Published OnlineFirst February 9, 2016; DOI: 10.1158/1078-0432.CCR-15-1709 Cancer Therapy: Preclinical Clinical Cancer Research Olfactory Receptor Family 7 Subfamily C Member 1 Is a Novel Marker of Colon Cancer–Initiating Cells and Is a Potent Target of Immunotherapy Rena Morita1,Yoshihiko Hirohashi1, Toshihiko Torigoe1, Satoko Ito-Inoda1, Akari Takahashi1, Tasuku Mariya1, Hiroko Asanuma1, Yasuaki Tamura1, Tomohide Tsukahara1, Takayuki Kanaseki1, Terufumi Kubo1, Goro Kutomi2, Toru Mizuguchi2, Takeshi Terui3, Kunihiko Ishitani3, Satoshi Hashino4, Toru Kondo5, Nozomi Minagawa6, Norihiko Takahashi6, Akinobu Taketomi6, Satoru Todo6, Masahiro Asaka4, and Noriyuki Sato1 Abstract Purpose: Cancer-initiating cells (CICs) are thought to be the antitumor effect was addressed by mice adoptive transfer essential for tumor maintenance, recurrence, and distant metas- model. tasis, and they are therefore reasonable targets for cancer Results: OR7C1 has essential roles in the maintenance of colon therapy. Cancer immunotherapy is a novel approach to target CICs, and the OR7C1-positive population showed higher tumor- cancer. In this study, we aimed to establish novel CIC-targeting igenicity than that of the OR7C1-negative population, indicating immunotherapy. that OR7C1 is a novel functional marker for colon CIC. Immu- Experimental Design: Colorectal cancer (CRC) CICs were nohistochemical staining revealed that OR7C1 high expression isolated as side population (SP) cells. The gene expression profile was correlated with poorer prognosis in CRC patients. OR7C1- of CRC CICs was analyzed by cDNA microarray and RT-PCR. derived antigenic peptide-specific CTLs showed specific cytotox- Protein expression of olfactory receptor family 7 subfamily C icity for CICs, and an OR7C1-specific CTL clone showed a greater member 1 (OR7C1) were analyzed by Western blot and immu- antitumor effect than did a CTL clone targeting all cancer cells in a nohistochemical staining. The functions of OR7C1 were analyzed CTL adoptive transfer mouse model. by gene overexpression and gene knockdown using siRNAs. Conclusions: OR7C1 is a novel marker for colon CICs and can OR7C1-positive cells were isolated by a flow cytometer and be a target of potent CIC-targeting immunotherapy. Clin Cancer Res; analyzed. CTLs specific for OR7C1 peptide were generated, and 22(13); 3298–309. Ó2016 AACR. Introduction with recurrence or distant metastasis is usually incurable and has an unfavorable prognosis (2, 3). Cancer stem–like cells/ Colorectal cancer (CRC) is one of the most common malig- cancer-initiating cells (CSC/CIC) are defined by their ability of nancies throughout the world, with more than 140,000 new tumor initiation, self-renewal, and differentiation (4). CICs cases every year in United States (1). Recent progress in CRC are resistant to standard cancer therapies including chemo- treatments has improved the prognosis, but advanced disease therapy and radiotherapy, and they are therefore thought to be responsible for tumor recurrence and metastasis (5,6). CICs are a reasonable target for cancer treatment, and elimination 1 Department of Pathology, Sapporo Medical University School of of CICs is essential to cure cancer. Medicine, Chuo-Ku, Sapporo, Japan. 2Department of Surgery, Sap- poro Medical University School of Medicine,Chuo-Ku, Sapporo, Japan. Cancer immunotherapy is a novel approach to target cancer 3Higashi-Sapporo Hospital, Shiroishi-ku, Sapporo, Japan. 4Depart- and is expected to become the fourth most widely used cancer ment of Gastroenterology and Hematology, Hokkaido University fi 5 therapy after surgery, chemotherapy, and radiotherapy. Identi - Graduate School of Medicine, Kita-Ku, Sapporo, Japan. Division of fi Stem Cell Biology, Institute for Genetic Medicine, Hokkaido University, cation of the rst human tumor-associated antigens (TAAs) has Kita-Ku, Sapporo, Japan. 6Department of Gastroenterological Surgery enabled cancer immunotherapy to be performed using antigenic I, Hokkaido University Graduate School of Medicine, Kita-Ku, Sapporo, peptides derived from TAAs (7–10). Clinical trials using antigenic Japan. peptides derived from TAAs are now being conducted in several Note: Supplementary data for this article are available at Clinical Cancer facilities, and some trials have shown fascinating and promising Research Online (http://clincancerres.aacrjournals.org/). results; however, the results of most studies using antigenic Corresponding Authors: Yoshihiko Hirohashi, Sapporo Medical University, peptide vaccination have been disappointing (11). Recently, South-1 West-17, Chuo-ku, Sapporo 060-8556, Japan. Phone: 811-1613-8374; melanoma-initiating cells (melanoma stem cells) have been þ þ Fax: 816-432-310; E-mail: [email protected]; and Toshihiko Torigoe, isolated as CD271 cells, and CD271 melanoma cells lack the [email protected] expression of melanoma antigens including TYR, MART1, and doi: 10.1158/1078-0432.CCR-15-1709 MAGEs. Low expression levels of melanoma antigens in mela- Ó2016 American Association for Cancer Research. noma-initiating cells are thought to be the reason for resistance to 3298 Clin Cancer Res; 22(13) July 1, 2016 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst February 9, 2016; DOI: 10.1158/1078-0432.CCR-15-1709 Cancer-Initiating Cell Targeting Immunotherapy G418 (Life Technologies, Inc.). No specific authentication of Translational Relevance K562 cells and T2-A24 cells were performed. Cancer stem–like cells (CSC)/cancer-initiating cells (CIC) Three pairs of frozen colorectal adenocarcinoma and corre- are a small subpopulation of cancer cells endowed with higher sponding non-neoplastic colorectal tissues, which were used for tumorigenicity and are resistant to standard therapy. Cancer RT-PCR, were obtained from surgically resected tissues removed at immunotherapy is a novel approach to target cancer, and we Kushiro City General Hospital. Sixty-seven specimens, used for aimed to establish CIC-targeting immunotherapy. OR7C1 is a immunohistochemical staining of OR7C1, were obtained from novel marker of CRC CICs and has an essential role in the surgical resection tissues at Department of Gastroenterological maintenance of CICs. OR7C1 is a novel cancer testis antigen, Surgery I, Hokkaido University Graduate School of Medicine and can be a target of CTLs. Targeting CICs by OR7C1-specific (Sapporo, Japan). Normal tissues used for immunohistochemical CTLs clones showed potent antitumor effects. These observa- staining of OR7C1 were obtained from pathologic dissection at tions indicate that treatment-resistant CICs can be targeted by the Department of Pathology, Sapporo Medical University School CTLs and antigenic peptides derived from OR7C1 are prom- of Medicine (Sapporo, Japan). To obtain CTL clones, we used ising candidates for CIC-targeting immunotherapy. blood from three healthy volunteer donors and three colon cancer patients at Higashi Sapporo Hospital (Sapporo, Japan). We obtained informed consent from all patients and volunteer donors according to the guidelines of the Declaration of Helsinki. peptide vaccination therapies using melanoma antigens (12). fl And recent studies indicate that CICs may contribute to make SP analysis and ow cytometry analysis of OR7C1 fl immunosuppressive microenvironment (13). We therefore SP analysis and ow cytometry analysis of OR7C1 were per- hypothesized that CIC-targeting immunotherapy might improve formed as described previously and as described in the Supple- the efficiency of cancer immunotherapy. To address the feasibility mentary Materials and Methods (14,17). of CIC-targeting immunotherapy, we isolated CRC CICs as side population (SP) cells by Hoechst 33342 staining and found that Xenograft model SP cells are susceptible to CTLs as are non-CIC main population Xenograft transplantation model was performed as described (MP) cells (14). We also reported that CICs of malignant fibrous previously (18). Cancer stem cell frequency was calculated by web histiocytoma could be recognized by autologous CTLs derived program Extreme Limiting Dilution Analysis (ELDA; http:// from tumor-infiltrating lymphocytes (15). Thus, CTLs can recog- bioinf.wehi.edu.au/software/elda/) software (19). nize treatment-resistant CICs. In this study, we identified a novel CIC antigen in CRC, cDNA microarray analysis olfactory receptor family 7 subfamily C member 1(OR7C1). A Total RNAs (3 mg) derived from SW480 SP cells and SW480 MP gene knockdown study using siRNAs and an OR7C1 overexpres- cells were reverse transcribed to generate double-stranded cDNA sion model showed that OR7C1 has a role in the maintenance of and labeled using Cy3-cytidine or Cy5-cytidine. Then the labeled CICs of CRC. A high expression level of OR7C1 was shown to be a cRNAs were hybridized to a oligonucleotide microarray (Pano- poor prognostic factor in the immunohistochemical staining of rama Human Micro Array, Sigma) and scanned by Genepix 4000B OR7C1. An OR7C1-specific CTL clone showed significantly stron- Microarray Scanner. Microarray raw data and processed data have ger antitumor effect than that of a shared antigen-specific CTL been deposited in the Array Express database (E-MTAB-2605). clone in an in vivo CTL adoptive transfer model. These results indicate that the CIC antigen OR7C1 is a novel marker for CRC RT-PCR analysis and quantitative RT-PCR analysis CICs and is a promising target for CIC-targeting immunotherapy. RT-PCR analysis was performed as described previously and