the IV9

peptide. Thus, IV9 is the natural CTL epitope and its presen-tation is limited to the colorectal CSC subset SP-H.

Colorectal CSCs express ASB4In accordance with the SP-H–specific detection of IV9 using

mass spectrometry, ASB4 was expressed in all three SP clones,but not in any of the MP clones we had established (Fig. 5A).Although MP-A is made up mostly of main-population cellswithout dedifferentiation, SP-H is composed of both side- andmain-population cells, suggesting that side-population cellsin SP-H constantly differentiate into main-population cells(Fig. 1A). We, therefore, re-sorted SP-H cells cultured for 3weeks into side and main populations again and determinedASB4 expression in each subset. Only the side-populationdescendants (SP-1, -2, and -3) but not the main-populationdescendants (MP-1, -2, and -3) expressed ASB4, suggesting thatside-population cells of SP-H ceased ASB4 expression whendifferentiated into main-population cells (Fig. 5B). Moreover,the ASB4 expression was detected in 4 out of 8 colorectal cancerlines derived from 3 different colorectal cancer patients (SW480and 620, Colo205, and HCT15; Fig. 3B). In fact, the expressionlevels increased to 1.9- to 3.0-fold when CSCs were enriched bysphere culture using ultra-low attachment plates without serum(Fig. 5C). Sphere culture did not influence the expression inColo320 and HCT116, both of which were ASB4-negative.

Figure 3.

The ASB4 gene is selectively expressed in tumor cells. A, The amino acid sequence of ASB4. Both isoform 1 (UniProt ID: Q9Y574-1, top) and isoform 2 (UniProtID: Q9Y574-2, bottom) encode the IV9 sequence (underlined). B, RT-PCR of indicated types of cancer cell lines. ASB4 and G3PDH bands appeared at 288and 452 bp, respectively. Data are representatives of three independent experiments. C, Quantitative-PCR of a panel of healthy adult and fetal tissues as wellas primary colorectal cancer tissues (T01-21). The y-axis, ASB4 expression relative to a healthy adult colon tissue. Dashed line indicates 5-fold expression. Dataare shown as mean þ SEM (n ¼ 3). The experiment was conducted in triplicate.






Sphere culture increased sphere formation in every colorectalcancer line as well as ASB4 expression in SW480 and HCT15using RT-PCR (Supplementary Fig. S3A–S3C). To further inves-tigate the enrichment of the IV9 epitope presentation by CSCs,we tested the SM4 responses. The amounts of IFNg produced bySM4 were increased by approximately 1.6-fold against SW480and HCT15 expressing b2m under sphere culture (Fig. 5D).These data demonstrate that ASB4 is the antigen of which theexpression and following CTL responses were linked to colo-rectal CSC subsets.

SM4 lyses colorectal CSCs but not non-CSCsWe aimed to identify CSC antigens that are exclusively pre-

sented by CSCs and elicit CTL responses lysing only CSCs. Inaccordance with the ELISPOT results, SM4 lysed T2-A24 cellspulsed with the synthetic IV9 peptide, but ignored control pep-tides or K562 cells (Fig. 6A). SM4 successfully lysed SP clones (SP-A, SP-B, and SP-H) and, aswe expected, leftMP clones (MP-A,MP-D, and MP-K) intact (Fig. 6B). The killing efficacy (LDH release(%)) against MP clones was near to zero, indicating that ASB4 is aCSC-specific antigen and the CTL activity discriminates tumori-

genic CSCs from non-CSCs. The result was consistent with the SP-H-specific IV9 peptide production observed in Fig. 4F. SP-H andunsorted SW480 contain approximately 30% and 1% to 2% ofside-population cells, respectively (Fig. 1A and SupplementaryFig. S4). We found that SM4 lysed SW480, besides SP-H, to as lowas 59.3% of SP-H at an effector/target (E/T) ratio of 9 (Fig. 6C). AsSM4 activity against such a small cell population was detectable,we tested other colorectal cancer lines without CSC enrichment.We found that SM4 recognized and lysed HCT15/b2m, whichexpresses ASB4 and is HLA-A24-positive, but left Colo205andColo320 intact, both ofwhich lackHLA-A24 (SupplementaryFig. S5). The CTL-mediated lysis of SW480 was blocked by thepresence of the pan-HLA class I W6/32 mAb as well as the HLA-A24–specific C7709A2 mAb, but not by the HLA-DR–specificL243 mAb, ensuring that the response was restricted to HLA-A24(Fig. 6D).

Adoptive transfer of SM4 CTLs suppressed tumor growth in vivoFinally, we investigated the effects of SM4 on colorectal

cancer using in vivo models. A randomly generated CTL clonewas prepared as a control, which did not respond to an IV9-

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IV9 is a natural CD8þ T-cell epitope processed and presented only by SP-H.A, Peptide binding assay using T2-A24 cells pulsed with the indicated synthetic peptidesand an HLA-A24–specific antibody (C7709A2). HIV and GK12 are representative HLA-A24–binding and –nonbinding peptides, respectively. The x-axis, theconcentration of pulsed synthetic peptides; the y-axis, the difference between samples and negative controls without primary mAb in units of mean fluorescenceintensity (DMFI). Data are shown asmeanþ SEM (n¼ 3) and P valueswere calculated using a two-tailed t test (�� , P <0.01).B, Flow cytometry of an IV9-specific CTLclone (SM4) stained with an IV9-HLA-A24 tetramer together with an HIV-HLA-A24 tetramer. The number inside the plot indicates the percentage cell populationsurrounded by the rectangle. The plot is a representative of three independent experiments. C, IFNg ELISPOT assay of SM4. Target cells were T2-A24 cells pulsedwith indicated synthetic peptides, SP-H, MP-A, or SW480 cells. The bar chart summarizes the numbers of IFNg spots per well. Data are shown asmeanþ SEM (n¼ 3)and P values were calculated using a two-tailed t test (�� , P < 0.01). Photographs are representative IFNg spots in each condition. D, RT-PCR of MP-A stablyexpressing an empty vector or ASB4 aswell as SP-H. Data are representatives of three independent experiments.E, IFNg ELISPOT assayof SM4. Target cellswere SP-H or MP-A stably expressing an empty vector or ASB4. Data are shown as mean þ SEM (n¼ 3) and P values were calculated using a two-tailed t test (�� , P < 0.01).F, Peptide fractionation and quantification using RP-HPLC. Samples were fractionated and dried in a vacuum. The amounts of IV9 peptides contained in eachfractionwere thenmeasuredbasedonSM4 responses in thepresenceof T2-A24 cells using IFNg ELISPOTassay. Sampleswere 10 fmol of IV9 synthetic peptides (left),a cell extract of 2.0� 108 SP-H (middle), and a cell extract of 2.0� 108MP-A (right). Mock indicates the results of RP-HPLC buffer alone,measured in-between sampleruns. Data are representatives of two independent experiments.

Miyamoto et al.





HLA-A24 tetramer. This clone recognized neither SP-H norSW480 or MP-A (Supplementary Fig. S6). ImmunodeficientNSG mice were implanted with 1 � 103 SW480 on day 0. SM4(5 � 105) was then intravenously injected before (on day �3)or after tumor implantation (on days 35 and 42; Fig. 7A). Weused both models to evaluate antitumor CTL effects at earlystages before tumors developed into large masses. In contrast tothe control CTL clone, the adoptive transfer of SM4 significantlyprevented tumor growth in both CTL injection models "beforeand after" tumor implantation (Fig. 7B and C, respectively).Even at the end of the time courses on day 56, both adoptivetransfer models controlled tumor sizes such that implantedtumors were not palpable. Hence, SM4, which targeted only anSP subset of SW480, prevented tumor formation of SW480,which was composed of 1% to 2% of SP cells among otherwise

MP cells. This result suggests that the CSC subset is a necessaryand sufficient target in preventing colorectal cancer formationat the early stages.

We also prepared mice-bearing established masses of SW480and counted the number of tumor-infiltrating CTLs 2 days afterthe adoptive transfer of SM4 (Supplementary Fig. S7A). The IHCresults showed that SM4, but not control CTLs, was present insideSW480 tumors, whereas both CTLs were found inside the spleens(Fig. 7D–F). We estimate that the in vivo SW480 tumors werecontained 2% or fewer SP cells, as is observed for in vitro cultures(26), because a similar analysis with SP-B tumors revealed thatthe percent of SP cells was stable in vivo (Supplementary Fig. 7B).The data demonstrated that SM4 was capable of homing toIV9-HLA-A24 complexes and migrating into tumors in search fora small cell population of CSCs.

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TheASB4 antigen is enriched in colonCSC subsets.A,Quantitative-PCRof indicated SP andMP clones derived fromSW480. The y-axis, ASB4 expression relative to ahealthy adult colon tissue. Data are shown as mean þ SEM (n¼ 3). B, RT-PCR of clones derived from SP-H. The clones were prepared by re-sorting SP-H into side-population (SP) and main-population (MP) cells with Hoechst dye staining. Data are representative of three independent experiments. C, Quantitative-PCR ofcolorectal cancer cell lines. The cells were cultured under a regular condition (serum) or without serum using ultra-low attachment plates (sphere). The y-axis,ASB4 expression relative to SW480 in the serum condition. Data are shown asmeanþ SEM (n¼ 3) andP valueswere calculated using a two-tailed t test (� , P <0.05).D, IFNg ELISPOT assay of SM4. Target colorectal cancer cells were cultured under a regular condition (serum) or without serum using ultra-low attachment plates(sphere). The bar chart summarizes the numbers of IFNg spots per well. Data are shown asmeanþ SEM (n¼ 3) and P values were calculated using a two-tailed t test(� , P < 0.05 and �� , P < 0.01).

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SM4 specifically lyses colon CSCs but not non-CSCs. A–D, LDH-release cytotoxicity assay using SM4 as effector cells. Target cells releasing LDH were T2-A24or K562 pulsed with indicated synthetic peptides (A), SP and MP clones derived from SW480 (B), SP-H and SW480 (C), and SP-H incubated with anti-HLAclass I (W6/32), anti-HLA-A24 (C7709A2), or anti–HLA-DR (L243)mAbs (D). The x-axis, effector/target ratio (E/T;A–C) or at E/T¼9 (D); the y-axis, LDH release (%).The cytotoxicity indicates the ratio of LDH released by target cells to the ones that underwent Triton X-100–induced cell death. Data are shown asmeanþ SEM [n¼ 3(A, B, and C) or 4 (D)] and P values were calculated using a two-tailed t test (� , P < 0.05 and �� , P < 0.01).






Immune surveillance of IV9-specific CD8þ T cells in patientswith colorectal cancer

Because the IV9 peptide identified in this study is displayed byHLA-A24, which is themost commonHLA-A type among the EastAsian population, we focused on HLA-A24–positive colorectalcancer patients and assessed the frequency of IV9-specific CD8þ Tcells in their PBMCs. All 6 patients were histologically diagnosedwith colorectal adenocarcinoma and the group consisted of 3menand3women, ages between 59 and 98 (Supplementary Table S2).The primary lesion had been surgically removed in three patients,and five patients had received chemotherapy or radiotherapy. Thegroup included five patients at stage IV and one patient at stage II.PBMCs from the patients were randomly fractionated and stim-ulated with 20 mg/mL of IV9 peptides for 14 days. Induced IV9-specific CD8þ cells were detected and counted using an IV9-HLA-A24 tetramer (Fig. 8A). We regarded the cell fraction labeled with0.03% or more proportions as tetramer positive (32). As a result,T-cell induction was detected in 5 out of 6 patients, and thefrequency of IV9-specific T-cell precursors ranged from4.4� 10�7

to 4.1� 10�6 of CD8þ cells (Fig. 8B). The lower limit of detectionin this assay was around 2.0 � 10�7 and the frequency in onepatient (B05)was under the limit. Three of the patients (B01, B03,and B06) at stage IV reached long survival (>8 years); for thesethree patients, we detected IV9-tetramer–positive CD8þ T cells inmultiple wells. Thus, CD8þ T cells of colorectal cancer patients arenot immunologically tolerant to the ASB4 antigen and IV9 pep-

tide stimulation elicited specific T-cell responses in patients withcolorectal cancer.

DiscussionCSC expression is prioritized in selection of antigens appro-

priate for cancer vaccination. Antigen expression in a CSC subsetmight influence antitumor CTL effects (33). In our study, not onlyis the IV9 peptide a natural CTL epitope directly identified usingHLA-ligandome analysis, but also it is presented by an SP but notan MP subset of SW480 cells. Repeated detection by biochemicalanalysis also implies that the IV9peptide is presented byHLA-A24of CSCs, rendering it a target of CTL immune surveillance.IV9-CTLs indeed discriminated CSCs from non-CSCs, lysing onlythe CSC subset. The IV9-CTLs belong to a group of CTLs thatrecognize CSCs, but are capable of ignoring non-CSCs. Theadoptive transfer of the CTLs prevented tumor formation in NSGmice implanted with 1� 103 unsorted SW480 cells that consistedof 1% to 2% of SP and the majority of MP cells. This resultdemonstrated that most SW480 cells were not the target, andthe ability of IV9-CTLs to ignore non-CSCs did not harnessthe antitumor effect in vivo. Although non-CSCs are predominantin number in many tumors, only CSCs are able to sustaintumorigenesis (34). In fact, NSG mice implanted with 1 � 104

SW480-MP cells did not develop tumors even after a month,whereas 1� 103 SW480-SP cells readily generated tumor masses.

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The adoptive transfer of SM4 CTLs prevents tumor development in vivo. A, Time courses of tumor implantation and CTL adoptive transfer. NSG mice weresubcutaneously injected with 1.0 � 103 SW480 (tumor) on day 0, and received 5.0 � 105 SM4 (CTL) either before (on day �3) or after (on days 35 and 42) tumorinjection. Tumor growth rates in the adoptive CTL-transfermodels are shown inB andC. The x-axis and the y-axis indicate days after tumor implantation and the sizesof tumors, respectively. Data are shown as mean þ SD [n ¼ 7 (B) or 5 (C)] and P values were calculated using a two-tailed t test (�� , P < 0.01). D–F,Immunohistochemistry of tissues fromNSGmice, which bore SW480 tumors and receivedCTL transfers (Supplementary Fig. S7).D,Representative results of humanCD8 staining. Adoptively transferred SM4 CTLswere detected in both tumor and spleen sections, while control CTLswere detected only in the spleen. Magnification,�200. E and F, Summary of CTL infiltration in tumors (E) and spleens (F). All NSG mice were implanted with SW480. Six individuals (#1–6) received SM4injection and the others (#7–12) received control CTLs injection. The x-axis, individual numbers; the y-axis, the highest numbers of CD8-positive cells/HPF.

Miyamoto et al.





These data together suggest that the CSC subset is a useful target ofimmunotherapy despite of its rarity.

The IV9 peptide is encoded by ASB4, the gene expressionof which was not detected using RT-PCR in an array of normalfetal and adult tissues, including mesenchymal stem cells ofthe bone marrow. Expression increased in colorectal cancertissues, and moreover, 28.6% (6 out of 21) of cases showedgreater than a 5-fold increase compared with normal colontissue without enrichment. Considering that the sphere cultureof SW480, Colo205, and HCT15 enriched ASB4 gene expres-sion, each primary colorectal cancer tissue is composed ofvarying proportions of the CSC subset, and in some cases,CSCs might be already enriched through their clinical courses.The exact molecular function of ASB4 in colorectal CSC func-tion is unclear. In mice, the expression of the ASB4 gene hasbeen reported in the developing placenta and the testis; how-ever, adult tissues cease ASB4 gene expression, suggesting ASB4functions early in development, perhaps at trophoblast differ-

entiation (35–37). On the contrary, its increased expression hasbeen reported in hepatocellular carcinomas and adrenal glandtumors, which may suggest another role of ASB4 in metastasisor tumorigenesis (38, 39). We quantitatively compared sphereformation of SP-H in the presence and absence of ASB4 geneexpression; however, silencing ASB4 gene expression did notdecrease the number of spheres. More persistent gene silencingother than siRNA might be necessary to observe the phenotypechange, or this result could suggest that ASB4 does not serve as adriver of sphere formation. Nevertheless, the findings of thepresent study demonstrate that the presence of the ASB4 anti-gen is linked to a CSC subset of colorectal cancer. ASB4 mayhold promise as a therapeutic target of CTL-based immuno-therapy in colorectal cancer patients. Here, it was possible todetect the IV9-CD8þ T-cell precursors in 5 out of 6 colorectalcancer patients, and we knew that the remaining patient (B05)had received dexamethasone for 6 months before blood sam-pling, being in a clinical state of immune suppression. All three

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Figure 8.IV9-specific CD8þ T cells are frequently found in HLA-A24colorectal cancer patients.A, Estimation of the frequency ofIV9-specific CD8þ T cells in a representative colorectalcancer patient. PBMCs from colorectal cancer patients weredistributed in 24 wells and stimulated with 20 mmol/L of IV9peptides according to the procedure described in MaterialsandMethods. Staining results of CD8þ cells in eachwell withIV9-HLA-A24 and control HIV-HLA-A24 tetramers areshown. The numbers insideplots indicate percentageof IV9-HLA-A24 single-positive cells (surrounded by rectangles).The frequencywas calculated as follows: number of positivewells (positivity rate >0.02%)/(24 � the initial number ofCD8-positive cells per well). Tetramer-positive wells areshown in red. B, Summarized frequency in 6 patients. Allpatients were HLA-A24–positive and the patientinformation is provided in Supplementary Table S2. Theprecursors were detected in 5 out of 6 cases.






patients with long-term survival over 8 years showed higherIV9-CD8þ T-cell frequency in contrast to the others, suggestinga relationship between patient survival and CD8þ T-cellsurveillance against the ASB4 antigen.

Clinical success of immune checkpoint blockades both inreducing the sizes of cancers and in prolonging cancer patientsurvival ensured that patients' T-cell responses are able to copewith malignancies (40). Accumulating evidence suggests thatmutational load is positively correlated with clinical effects,and has sparked further research on screening neoantigens thatarise from gene mutations (41–44). CTLs targeting a neoanti-gen discriminate it from their wild-type counterpart, and exhib-it high cytotoxicity against tumors carrying the responsible genemutation (27). However, most colorectal cancers belong to amicrosatellite-stable tumor type, being an exception that isrefractory to immune checkpoint blockade despite their prev-alence (43). Individual variation in gene mutations also call forpersonalized immunotherapy, which requires detection ofantigens for each patient (45). In this study, the antigen wefocused on was, conversely, expressed only in a subset ofcolorectal cancer cells. In our SW480-derived SP- and MP-clonemodels, certain descendants of SP clones differentiated into MPcells during culture, keeping the proportion of SP-cells low.Therefore, we consider that eliminating such a small subset ofcancer cells may not be effective in reducing the sizes of alreadydeveloped bulky tumors in vivo. Instead, the CSCs responsiblefor the onset of tumor formation are shared among patients,generating a practical target for vaccination. The capability ofIV9-CTLs to home to the target may help to identify tumor budsscattered over the body of patients at an early stage. We thusdeduce that CSC-specific CTL surveillance could be usefulto colorectal cancer patients with cured primary lesions inpreventing relapse or metastasis.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: T. Kanaseki, N. Sato, T. TorigoeDevelopment of methodology: S. Miyamoto, V. Kochin, T. Kanaseki,Y. HirohashiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Miyamoto, V. Kochin, A. Hongo, S. Tokita,Y. Kikuchi, A. Takaya, T. Terui, K. Ishitani, F. HataAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Miyamoto, V. Kochin, T. Kanaseki, T. Tsukahara,I. Takemasa, A. Miyazaki, H. Hiratsuka, T. TorigoeWriting, review, and/or revision of the manuscript: S. Miyamoto, T. KanasekiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. TorigoeStudy supervision: T. Kanaseki, N. Sato, T. Torigoe

AcknowledgmentsThe authors thank Dr. Goto (Sumitomo Dainippon Pharma) for providing

tetramers and Mr. Matsuo (Sapporo Clinical Laboratory) for technical supporton IHC. This work was supported by Japan Society for the Promotion of Science(JSPS) to T. Kanaseki, Suhara Kinen Zaidan to T. Kanaseki, Practical Research forInnovative Cancer Control from Japan Agency for Medical Research andDevelopment (AMED) to T. Torigoe, Grants-in-Aid of Ono Cancer ResearchFund and to T. Torigoe. Grant-in-Aid for Scientific Research from theMinistry ofEducation, Culture, Sports, Science and Technology of Japan to N. Sato, and aprogram for developing the supporting system for upgrading education andresearch from the Ministry of Education, Culture, Sports, Science and Technol-ogy of Japan to N. Sato.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 14, 2017; revised November 9, 2017; accepted January12, 2018; published OnlineFirst January 25, 2018.

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2018;6:358-369. Published OnlineFirst January 25, 2018.Cancer Immunol Res Sho Miyamoto, Vitaly Kochin, Takayuki Kanaseki, et al. CTL Immunotherapy of Colorectal CancerThe Antigen ASB4 on Cancer Stem Cells Serves as a Target for

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