CCL9 Induced by Tgfb Signaling in Myeloid Cells Enhances Tumor Cell Survival in the Premetastatic Organ Hangyi H

Published OnlineFirst October 19, 2015; DOI: 10.1158/0008-5472.CAN-15-2282-T Cancer Microenvironment and Immunology Research CCL9 Induced by TGFb Signaling in Myeloid Cells Enhances Tumor Cell Survival in the Premetastatic Organ Hangyi H. Yan1, Jian Jiang1,2, Yanli Pang3, B.R. Achyut1, Michael Lizardo4, Xinhua Liang2, Kent Hunter1, Chand Khanna4, Christine Hollander1, and Li Yang1 Abstract Tumor cell survival in the hostile distant organ is a rate- cells lacking TGFb signaling rescued the tumor metastasis limiting step in cancer metastasis. Bone marrow–derived mye- defect observed in mice with myeloid-specific Tgfbr2 deletion. loid cells can form a premetastatic niche and provide a tumor- The expression level of CCL23, the human orthologue for promoting microenvironment. However, it is unclear whether CCL9, in peripheral blood mononuclear cells correlated with these myeloid cells in the premetastatic site have any direct progression and survival of cancer patients. Our study demon- effect on tumor cell survival. Here, we report that chemokine strates that CCL9 could serve as a good candidate for anti- þ þ CCL9 was highly induced in Gr-1 CD11b immature myeloid metastasis treatment by targeting the rate-limiting step of cells and in premetastatic lung in tumor-bearing mice. Knock- cancer cell survival. In addition, targeting CCL9 may avoid down of CCL9 in myeloid cells decreased tumor cell survival the adverse effects of TGFb-targeted therapy. Cancer Res; 75(24); and metastasis. Importantly, CCL9 overexpression in myeloid 5283–98. Ó2015 AACR. þ þ þ cells that consist of Ly6G CD11b granulocytes, Ly6C Introduction þ þ þ CD11b monocytes and F4/80 CD11b macrophages (9). Tumor cell survival is a rate-limiting step in cancer metastasis þ þ Gr-1 CD11b cells are overproduced in cancer patients, neg- (1). The majority of the cancer cells die in the peripheral blood atively regulate host antitumor immunity (10–12), and pro- and distant hostile organs during the multiple-step processes, mote tumor angiogenesis (13). These immature myeloid cells including invasion, intravasation, extravasation, and coloniza- are also found in premetastatic organ of tumor-bearing mice to tion (1). Recent evidence strongly suggests a critical role of host modulate host inflammatory/immune microenvironment cells and tumor microenvironment in supporting tumor cell (7, 14), including NK (natural killer) cell activities (15, 16) survival and metastasis. One mechanism is through formation and promoting mesenchymal-to-epithelial transition of tumor of premetastatic niche (2, 3). Myeloid cells play a critical role in cells (17). However, it is not known whether these myeloid this process (2, 4). These include VEGFR1 myeloid progenitor þ þ þ cells can directly support tumor cell survival in the premeta- cells (5), Mac-1 myeloid cells (6), and Gr-1 CD11b mye- þ static organs. loid–derived suppressor cells (MDSC; ref. 7) as well as Ly6G þ þ þ One important molecular mechanism underlying metastasis- Ly6C granulocytes (8). Gr-1 CD11b immature myeloid cells promoting function of myeloid cells is myeloid-specific TGFb constitute the majority of the immature myeloid cells under signaling (18). TGFb is a pleiotropic molecule and it possesses tumor conditions. They are heterogeneous immature myeloid both tumor-suppressor and tumor-promoter functions (19, 20). The mechanisms underlying the dual role of TGFb are very intricate and are poorly understood. Recent work reveals that 1Laboratory of Cancer Biology and Genetics, National Cancer Institute, NIH, Bethesda, Maryland. 2State Key Laboratory of Oral Diseases,West TGFb regulates production of chemokine/chemokine receptors in China College of Stomatology, Sichuan University, Chengdu, Sichuan, tumor cells that are important for inflammatory cell recruitment 3 P.R. China. Department of Physiology & Pathophysiology, Peking in the tumor microenvironment (21–23). This includes stromal- University Health Science Center, Beijing, P.R. China. 4Pediatric Oncol- ogy Branch, National Cancer Institute, NIH, Bethesda, Maryland. derived factor 1 (SDF-1 or CXCL12; ref. 24) and CCL9 (25). CXCL12 mediates its effects through CXCR4, a receptor that is Note: Supplementary data for this article are available at Cancer Research highly expressed on putative stem and progenitor cells (26, 27). Online (http://cancerres.aacrjournals.org/). þ CCL9 signals through CCR1 and recruits myeloid progenitor Current address for Y. Pang: Center for Reproductive Medicine, Department of cells for tumor cell invasion (25). In addition, TGFb suppresses Obstetrics and Gynecology, Peking University Third Hospital, Beijing 100191, P.R. China. CXCL1 and CXCL5, and deletion of TGFb signaling in cancer cells significantly increases the expression of both chemokines J. Jiang and Y. Pang share co-second authorship for this article. (21). These chemokines are responsible for the recruitment of þ þ Corresponding Author: Li Yang, National Cancer institute, 37 Convent Drive, Gr-1 CD11b myeloid cells to the tumor microenvironment Bethesda, MD 20892. Phone: 301-496-5260; Fax: 301-402-1031; E-mail: (21), where they produce large quantities of matrix metallopro- [email protected] teases (21). A chemokine signature generated due to a loss of doi: 10.1158/0008-5472.CAN-15-2282-T TGFb signaling correlates with poor prognosis of human breast Ó2015 American Association for Cancer Research. cancer (22). www.aacrjournals.org 5283 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst October 19, 2015; DOI: 10.1158/0008-5472.CAN-15-2282-T Yan et al. CCL9, a chemokine also known as macrophage inflammatory Single-cell videomicroscopy for evaluating tumor cell survival protein-1 gamma (MIP-1g; refs. 25, 28–30), is often produced in Mice were euthanized and lungs were removed 6 hours after tail macrophages, osteoclasts, and functions as a cell survival factor vein injection of GFP-labeled tumor cells (5 Â 105) together with þ þ (31, 32). CCL9/CCR1 signaling has been shown to be important Gr-1 CD11b (1.5 Â 106) or RAW264.7 cells (2 Â 105). Images for the recruitment of myeloid progenitors to intestinal tumors, were normalized to the basal signal obtained from lungs 1 hour leading to enhanced invasion (25, 30). However, it remains to be after tail vein injection. Lungs were observed under the fluorescent determined whether CCL9 promotes cancer cell survival, and microscope, 10 random pictures under Â10 magnification were whether TGFb regulates CCL9 production. Here, we report that taken and further analyzed as fluorescence signal per field by CCL9 is highly induced in immature myeloid cells in the pre- OpenLab software (Improvision) or ImageJ. For extravasation metastatic lung of tumor-bearing mice or in coculture with tumor experiment, mice were injected with 100 mL of 12.5 mg/mL 70,000 cells, and it plays a critical role in tumor cell survival at the MW tetramethylrhodamine (Invitrogen). Lungs were imaged metastatic site. We further identify CCL9 as a critical mediator under Zeiss 510 NLO confocal microscope. Images were analyzed of the metastasis-promoting effects of myeloid-specific TGFb with Zeiss ZEN software. signaling. Our study provides insight for understanding TGFb cancer biology and suggests a new target for cancer treatment. Ex vivo pulmonary metastasis assay GFP-labeled tumor cells (5 Â 105) were coinjected with sorted þ þ Materials and Methods Gr-1 CD11b cells (1.5 Â 106) or RAW264.7 cells (2 Â 105) Cells and mice through the tail vein. Mice were euthanized 5 minutes after Murine mammary tumor cell lines 67NR, 168FARN, 4T07, 4T1, injection, and the lungs were infused with an agarose medium 67NR-GFP, 4T1-GFP, 4T1-luciferase and melanoma B16F1, mixture as described previously (35). Lung sections (1–2-mm- B16BL6, B16F10, murine macrophage cell line RAW264.7 and thick) were placed on Gelfoam (Pfizer-Pharmacia & Upjohn Co.) neutrophil cell line 32DCl3, as well as human breast tumor cell for culture for 1 to 2 weeks. LEICA-DM IRB fluorescent inverted line MDA-MB-231 and human melanoma cell lines A375S2, SK- microscope (Leica) and Retiga-EXi Fast 1394 Mono Cooled CCD MEL-28 were maintained per standard cell culture techniques. camera (QImaging) were used to capture GFP-positive cells at Cells labeled with GFP were obtained through pSico-GFP virus Â10 or Â2.5 magnification. The GFP fluorescence pixels were þ infection as published (33) and followed by GFP cell sorting. obtained and analyzed using OpenLab software (Improvision) or B16F1 and B16F10 were provided by Drs. Glenn Merlino and ImageJ (35). The fluorescence intensity per field was quantified þ þ Shioko Kimura, NCI. All Gr-1 CD11b cells were sorted through and normalized to day 0 signal and presented as metastasis FACS or MACS (magnetic-activated cell sorting) from spleens of survival index. Three to six lung sections for each mouse, and a tumor-bearing wild-type, Tgfbr2MyeKO and Tgfbr2Flox mice unless total of 3 to 4 mice were evaluated for each experimental group. otherwise specified. Tgfbr2MyeKO and Tgfbr2Flox control mice were produced as previously described (18). p38 dominant–negative Flow cytometry and cell sorting mice are precious gifts from Dr. Albert J. Fornace, Jr. (Georgetown Single cell suspensions were made from spleens or peripheral University). All animal studies are approved by the National blood of normal and 4T1 tumor-bearing mice (13), as well as lung Cancer Institute Animal Care and Use Committee. tissues (36). Cells were labeled with fluorescence-conjugated antibodies: Gr-1, CD11b, Ly6G, Ly6C, F4/80, Annexin V, 7AAD Spontaneous and experimental metastasis (BD Pharmingen), and CCR1 (R&D Systems). For flow-cytometry For orthotopic metastasis, mammary tumor 4T1 cells (5 Â 105) analysis, cells were run on a FACS Calibur or Fortessa flow were injected into the #2 mammary fat pad (MFP); B16F10 cytometer (BD Biosciences) and analyzed on FlowJo. For sorting, þ þ þ þ þ þ melanoma cells (1 Â 106) were injected s.c.

Load more