of colorectal cancer · 1/23/2018 · samples were then loaded into a nano-flow uhplc (easy-nlc...

The antigen ASB4 on cancer stem cells serves as a target for CTL immunotherapy

of colorectal cancer

Sho 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, Toshihiko Torigoe1

1 Department of Pathology, Sapporo Medical University, Sapporo, Japan

2 Department of Oral Surgery, Sapporo Medical University, Sapporo, Japan

3 Higashi-Sapporo Hospital, Sapporo, Japan

4 Sapporo Dohto Hospital, Sapporo, Japan

5 Department of Surgery, Sapporo Medical University, Japan

Correspondence: Takayuki Kanaseki

Dept. of Pathology, Sapporo Medical University

Sapporo, Japan 060-8556

Tel: + 81-11-611-2111 (Ext. 25510)

FAX: + 81-11-643-2310

E-mail: [email protected]

on September 27, 2020. © 2018 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 25, 2018; DOI: 10.1158/2326-6066.CIR-17-0518

mailto:[email protected]

http://cancerimmunolres.aacrjournals.org/

Running title: A cancer stem cell antigen for CTL immunotherapy

Keywords: Cancer stem cells, Tumor-associated antigen, HLA-

class I, CTL, Immunotherapy

Disclosure of COI: The authors declare no competing financial interests

Abbreviations

CTL Cytotoxic T lymphocyte

CSC Cancer stem Cell

LC Liquid Chromatography

MS Mass Spectrometry

IHC Immunohistochemistry

mAb Monoclonal antibody




Abstract

Colorectal cancer consists of a small number of cancer stem cells (CSCs) and many

non-CSCs. Although rare in number, CSCs are a target for cancer therapy, because

they survive conventional chemo- and radiotherapies and perpetuate tumor formation in

vivo. In this study, we conducted an HLA ligandome analysis to survey HLA-A24

peptides displayed by CSCs and non-CSCs of colorectal cancer. The analysis identified

an antigen, ASB4, which was 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. Since ASB4 was not expressed by normal

tissues, its peptide epitope elicited CD8+ cytotoxic T-cell (CTL) responses, that lysed

CSCs of colorectal cancer and left non-CSCs intact. Therefore, ASB4 is a tumor-

associated antigen that can elicit CTL responses specific to CSCs and can discriminate

between two cellular subsets of colorectal cancer. Adoptively transferred CTLs specific

for the CSC antigen ASB4 could infiltrate implanted colorectal cancer cell tumors and

effectively prevented tumor growth in a mouse model. As the cancer cells implanted in

these mice contained very few CSCs, the elimination of a CSC subset could be the

condition necessary and sufficient to control tumor formation in vivo. These results

suggest that CTL-based immunotherapies against colorectal CSCs might be useful for

preventing relapses.




Introduction

Solid tumors are heterogeneous, consisting of a variety of cell types. One of these cell

types, the cancer stem cells (CSCs), or cancer-initiating cells, comprise a small subset

of tumor cells and are responsible for tumorigenesis1-3. The existence of CSCs was first

reported in a hematologic tumor, and thereafter in a variety of solid tumors, including

colon, breast, head and neck, uterine, brain, pancreas, and prostate cancers4-10.

Intratumoral heterogeneity and the presence of CSC subsets have been established in

primary solid-cancer tissues11. CSCs are resistant to conventional chemotherapy and

radiotherapy, most likely due to their quiescent status. CSCs may also have

mechanisms to activate drug transporters or DNA-damage checkpoint responses12-14.

Leukemic stem cells are resistant to a targeted agent, imatinib15. With these attributes,

CSCs may contribute to relapse or metastasis in clinical settings, increasing the

demand for a therapy focused on CSCs.

Our group and others have proposed that cytotoxic CD8+ T-lymphocytes (CTL)

could be used for the immunotherapeutic targeting of CSCs. Indeed, CTL responses

against CSCs have been demonstrated in the context of colon, kidney, cervical, brain,

head and neck, and breast cancers16-24. Thus, host CTLs may target CSCs, thereby

protecting against tumor growth in vivo. Meanwhile, it remains unclear whether CSCs

are an important therapeutic target. In some studies, CTLs responding to CSC antigens

also recognized or lysed non-CSC counterparts, which account for the majority of cells

in solid cancers. To disambiguate effects of CTLs on CSCs from effects on non-CSCs,

we searched colorectal cancer cells for CSC-specific antigens that were naturally

processed and elicited CTL responses only to CSCs, leaving non-CSCs intact.

In this study, we took advantage of a pair of cell lines that arose from side-

population (SP) and main-population (MP) cells of colorectal cancer, which provided us




with a consistent source of CSCs26. We then mapped the HLA-A24 peptide landscapes

of the CSCs and non-CSCs with HLA ligandome analysis using mass spectrometry. The

comparison between those two cellular subsets revealed that many HLA-A24 peptides

were expressed by both cell types. A few peptides were identified only in CSCs. We

further analyzed one of these peptides: ankyrin repeat and SOCS box protein 4 (ASB4).

CSCs but not non-CSCs expressed the gene and the peptide. Here, we found that the

CTLs responding to the ASB4 antigen discriminate CSCs from non-CSCs of colorectal

cancer and controlled colorectal cancer growth in vivo. These data may suggest that

control of CSCs is required to prevent colorectal cancer growth. The CSC-specific

antigen ASB4 may be useful as a therapeutic target in immunotherapy against

colorectal cancer.




Materials and Methods

Cell lines

Cell lines were maintained in RPMI 1640 or DMEM supplemented with 10% FBS and

1% antibiotics. The human cell lines used in this study were as follows: colon carcinoma

(SW480, SW620, Colo205, HCT15, Colo320, HCT116, HT29, and CRC21), lung

carcinoma (A549, SBC1, SBC5, and Lc817), kidney carcinoma (Caki-1 and ACHN),

liver carcinoma (HepG2), breast carcinoma (MCF7 and MDA-MB-468), ovary carcinoma

(ES2), prostate carcinoma (PC3), pancreas carcinoma (panc1), melanoma 1102-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, K562

were purchased from American Type Culture Collection. SBC1, SBC5, Lc817 were

purchased from the Japanese Cancer Research Resources Bank (JCRB, Osaka,

Japan). 1102-MEL was a gift from Dr. F.M. Marincola (National Cancer Institute,

Bethesda, MD). T2-A24 was a gift from Dr. K. Kuzushima (Aichi Cancer Center

Research Institute). CRC21 was established in our lab. The cell lines were immediately

frozen in batches on arrival, and the culture period of each batch was limited to a

maximum of 8 weeks. Because the HCT-15 line lacks beta 2-microglobulin (β2m)

expression, we used HCT-15 cells stably expressing β2m throughout the whole study.

Antibodies




Hybridomas for anti-HLA-A24 (C7709A2, a gift from Dr. P.G. Coulie), pan HLA-class I

(W6/32, ATCC), and anti–HLA-DR (L243, ATCC) were cultured in Hybridoma-SFM

(Gibco) supplemented with 1% penicillin/streptomycin in CELLine bioreactor flasks

(Corning). Produced mAbs were condensed and collected through a semipermeable

membrane during cell culture.

Side population assay

The original protocol has been previously described46, 47. Briefly, cells were labeled with

5 µg/ml Hoechst 33342 dye (Lonza, Walkersville, MD) for 90 minutes in the presence or

absence of 50 µmol/L verapamil (Sigma-Aldrich). Verapamil was used to inhibit ABCG2

activity. Approximately 1×106 viable cells were then analyzed and sorted using a

FACSAria II (BD Biosciences, Franklin Lakes, NJ). The Hoechst dye was excited with

the UV laser at 355 nm and its fluorescence was measured using a 450/20 nm band-

pass filter (Hoechst Blue) and a 670 nm long-pass filter (Hoechst Red). Dead cells were

labeled with 1 μg/mL propidium iodide and excluded from the analysis.

Sphere formation assay

Cells were cultured in ultra-low attachment plates (Corning Incorporated Life Sciences,

Acton, MA, USA) in serum-free Dulbecco’s modified Eagle’s medium/F12 medium (Life

Technologies) supplemented with 20 ng/ml human recombinant epidermal growth factor,

10 ng/ml human recombinant basic fibroblast growth factor (R&D Systems, Minneapolis,

MN, USA), and 1% N2 supplement (Invitrogen). On day 7, we collected spheres or

counted the number of spheres of which the diameter was >100 µm.




HLA-A24 ligandome analysis

The peptides bound to HLA-A24 of SP-H and MP-A cells were isolated and sequenced

using mass spectrometry. We used two different mass spectrometers, Q-Exactive Plus

(Thermo) and 4800 Plus MALDI-TOF/TOF Analyzer (AB Sciex), and the detected

peptides in three technical replicates each for SP-H and MP-A were collectively

analyzed. The precise workflow and procedure have been previously described27, 48.

Cell lysates were prepared from approximately 1.0×109 SP-H or MP-A cells with lysate

buffer containing 0.6% CHAPS (DOJINDO) and 1×complete protease inhibitor (Roche)

in PBS. The peptide-HLA-A24 complexes included in the samples were then isolated

using affinity chromatography of an HLA-A24 specific C7709A2 mAb coupled to

cyanogen bromide-activated Sepharose 4B (GE Healthcare). The HLA-bound peptides

were then eluted with 0.2% TFA and purified using a 10 kDa ultra-centrifugal filter

(Millipore), desalted using C18 ZipTip (Millipore), and condensed by vacuum

centrifugation.

Samples were then loaded into a nano-flow UHPLC (Easy-nLC 1000 system,

Thermo) online-coupled to an Orbitrap mass spectrometer equipped with a nanospray

ion source (Q-Exactive Plus, Thermo). We separated the samples using a 75 µm x 20

cm capillary column with a particle size of 3 µm (NTCC-360, Nikkyo Technos) by

applying a linear gradient ranging from 3% to 30% buffer B (100% acetonitrile and 0.1%

formic acid) at a flow rate of 300 nl/min for 80 min. In mass spectrometry analysis,

survey scan spectra were acquired at a resolution of 70,000 at 200 m/z with a target

value of 3e6 ions, ranging from 350 to 2000 m/z with charge states between 1+ and 4+.

We applied a data-dependent top 10 method, which generates high-energy collision

dissociation (HCD) fragments for the 10 most intense precursor ions per survey scan.

MS/MS resolution was 17,500 at 200 m/z with a target value of 1e5 ions.




For MS/MS data analysis, we used the Sequest HT (Thermo) and Mascot ver 2.5

(Matrix Science) algorithms embedded in the Proteome Discoverer 2.1 platform

(Thermo), and the peak lists were searched against the UniProt human databases. The

tolerance of precursor ions and fragment ions were set to 10 ppm and 0.02 Da,

respectively. More than 80% of the peptides including IV9 listed in Supplementary Table

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 to avoid overlooking potential CSC antigens.

RT-PCR and quantitative PCR

Total 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

instructions. cDNA was synthesized from 2μg of total RNA by reverse transcription with

Superscript III and oligo(dT) primers (Life Technologies). cDNA from human fetal and

adult tissues was purchased from Clontech and Bio Chain. RT-PCR mixtures were

initially incubated at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 15

s, annealing at 63°C for 30 s, and extension at 72°C for 30 s. Primer pairs were as

follows: ASB4, 5′-CTGTCTTGTTTGGCCATGTG-3′ and 5′-

GCGTCTCCTCATCTTGGTTG-3′ (product size 288 bp); G3PDH, 5′-

ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ (product size

452 bp).

Gene expression was also quantified using a StepOne Real-Time PCR System

(Applied Biosystems) with PowerUp SYBR Green Master Mix (Thermo). An initial

denaturation step of 95°C for 10 min was followed by 40 cycles of denaturation at 95°C

for 15 s and annealing/extension at 60°C for 60 s. Primer pairs for qPCR were as

follows: ASB4, 5′-CTGTCTTGTTTGGCCATGTG-3′ and 5′-




GCGTCTCCTCATCTTGGTTG-3′; G3PDH, 5′-GGATTTGGTCGTATTGGG-3′ and 5′-

GGAAGATGGTGATGGGATT-3′. Each sample was analyzed in triplicate and the

threshold cycle values (Ct) of ASB4 were normalized according to those of G3PDH.

Synthetic peptides and binding assay

Synthetic peptides of the following purities were purchased from Sigma Genosis (IV9

(IYPPQFHKV), 91.7%; HIVenv584-594 (RYLRDQQLL), 81.5%; GK12 (GYISPYFINTSK),

89.7%). Peptides in a range of the indicated concentrations were pulsed onto T2-A24

cells, incubated for 3 h at 27 °C, and then incubated for 2.5 h at 37 °C. Cells were

incubated with C7709A2, followed by a secondary FITC-conjugated antibody, and then

analyzed using flow cytometry. The difference in mean fluorescence intensity (ΔMFI)

indicates the difference in MFI values between samples with and without the primary

antibody.

CTL induction and cloning

We used the established method for CTL induction49. Briefly, CD8+ cells were

isolated from PBMCs of HLA-A*24:02-positive healthy donors or colorectal cancer

patients using anti-CD8 coupled to magnetic microbeads (Miltenyi Biotec). Remaining

CD8- cells were PHA-activated and used as PHA blasts. CD8+ cells were cultured in

AIM-V medium (Life Technologies) containing 10% human serum (kindly provided by Dr.

Takamoto, Japanese Red Cross Hokkaido Block Blood Center), 20 U/ml human IL-2

(kindly provided by Takeda Pharmaceutical), and 10 ng/ml IL-7 (R&D systems) for 4

weeks. The CD8+ cells were repeatedly stimulated with autologous PHA-blasts pulsed

with 20 µM of the IV9 peptide. To generate CD8+ T-cell clones, single cells binding both




anti-CD8 (Beckman Coulter) and an IV9-HLA-A24 tetramer (MBL, Japan), as well as the

single cells positive for CD8 but negative for binding to the tetramer, were isolated using

FACS Aria II (BD). They were expanded in AIM-V medium containing 100 U/ml IL-2, 1

μg/ml PHA, and X-ray-irradiated PBMCs from healthy volunteers. The SM4 and control

CTL clones were selected from the tetramer-positive and negative clones, respectively.

ELISPOT IFNγ assay

Tetramer-positive CTLs were added to ELISPOT plates coated with anti-Human IFNγ

(BD Biosciences) at 5.0×104 cells/ml per well. T2-A24 or the indicated cancer lines at

5.0×104 was added to the corresponding wells. T2-A24 was pre-incubated at room

temperature for 2 h with 20 μM of IV9 or irrelevant HIV peptide. After incubation in a 5%

CO2-incubator at 37 °C for 24 h, the wells were incubated with a biotinylated anti-human

IFNγ antibody for 2 h at room temperature, followed by the ELISPOT Streptavidin-HRP

antibody for 1 h. Spots were visualized using the ELISPOT AEC Substrate Set

according to the manufacturer’s instructions (BD Biosciences).

Biochemical analysis of naturally processed peptides using RP-HPLC

This analysis followed the original protocol with modifications50. Peptide extracts of

2×108 SP-H or MP-A were prepared by acid extraction using 10% formic acid in the

presence of 2 µM of an irrelevant peptide followed by filtration using a <10k Da cut-off

spin-column (Amicon). The samples were fractionated using RP-HPLC equipped a

2.1×250 mm C18 column with a particle size of 2 µm (ZORBAX 300-SB C18, Agilent) by

applying a linear gradient ranging from 23% to 45% buffer B for 45 min. Each fraction

was then dried using vacuum centrifugation overnight. Each fraction was incubated with




5×104 SM4 and 5×104 T2-A24. The IFNγ produced by SM4 responding to its naturally

processed epitope was detected using the ELISPOT assay.

LDH cytotoxicity assay

The amount of lactate dehydrogenase (LDH) released from lysed target cells was

measured using an LDH cytotoxicity detection kit according to the manufacturer’s

instructions (TaKaRa, Japan). Target cells (1.0×104) were co-incubated with the

indicated numbers (E/T ratio) of CTLs at 37˚C for 6 h. The percentage of LDH released

(cytotoxicity) was calculated as follows: % LDH release = 100 × (experimental LDH

release - spontaneous LDH release)/(maximal LDH release - spontaneous LDH

release). LDH values from CTL alone and target cells treated with 2% Triton X-100

(Sigma) were used to estimate spontaneous and maximal LDH releases, respectively. In

the HLA-blocking assay, target cells were pre-incubated for an hour with 100 μg/mL of

anti-HLA-class I (W6/32), anti-HLA-2402 (C7709A2) or anti-HLA-DR (L243).

Mice and xenograft models

NSG mice were purchased from the Jackson Laboratory. The mice were maintained in

the animal facility of Sapporo Medical University and all procedures were performed in

accordance with the institutional animal care guidelines. To evaluate the tumorigenicity

of SP-H and MP-A, NSG mice were subcutaneously injected 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

subcutaneously injected, followed by the adoptive intravenous transfer of 5.0×105 CTLs

at the indicated time points. The major (x) and minor (y) axes of the tumors were

routinely measured. Tumor volume was calculated as follows: volume = xy2/2. To




assess CTL infiltration into tumors, NSG mice were subcutaneously injected with

1.0×106 SW480 cells. Subsequently, the mice were intravenously injected with 1.0×105

and 2.0×106 CTLs, on day 28 and 29, respectively. The tumors and spleens were

removed and fixed with 10% formalin on day 31. The paraffin-embedded tissues were

immunohistochemically stained with a human CD8 antibody (DAKO). The numbers of

CD8+ cells per HPF were manually counted.

Patients

The study was performed with approval of the Institutional Review Board of Sapporo

Medical University. Clinical samples of patients with colorectal cancer were included in

this study, with informed consent according to the guidelines of the Declaration of

Helsinki. PBMCs were isolated using Lymphoprep (Nycomed) from whole blood

samples of patients and healthy donors.

IV9-specific CD8+ T-cell precursor frequency of colorectal cancer patients

PBMCs from HLA-A24-positive colorectal cancer patients were distributed over 24 wells

per patient and cultured in AIM-V medium supplemented with 5% human serum and 50

U/ml IL-2. PBMCs were stimulated with 20 µM IV9 peptide on day 0 and day 8, and then

stained with anti-CD8, an IV9-HLA-A24 tetramer, and an irrelevant HIV-HLA-A24

tetramer on day 15. The frequency of anti-IV9 CD8+ T-cell precursors was calculated as

follows: frequency = the number of wells positive to IV9-HLA-A24/(24 × the initial

number of CD8+ cells per well). Samples showing >0.02% positivity were counted as

positive to the IV9-HLA-A24 tetramer.




Results

A clone derived from human colorectal cancer cells shows CSC phenotypes

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

challenging28. To address this issue, we generated both CSC and non-CSC lines from a

single-cell clone of human colorectal cancer SW480 cells26. In that study, we isolated

both side and main population cells using Hoechst 33342 dye staining, and

demonstrated that both CSC-like (SW480-SP-A, SP-B, and SP-H) and non-CSC-like

(SW480-MP-A, MP-D, and MP-K) phenotypes were sustained in culture. Although 1-2%

of the parent SW480 cell lines were side-population cells, a side-population assay

demonstrated that the representative clone SP-H was 32.6% side-population cells, and

the enriched side-population cells were maintained after 4 months in vitro serum culture

(Fig. 1A). Likewise, <0.1% of a representative clone of MP-A were side-population cells

and this clone never generated new side-population cells (Fig. 1B). The number of

spheres formed in non-serum culture was approximately 10.6 times higher in SP-H than

MP-A (Fig. 1C). In addition, SP-H formed significantly larger masses in mouse xenograft

models in vivo (Fig. 1 D-F). Tumor masses were palpable on day 28 and 20 of 1.0×103

and 1.0×104 SP-H implantation, respectively, whereas MP-A never formed palpable

tumors by day 42. These data indicate that the tumor-initiating ability of SP-H was

higher than that of MP-A both in vitro and in vivo. We conclude that SP-H and MP-A

clones represent CSC and non-CSC phenotypes. Both clones maintained stable

phenotypes.




HLA-A24 ligandome analysis identified a CSC-specific peptide, IV9

CSCs are long-lived in the host environment and harbor tumor-initiating ability, hence

are a target for immunotherapy29. Since antigen processing of CSCs is not fully

understood, we directly surveyed the HLA-A24 peptide landscape of SP-H and MP-A

(Fig. 2A). Briefly, 1×109 cells of SP-H or MP-A were lysed, and a mixture of peptide-

HLA-A24 complexes were immunoprecipitated using an HLA-A24-specific antibody. The

bound peptides were then eluted and sequenced using mass spectrometry coupled with

nano-flow liquid chromatography. The analysis identified 178 sequences of non-

redundant HLA-A24 peptides from SP-H and MP-A (Supplementary Table S1). The

average length of the detected sequences was approximately 9.1 amino acids (Fig. 2B),

and both tyrosine (Y) at P2 and phenylalanine (F) or leucine (L) at PΩ were conserved

across the sequences (Fig. 2C). These peptide profiles ensured that our ligandome

analysis identified HLA-A24 ligands from SP-H and MP-A30.

Although most of the peptides were MP-A-specific (86 peptides) or shared

between SP-H and MP-A (57 peptides), 35 peptides were detected only in SP-H (Fig.

2D). Removal of peptides for which the source gene was expressed in normal organ

tissues left a 9-mer peptide (IYPPQFHKV, “IV9”) (Fig. 2E). This peptide, IV9, was

repeatedly isolated from SP-H samples but never detected in MP-A samples. Although

both SP and MP clones expressed HLA-A24 on the cell surface, MP clones expressed

more HLA-A24, potentially explaining a difference in the number of isolated HLA-A24

peptides between SP and MP clones (Supplementary Fig. S1).

ASB4 is expressed by variety of cancers but not by normal tissues

IV9 is encoded by both of two known isoforms of ASB4 (UniProt ID: Q9Y574), a

potential component of the E3 ubiquitin ligase complex (Fig. 3A)31. The ASB4 gene was




expressed by a variety of cancer cell lines, including lines derived from colorectal

cancers (SW480, SW620, colo205, and HCT15), lung cancers (A549, SBC1, and

SBC5), and a kidney cancer (Caki-1), as well as a liver cancer (HepG2) (Fig. 3B).

Expression was higher in clinically dissected colorectal cancer tissues, greater than 5-

fold higher in 6 out of 21 cases (Fig. 3C). The ASB4 gene was little expressed by a

panel of normal adult and fetal tissues as well as cultured mesenchymal stem cells

derived from bone marrow (Fig. 3C and Supplementary Fig. S2). Thus, expression of

the ASB4 gene was tumor-specific in both cell lines and primary colorectal cancer

tissues.

IV9 is a natural CD8+ T-cell epitope processed and presented only by SP-H

The synthetic IV9 peptide harboring Y at P2 and V at P9 stabilizes expression of HLA-

A24 on T2-A24 cells (Fig. 4A). We stimulated peripheral blood mononuclear cells

(PBMCs) derived from a healthy donor with the IV9 peptide. From a cell population

responding to an IV9-HLA-A24 tetramer, we established the CD8+ T-cell clone line

(SM4), 98.8% of which was positive for the tetramer (Fig. 4B). The SM4 line responded

to SW480 as well as to T2-A24 pulsed with 2 µM IV9 peptides, producing IFNγ (Fig.

4C). SM4 produced IFNγ in response to SP-H and SW480, but not in response to MP-A

cells. The SM4 line produced more IFNγ against the MP-A cells expressing ASB4,

demonstrating that ASB4 was the responsible antigen encoding the CTL epitope (Fig.

4D and E).

Next, we biochemically evaluated and quantified the natural T-cell epitope

produced in live SP-H and MP-A cells. We fractionated cell extracts from 2×108 SP-H

and MP-A using RP-HPLC, then incubated these extracts with SM4 in the presence of

T2-A24 as antigen-presenting cells. We measured production of IFNγ using an




ELISPOT assay (Fig. 4F). We found that the SP-H extract contained the natural SM4

epitope at fraction #5, the same fraction as synthetic IV9 peptide. Since this assay used

a consistent set of T cells and APCs across samples, only the amount of the IV9 peptide

included in samples influenced the number of IFNγ spots. Therefore, we estimated the

amount of IV9 in 2×108 SP-H at approximately 12 fmol or more, because the number of

positive spots at #5 was increased by 1.28-fold (96/75) compared to that of the 10 fmol

synthetic peptide. MP-A extract did not contain IV9. Considering that ASB4 gene

overexpression restored SM4 responses against MP-A, we assumed that the antigen

processing machinery of MP-A was capable of presenting the IV9 peptide. However,

loss of the ASB4 gene expression resulted in loss of the IV9 peptide. Thus, IV9 is the

natural CTL epitope and its presentation is limited to the colorectal CSC subset SP-H.

Colorectal CSCs express ASB4

In 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 cells without

dedifferentiation, SP-H is composed of both side- and main-population cells, suggesting

that side-population cells in SP-H constantly differentiate into main-population cells (Fig.

1A). We therefore re-sorted SP-H cells cultured for 3 weeks into side and main

populations again and determined ASB4 expression in each subset. Only the side-

population descendants (SP-1, -2, and -3) but not the main-population descendants

(MP-1, -2, and -3) expressed ASB4, suggesting that side-population cells of SP-H

ceased ASB4 expression when differentiated into main-population cells (Fig. 5B).

Moreover, the ASB4 expression was detected in 4 out of 8 colorectal cancer lines

derived from 3 different colorectal cancer patients (SW480 and 620, Colo205, and




HCT15) (Fig. 3B). In fact, the expression levels increased to 1.9-3.0-fold when CSCs

were enriched by sphere culture using ultra-low attachment plates without serum (Fig.

5C). Sphere culture did not influence the expression in Colo320 and HCT116, both of

which were ASB4-negative. Sphere culture increased sphere formation in every

colorectal cancer line as well as ASB4 expression in SW480 and HCT15 using RT-PCR

(Supplementary Fig. S3A-C). To further investigate the enrichment of the IV9 epitope

presentation by CSCs, we tested the SM4 responses. The amounts of IFNγ produced

by SM4 were increased by approximately 1.6-fold against SW480 and HCT15

expressing β2m under sphere culture (Fig. 5D). These data demonstrate that ASB4 is

the antigen of which the expression and following CTL responses were linked to

colorectal CSC subsets.

SM4 lyses colorectal CSCs but not non-CSCs

We aimed to identify CSC antigens that are exclusively presented by CSCs and elicit

CTL responses lysing only CSCs. In accordance with the ELISPOT results, SM4 lysed

T2-A24 cells pulsed with the synthetic IV9 peptide, but ignored control peptides or K562

cells (Fig. 6A). SM4 successfully lysed SP clones (SP-A, SP-B, and SP-H) and, as we

expected, left MP 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 a CSC-

specific antigen and the CTL activity discriminates tumorigenic CSCs from non-CSCs.

The result was consistent with the SP-H-specific IV9 peptide production observed in

Fig. 4F. SP-H and unsorted SW480 contain approximately 30% and 1-2% of side-

population cells, respectively (Fig. 1A and Supplementary Fig. S4). We found that SM4

lysed SW480, besides SP-H, to as low as 59.3% of SP-H at an effector/target (E/T) ratio

of 9 (Fig. 6C). Since SM4 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/β2m, which expresses ASB4 and is HLA-A24-positive, but

left Colo205 and Colo320 intact, both of which lack HLA-A24 (Supplementary Fig. S5).

The CTL-mediated lysis of SW480 was blocked by the presence of the pan-HLA class I

W6/32 mAb as well as the HLA-A24-specific C7709A2 mAb, but not by the HLA-DR-

specific L243 mAb, ensuring that the response was restricted to HLA-A24 (Fig. 6D).

Adoptive transfer of SM4 CTLs suppressed tumor growth in vivo

Finally, we investigated the effects of SM4 on colorectal cancer using in vivo models. A

randomly generated CTL clone was prepared as a control, which did not respond to an

IV9-HLA-A24 tetramer. This clone recognized neither SP-H nor SW480 or MP-A

(Supplementary Fig. S6). Immunodeficient NSG 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 day 35 and 45) (Fig. 7A). We used both models to evaluate

anti-tumor CTL effects at early stages before tumors developed into large masses. In

contrast to the control CTL clone, the adoptive transfer of SM4 significantly prevented

tumor growth in both CTL injection models “before and after” tumor implantation (Fig. 7B

and C, respectively). Even at the end of the time courses on day 56, both adoptive

transfer models controlled tumor sizes such that implanted tumors were not palpable.

Hence, SM4, which targeted only an SP subset of SW480, prevented tumor formation of

SW480, which was composed of 1-2% of SP cells among otherwise MP cells. This

result suggests that the CSC subset is a necessary and sufficient target in preventing

colorectal cancer formation at the early stages.

We also prepared mice bearing established masses of SW480 and counted the

number of tumor-infiltrating CTLs 2 days after the adoptive transfer of SM4




(Supplementary Fig. S7A). The IHC results showed that SM4, but not control CTLs, was

present inside SW480 tumors, whereas both CTLs were found inside the spleens (Fig.

7D-F). We estimate that the in vivo SW480 tumors were contained 2% or fewer SP

cells, as is observed for in vitro cultures26, because a similar analysis with SP-B tumors

revealed that the percent of SP cells was stable in vivo (Supplementary Fig. 7B). The

data demonstrated that SM4 was capable of homing to IV9-HLA-A24 complexes and

migrating into tumors in search for a small cell population of CSCs.

Immune surveillance of IV9-specific CD8+ T cells in patients with colorectal

cancer

Because the IV9 peptide identified in this study is displayed by HLA-A24, which is the

most common HLA-A type among the East Asian population, we focused on HLA-A24-

positive colorectal cancer patients and assessed the frequency of IV9-specific CD8+ T

cells in their PBMCs. All 6 patients were histologically diagnosed with colorectal

adenocarcinoma and the group consisted of 3 men and 3 women, aged 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. The group

included five patients at stage IV and one patient at stage II. PBMCs from the patients

were randomly fractionated and stimulated with 20 µg/ml of IV9 peptides for 14 days.

Induced IV9-specific CD8+ cells were detected and counted using an IV9-HLA-A24

tetramer (Fig. 8A). We regarded the cell fraction labeled with 0.03% or more proportions

as tetramer positive32. As a result, T-cell induction was detected in 5 out of 6 patients,

and the frequency of IV9-specific T-cell precursors ranged from 4.4×10-7 to 4.1×10-6 of

CD8+ cells (Fig. 8B). The lower limit of detection in this assay was around 2.0×10-7 and

the frequency in one patient (B05) was under the limit. Three of the patients (B01, B03,




and B06) at stage IV reached long survival (>8 years); for these three patients we

detected IV9-tetramer positive CD8+ T-cells in multiple wells. Thus, CD8+ T cells of

colorectal cancer patients are not immunologically tolerant to the ASB4 antigen and IV9

peptide stimulation elicited specific T-cell responses in patients with colorectal cancer.




Discussion

CSC expression is prioritized in selection of antigens appropriate for cancer vaccination.

Antigen expression in a CSC subset might influence anti-tumor CTL effects33. In our

study, not only is the IV9 peptide a natural CTL epitope directly identified using HLA-

ligandome analysis, but also it is presented by an SP but not an MP subset of SW480

cells. Repeated detection by biochemical analysis also implies that the IV9 peptide is

presented by HLA-A24 of CSCs, rendering it a target of CTL immune surveillance. IV9-

CTLs indeed discriminated CSCs from non-CSCs, lysing only the CSC subset. The IV9-

CTLs belong to a group of CTLs that recognize CSCs, but are capable of ignoring non-

CSCs. The adoptive transfer of the CTLs prevented tumor formation in NSG mice

implanted with 1×103 unsorted SW480 cells that consisted of 1-2% of SP and the

majority of MP cells. This result demonstrated that most SW480 cells were not the

target, and the ability of IV9-CTLs to ignore non-CSCs did not harness the anti-tumor

effect in vivo. Although non-CSCs are predominant in number in many tumors, only

CSCs are able to sustain tumorigenesis34. 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. These data together suggest that the CSC

subset is a useful target of immunotherapy despite of its rarity.

The IV9 peptide is encoded by ASB4, the gene expression of which was not

detected using RT-PCR in an array of normal fetal and adult tissues, including

mesenchymal stem cells of the bone marrow. Expression increased in colorectal cancer

tissues, and moreover, 28.6% (6 out of 21) of cases showed greater than a 5-fold

increase compared to normal colon tissue without enrichment. Considering that the

sphere culture of SW480, Colo205, and HCT15 enriched ASB4 gene expression, each

primary colorectal cancer tissue is composed of varying 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 function is unclear. In mice, the

expression of the ASB4 gene has been reported in the developing placenta and the

testis; however, adult tissues cease ASB4 gene expression, suggesting ASB4 functions

early in development, perhaps at trophoblast differentiation35-37. On the contrary, its

increased expression has been reported in hepatocellular carcinomas and adrenal

gland tumors, which may suggest another role of ASB4 in metastasis or

tumorigenesis38, 39. We quantitatively compared sphere formation of SP-H in the

presence and absence of ASB4 gene expression; however, silencing ASB4 gene

expression did not decrease the number of spheres. More persistent gene silencing

other than siRNA might be necessary to observe the phenotype change, or this result

could suggest that ASB4 does not serve as a driver of sphere formation. Nevertheless,

the findings of the present study demonstrate that the presence of the ASB4 antigen is

linked to a CSC subset of colorectal cancer. ASB4 may hold promise as a therapeutic

target of CTL-based immunotherapy in colorectal cancer patients. Here, it was possible

to detect the IV9-CD8+ T-cell precursors in 5 out of 6 colorectal cancer patients, and we

knew that the remaining patient (B05) had received dexamethasone for 6 months before

blood sampling, being in a clinical state of immune suppression. All three patients with

long-term survival over 8 years showed higher IV9-CD8+ T-cell frequency in contrast to

the others, suggesting a relationship between patient survival and CD8+ T-cell

surveillance against the ASB4 antigen.

Clinical success of immune checkpoint blockades both in reducing the sizes of

cancers and in prolonging cancer patient survival ensured that patients’ T-cell

responses are able to cope with malignancies40. Accumulating evidence suggests that

mutational load is positively correlated with clinical effects, and has sparked further

research on screening neoantigens that arise from gene mutations41-44. CTLs targeting




a neoantigen discriminate it from their wild-type counterpart, and exhibit high cytotoxicity

against tumors carrying the responsible gene mutation27. However, most colorectal

cancers belong to a microsatellite-stable tumor type, being an exception that is

refractory to immune checkpoint blockade despite their prevalence43. Individual variation

in gene mutations also call for personalized immunotherapy, which requires detection of

antigens for each patient45. In this study, the antigen we focused on was, conversely,

expressed only in a subset of colorectal cancer cells. In our SW480-derived SP- and

MP-clone models, certain descendants of SP-clones differentiated into MP cells during

culture, keeping the proportion of SP-cells low. Therefore, we consider that eliminating

such a small subset of cancer cells may not be effective in reducing the sizes of already

developed bulky tumors in vivo. Instead, the CSCs responsible for the onset of tumor

formation are shared among patients, generating a practical target for vaccination. The

capability of IV9-CTLs to home to the target may help to identify tumor buds scattered

over the body of patients at an early stage. We thus deduce that CSC-specific CTL

surveillance could be useful to colorectal cancer patients with cured primary lesions in

preventing relapse or metastasis.




Acknowledgments and funding

We thank Dr. Goto (Sumitomo Dainippon Pharma) for providing tetramers and Mr.

Matsuo (Sapporo Clinical Laboratory) for technical support on IHC. This work was

supported by Japan Society for the Promotion of Science (JSPS) to T.K., Suhara Kinen

Zaidan to T.K., the Practical Research for Innovative Cancer Control from Japan Agency

for Medical Research and development (AMED) to T.T., Grants-in-Aid of Ono Cancer

Research Fund and to T.T. Grant-in-Aid for Scientific Research from the Ministry of

Education, Culture, Sports, Science and Technology of Japan to N.S., and a program

for developing the supporting system for upgrading education and research from the

Ministry of Education, Culture, Sports, Science and Technology of Japan to N.S. The

authors declare that they have no conflicts of interest.




Figure legends

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. Upper and

lower panels 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 numbers inside 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 µm) were microscopically detected and the

bar chart summarizes the numbers of wells containing at least one sphere among 96

wells. Data are shown as mean + SEM (n = 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 + SD (n = 9 in E, and n = 14 in F) and P

values were calculated using a two-tailed t-test (* P < 0.05 and ** P < 0.01).

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 analysis

comprehensively 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 ligands detected 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) Tandem mass (MS/MS) spectrum of IYPPQFHKV

(IV9). Detected b- (or a-) and y-ions are indicated in red and blue, respectively.

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

(UniProt ID: Q9Y574-2, bottom) encode the IV9 sequence (underlined). (B) RT-PCR of

indicated types of cancer cell lines. ASB4 and G3PDH bands appeared at 288 and 452

bp, respectively. Data are representatives of three independent experiments. (C)

Quantitative-PCR of a panel of healthy adult and fetal tissues as well as primary

colorectal cancer tissues (T01-21). The y-axis, ASB4 expression relative to a healthy

adult colon tissue. Dashed line indicates 5-fold expression. Data are shown as mean +

SEM (n = 3). The experiment was conducted in triplicate.

Figure 4. IV9 is a natural CD8+ T-cell epitope processed and presented only by

SP-H. (A) Peptide binding assay using T2-A24 cells pulsed with the indicated synthetic

peptides and an HLA-A24 specific antibody (C7709A2). HIV and GK12 are

representative HLA-A24-binding and -non-binding peptides, respectively. The x-axis, the

concentration of pulsed synthetic peptides; the y-axis, the difference between samples

and negative controls without primary mAb in units of mean fluorescence intensity

(ΔMFI). Data are shown as mean + SEM (n = 3) and P values were calculated using a




two-tailed t-test (** P < 0.01). (B) Flow cytometry of an IV9-specific CTL clone (SM4)

stained with an IV9-HLA-A24 tetramer together with an HIV-HLA-A24 tetramer. The

number inside the plot indicates the percentage cell population surrounded by the

rectangle. The plot is a representative of three independent experiments. (C) IFNγ

ELISPOT assay of SM4. Target cells were T2-A24 cells pulsed with indicated synthetic

peptides, SP-H, MP-A, or SW480 cells. The bar chart summarizes the numbers of IFNγ

spots per well. Data are shown as mean + SEM (n = 3) and P values were calculated

using a two-tailed t-test (** P < 0.01). Photographs are representative IFNγ spots in

each condition. (D) RT-PCR of MP-A stably expressing an empty vector or ASB4 as well

as SP-H. Data are representatives of three independent experiments. (E) IFNγ

ELISPOT assay of SM4. Target cells were SP-H or MP-A stably expressing an empty

vector or ASB4. Data are shown as mean + SEM (n = 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 each fraction were then measured based on SM4 responses in

the presence of T2-A24 cells using IFNγ ELISPOT assay. Samples were 10 fmol of IV9

synthetic peptides (left), a cell extract of 2.0×108 SP-H (middle), and a cell extract of

2.0×108 MP-A (right). Mock indicates the results of RP-HPLC buffer alone, measured in-

between sample runs. Data are representatives of two independent experiments.

Figure 5. The ASB4 antigen is enriched in colon CSC subsets. (A) Quantitative-

PCR of indicated SP and MP clones derived from SW480. The y-axis, ASB4 expression

relative to a healthy adult colon tissue. Data are shown as mean + SEM (n = 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 of colorectal

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 as mean + SEM (n = 3) and P values

were calculated using a two-tailed t-test (* P < 0.05). (D) IFNγ 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 IFNγ spots per well. Data are shown as mean + SEM (n = 3) and P values

were calculated using a two-tailed t-test (* P < 0.05 and ** P < 0.01).

Figure 6. 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-A24

or 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-HLA class 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/T = 9 (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 as mean + SEM (n = 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).

Figure 7. The adoptive transfer of SM4 CTLs prevents tumor development in vivo.

(A) Time courses of tumor implantation and CTL adoptive transfer. NSG mice were

subcutaneously 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 day 35 and 42) tumor injection. Tumor

growth rates in the adoptive CTL-transfer models are shown in (B) and (C). The x-axis




and the y-axis indicate days after tumor implantation and the sizes of tumors,

respectively. Data are shown as mean + SD (n = 7 (B) or 5 (C)) and P values were

calculated using a two-tailed t-test (** P < 0.01). (D-F) Immunohistochemistry of tissues

from NSG mice, which bore SW480 tumors and received CTL transfers (Supplementary

Fig. S7). (D) Representative results of human CD8 staining. Adoptively transferred SM4

CTLs were detected in both tumor and spleen sections, while control CTLs were

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 SM4 injection 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.

Figure 8. IV9-specific CD8+ T cells are frequently found in HLA-A24 colorectal

cancer patients. (A) Estimation of the frequency of IV9-specific CD8+ T cells in a

representative colorectal cancer patient. PBMCs from colorectal cancer patients were

distributed in 24 wells and stimulated with 20 µM of IV9 peptides according to the

procedure described in Materials and Methods. Staining results of CD8+ cells in each

well with IV9-HLA-A24 and control HIV-HLA-A24 tetramers are shown. The numbers

inside plots indicate percentage of IV9-HLA-A24 single-positive cells (surrounded by

rectangles). The frequency was calculated as follows: number of positive wells

(positivity rate > 0.02%)/(24 × the initial number of CD8-positive cells per well).

Tetramer-positive wells are shown in red. (B) Summarized frequency in 6 patients. All

patients were HLA-A24-positive and the patient information is provided in

Supplementary Table S2. The precursors were detected in 5 out of 6 cases.




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50. Kanaseki T, Shastri N. Biochemical analysis of naturally processed antigenic

peptides presented by MHC class I molecules. Methods Mol Biol 2013, 960: 179-

185.




150 200 250

32.6

%

29.5

%

Day 0

0.07

%

0.08

%

Hoechst red

Hoechst

blu

e

SP-H

MP

-A

0

50

Num

ber

of

sphere

-positiv

e w

ells *

SP-H MP-A

Figure 1

A C

D

40

30

20

10

50

100

150

200

250

01000 50 150 200 250

50

100

150

200

250

1000 500

Day 120

150 200

50

100

150

200

250

01000 50 250 150 200 250

50

100

150

200

250

1000 500

150 200 250

0.06

%

0.06

%

0.00

%

0.00

%

Hoechst red

B

50

100

150

200

250

01000 50 150 200 250

50

100

150

200

250

1000 500

150 200

50

100

150

200

250

01000 50 250 150 200 250

50

100

150

200

250

1000 500

MP-A

SP

-H

Verapamil (-) Verapamil (-)Verapamil (+) Verapamil (+)

**

SP-H 1.0x104

MP-A 1.0x104

F

400 50

2000

0

1000

3000

Days

302010

*

Days

Tum

or

volu

me (

mm

3)

0

SP-H 1.0x103

MP-A 1.0x103

E

1000

400 50302010

500




Figure 2

HLA-A24

Lysis

pHLA-A24

complexes

Peptide elution

LC-MS/MS

HLA-A24

HLA-A02

HLA-B

HLA-C

A

E

Inte

nsity c

ounts

(x10

3)

m/z

SP-H

MP-A

C

226.11833 854.47906735.39319

756.42523518.03937 638.34009

607.29907129.10220 853.47418

368.20041

136.07568

y₇⁺-NH₃

835.44592a₇²

y₈²

y₄²⁺-NH₃

257.13904

y₂⁺-NH₃

229.15509 b₄⁺

471.24179

y₅⁺

658.36365y₆²⁺-NH

y₁⁺

118.08611

y₅⁺-NH₃

641.34058

b₇²⁺

442.24500

y₇²⁺-NH₃

418.23401

y₄⁺

530.30902

y₃⁺

383.24060b₂⁺

277.15445

y₂⁺

246.18076

y₆²⁺

378.21338

y₆⁺

755.41876

y₇⁺

852.47205

y₇²⁺

426.73978

a₂⁺

249.15955

a₁⁺

86.09631

200 400 600 800 1000

m/z

0

20

40

60

80

100

120

140

Inte

nsity [

co

un

ts]

(10

^3

)

ST211_SW480_C7709A2.raw #24362 RT: 46.4711 min

FTMS, [email protected], z=+2, Mono m/z=564.81348 Da, MH+=1128.61968 Da, Match Tol.=0.02 Da

57

35

86

SP-H MP-A

D

B 150

100

50

0 8 9 10 11 12

SP-H MP-A

Num

ber

of

peptides

AA AA

150

100

50

0 8 9 10 11 12

I Y P P Q F H K V

b2 b4 b7

y1 y2 y3 y4 y5 y6 y7




Lu

ng

Liv

er

Skele

tal m

uscle

Leukocyte

Pro

sta

te

Pla

centa

Ovary

Testis

Bra

in

Kid

ney

Sm

all

inte

stine

Pancre

as

Heart

Sple

en

Thym

us

Colo

n

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nal gla

nd

Lu

ng

Liv

er

Skele

tal m

uscle

Bra

in

Kid

ney

Pancre

as

Heart

Sple

en

Colo

n

T01

T02

T03

T04

T05

T06

T07

T08

T09

T10

T11

T12

T13

T14

T15

T16

T17

T18

T19

T20

T21

Adult tissues

Colon cancer tissues

Fetal tissues

AS

B4 e

xpre

ssio

n (

fold

)

40

10

20

30

0

50

Figure 3

A B

C

MDGTTAPVTK SGAAKLVKRN FLEALKSNDF GKLKAILIQR QIDVDTVFEV

EDENMVLASY KQGYWLPSYK LKSSWATGLH LSVLFGHVEC LLVLLDHNAT

INCRPNGKTP LHVACEMANV DCVKILCDRG AKLNCYSLSG HTALHFCTTP

SSILCAKQLV WRGANVNMKT NNQDEETPLH TAAHFGLSEL VAFYVEHGAI

VDSVNAHMET PLAIAAYWAL RFKEQEYSTE HHLVCRMLLD YKAEVNARDD

DFKSPLHKAA WNCDHVLMHM MLEAGAEANL MDINGCAAIQ YVLKVTSVRP

AAQPEICYQL LLNHGAARIY PPQFHKVRLC PVVSRLRKIQ MAALSEISK

MDGTTAPVTK SGAAKLVKRN FLEALKSNDF GKLKAILIQR QIDVDTVFEV

EDENMVLASY KQGYWLPSYK LKSSWATGLH LSVLFGHVEC LLVLLDHNAT

INCRPNGKTP LHVACEMANV DCVKILCDRG AKLNCYSLSG HTALHFCTTP

SSILCAKQLV WRGANVNMKT NNQDEETPLH TAAHFGLSEL VAFYVEHGAI

VDSVNAHMET PLAIAAYWAL RFKEQEYSTE HHLVCRMLLD YKAEVNARDD

DFKSPLHKAA WNCDHVLMHM MLEAGAEANL MDINGCAAIQ YVLKVTSVRP

AAQPEICYQL LLNHGAARIY PPQFHKVIQA CHSCPKAIEV VVNAYEHIRW

NTKWRRAIPD DDLEKYWDFY HSLFTVCCNS PRTLMHLSRC AIRRTLHNRC

HRAIPLLSLP LSLKKYLLLE PEGIIY

SW

480

SW

620

Colo

205

HC

T15

Colo

320

HC

T116

HT

29

CR

C21

A549

SB

C1

SB

C5

LC

81

7

Caki1

AC

HN

HepG

2

MC

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MD

AM

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ES

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PC

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NC

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me

l

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S

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Colo

n c

a.

Lung c

a.

Kid

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Liv

er

ca

.

Bre

ast

ca

.

Ovary

ca

.

Pro

sta

te c

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ca

.

Oste

osarc

om

a




10

2

10

0

102 100

A24/HIV tetramer-FITC

0

2000

Figure 4

98.8%

A24/I

V9 t

etr

am

er-

PE

101 103 104

10

1

10

3

10

4

SP

-H

MP

-A

SW

480

（-） HIV IV9

T2-A24

**

**

IV9

300

200

100

0.3 1 3.3 10

ΔMFI

IFN

-γ s

pots

/ w

ell

400

0

600

200

800

(μM)

400

B A C

F

** **

IFN

-γ s

pots

/ w

ell

Fraction

HIV

GK12

0

80

100

60

40

20

1 2 3 4 5 6 7 8 9 10 11 12

IV9 peptide

Mock

0

80

100

60

40

20

1 2 3 4 5 6 7 8 9 10 11 12

MP-A

Mock

0

80

100

60

40

20

1 2 3 4 5 6 7 8 9 10 11 12

SP-H

Mock

Fraction Fraction

ASB4

G3PDH

+vec SP

-H

+ASB4

MP-A

D

**

+vec

1000

400

0

SP

-H

+ASB4

600

200

**

MP-A

800

E

IFN

-γ s

pots

/ w

ell




A

SP

-A

40

10

20

30

0

50

AS

B4 e

xpre

ssio

n (

fold

)

AS

B4 e

xpre

ssio

n Serum

Sphere

0

5

10

15

20

25

SW

480

Colo

205

HC

T15

Colo

320

HC

T116

*

*

*

Figure 5

MP

-A

SP

-B

SP

-H

MP

-D

MP

-K

300

0

200

100

SW

480

HC

T15

Colo

320

400 *

*

SP

-1

SP

-2

SP

-3

MP

-1

MP

-2

MP

-3

SP-H

G3PDH

ASB4

B C D

IFN

-γ s

pots

/ w

ell

Serum

Sphere




LD

H r

ele

ase (

%)

1 9 3

E/T

30

20

10

0

A

25

20

15

10

5

-5

0

W6/3

2

L2

43

C7709A

2

K562

(-)

SP-H

**

* SP-A

K562

SP-B

SP-H

MP-A

MP-D

MP-K 40

20

0

30

10

50

1 9 3

E/T

10

20

0

1 9 3

E/T

30

B C D

K562 + IV9

T2-A24 + HIV

T2-A24

T2-A24 + IV9

K562

SP-H

SW480

Figure 6




80

40

20

0

60

70

30

10

50

90

100

80

40

20

0

60

70

30

10

50

90

100

CD

8+

T c

ells

/ H

PF

SM4

Control CTL

Tumor Spleen

A

D E #

1

#2

#3

#4

#5

#6

#7

#8

#9

#1

0

#1

1

#1

2

#1

#2

#3

#4

#5

#6

#7

#8

#9

#1

0

#1

1

#1

2

Tum

or

volu

me (

mm

3)

Days

**

PBS 1000

500

0

30 50 0 40 60

control CTL

SM4

B C

F

Days

**

PBS 1200

600

0

30 50 0 40 60

control CTL

SM4

Figure 7

-3

Tumor

CTL

or

PBS

0

Days

Tumor

CTL

or

PBS

0 35 42

CTL injection on day -3

(before tumor implantation)

CTL injection on day 35 and 42

(after palpable tumor growth)

CTL CTL




Figure 8

IV9

-HLA

-A2

4 t

etr

am

er-

PE

HIV-HLA-A24 tetramer-FITC

0.08%

0.06% 0.03% 0.02% 0.00%

0.00% 0.01% 0.01% 0.01%

0.02% 0.01% 0.01%

0.00% 0.00% 0.00%

0.00%

0.02%

0.00% 0.01% 0.00%

0.00% 0.02% 0.00% 0.01%

A

B

Fre

quency o

f IV

9-s

pecific

CD

8+

T-c

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pre

curs

ors

10-6

10-7

10-5

CRC Pt.




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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