Microenvironment and Immunology Targeting FSTL1 Prevents Tumor Bone Metastasis and Consequent Immune Dysfunction

Bonemetastasis greatly deteriorates the quality of life in patientswith cancer. Althoughmechanisms have been widely investigated, the relationship between cancer bone metastasis and antitumor immunity in the host has been much less studied. Here, we report a novel mechanism of bone metastasis mediated by FSTL1, a follistatinlike glycoprotein secreted by Snailþ tumor cells, whichmetastasize frequently to bone.We found that FSTL1 plays a dual role in bonemetastasis—in oneway bymediating tumor cell invasion andbone tropismbut also in a second way by expanding a population of pluripotent mesenchymal stem-like CD45 ALCAMþ cells derived from bone marrow. CD45 ALCAMþ cells induced bonemetastasis de novo, but they also generated CD8 T cells with weak CTL activity in the periphery, which also promoted bone metastasis in an indirect manner. RNA interferencemediated attenuation of FSTL1 in tumor cells prevented bone metastasis along with the parallel increase in ALCAMþ cells andCD8 T cells. These effectswere accompanied byheightened antitumor immune responses in vitro and in vivo. In clinical specimens of advanced breast cancer, ALCAMþ cells increasedwith FSTL1 positivity in tumor tissues, but not in adjacent normal tissues, consistent with a causal connection between these molecules. Ourfindings define FSTL1 as an attractive candidate therapeutic target to prevent or treat bonemetastasis, which remains a major challenge in patients with cancer. Cancer Res; 73(20); 1–9. 2013 AACR. Introduction Bone metastasis of tumor cells is frequently seen in patients with cancer, particularly with breast cancers and prostate cancers, and greatly deteriorates the quality of life in patients, leading to poor prognosis (1). Chemokines and its receptors are one of the representative molecules regulating bone tropism of tumor cells. For examples, CXCR4þ tumor cells are attracted by CXCL12 secreted from stromal cells in bone marrow (2), and CXCL12maintains proliferation and survival of the tumor cells (3). Bone-derived TGFb and PDGF also promote tumor progression in bone marrow (4). CCL2 is known as another chemokine critical for bone metastatic mechanism in various cancers (5, 6). The excess of tumor growth in bone marrow results in disruption of skeletal integrity, and causes abnormal osteogenesis or osteolysis in patients with cancer (1). One of the most influential molecules is RANKL, which is highly expressed in normal mesenchymal cells including osteoblasts and stromal cells, and metastatic tumor cells undergoing epithelial-to-mesenchymal transition (EMT; ref. 7). A variety of therapeutics targeting these molecules has been developed for treating bone metastasis (4). However, their subjects are mostly postmetastasis events, but not premetastatic state. Elimination of the trigger for tumor seeding toward bone would be a higher priority of cancer therapy rather than treatment of tumor progression and osteo-imbalance in bone marrow. Moreover, although bone metastasis mechanisms have been widely investigated, the relationship with antitumor immunity has been rarely explored, although bone marrow is an essential organ for hematopoiesis and immune responses (8). We previously showed a close relationship between cancer metastasis and immunosuppression focusing on an EMT-governing transcriptional factor Snail (9). Snail expression in tumor cells promotes both tumor metastasis and induction of immunoregulatory cells simultaneously. Some immunoregulatory members such as mesenchymal stem cells (MSC; ref. 10) and myeloid-derived suppressor cells (MDSC; ref. 11) are originated from bone marrow. To understand the interaction between bone metastasis and antitumor immunity may provide a new insight into bone metastasis mechanism. In this study, we attempted to elucidate a new mechanism of cancer bone metastasis from the immunologic perspective. Materials and Methods Cell lines and mice Murine melanoma B16-B10 cells were kindly provided by Cell Resource Center for Biomedical Research at Tohoku University in Japan. Human tumor cells including melanoma HS294T and pancreatic cancer Panc1 were purchased from American Type Culture Collection, and were authenticated by Authors' Affiliation: Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Authors: Chie Kudo-Saito, Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Phone: 813-5363-3778; Fax: 81-3-5362-9259; E-mail: [email protected]; andYutaka Kawakami, E-mail: [email protected] doi: 10.1158/0008-5472.CAN-13-1364 2013 American Association for Cancer Research. Cancer Research www.aacrjournals.org OF1 Research. on April 11, 2017. © 2013 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst August 21, 2013; DOI: 10.1158/0008-5472.CAN-13-1364