CXCL14 is an autocrine growth factor for fibroblasts and acts as a multi-modal stimulator of prostate tumor growth Martin Augstena,1, Christina Ha¨ gglo¨ fa,1, Eleonor Olssonb, Claudia Stolza,2, Panagiotis Tsagozisa, Tetyana Levchenkoa, Mitchell J. Frederickc, Åke Borgb, Patrick Micked, Lars Egevada, and Arne Ostman¨ a,3 aDepartment of Oncology-Pathology, Karolinska Institutet, 171 76 Stockholm, Sweden; bDepartment of Oncology, Lund University, 221 85 Lund, Sweden; cDepartment of Head and Neck Surgery, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030; and dDepartment of Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden Communicated by George Klein, Karolinska Institutet, Stockholm, Sweden, January 5, 2009 (received for review May 6, 2008) This study explored the role of secreted fibroblast-derived factors of chemokines (7, 10–12). In breast cancer, both CXCL12 and in prostate cancer growth. Analyses of matched normal and tumor CXCL14 were found to be up-regulated in the tumor stroma (10, tissue revealed up-regulation of CXCL14 in cancer-associated fi- 13). Furthermore, CAFs of ovarian cancer over-express the che- broblasts of a majority of prostate cancer. Fibroblasts over- mokine CXCL1/GRO1 (11). expressing CXCL14 promoted the growth of prostate cancer xeno- In this study, characterization of human prostate CAFs led to the grafts, and increased tumor angiogenesis and macrophage identification of CXCL14 as a novel CAF-derived tumor- infiltration. Mechanistic studies demonstrated that autocrine stimulatory factor. CXCL14-stimulation of fibroblasts stimulate migration and ERK- dependent proliferation of fibroblasts. CXCL14-stimulation of Results monocyte migration was also demonstrated. Furthermore, CXCL14-producing fibroblasts, but not recombinant CXCL14, en- CXCL14 Is Up-Regulated in CAFs of Human Prostate Cancer. Fibro- hanced in vitro proliferation and migration of prostate cancer cells blast-enriched stroma was isolated from 4 matched sets of normal and in vivo angiogenesis. These studies thus identify CXCL14 as a and tumor tissue by laser capture microdissection. Array-based MEDICAL SCIENCES novel autocrine stimulator of fibroblast growth and migration, comparison of amplified mRNA identified 266 transcripts that were with multi-modal tumor-stimulatory activities. In more general at least 2-fold up-regulated in tumor stroma, 15 of which encoded terms, our findings suggest autocrine stimulation of fibroblasts as secreted proteins. Among them were the chemokine CXCL14 a previously unrecognized mechanism for chemokine-mediated which was Ϸ40-fold up-regulated in tumor stroma. stimulation of tumor growth, and suggest a novel mechanism qRT-PCR analyses of 8 matched normal and tumor tissues whereby cancer-associated fibroblasts achieve their pro-tumorigenic confirmed stromal up-regulation of CXCL14 in 6 out of 8 cases (Fig. phenotype. 1A). qRT-PCR with primers specific for endothelial cells (CD31), macrophages (CD163) and leukocytes (CD45) indicated a moder- cancer-associated fibroblasts ͉ prostate cancer ͉ tumor stroma ate, 1.5- to 3-fold increase in relative content of these cell types in the tumor stroma preparations. Since these changes were too small hemokines are a family of secreted proteins that stimulate to explain the increased CXCL14 levels, it was concluded that the Cchemotaxis and cell growth. More than 50 chemokines have up-regulation of CXCL14 occurred in the prostate CAFs. been identified and are divided into the CXC, CC, C, and CX3C Immunohistochemical analyses of 27 matched pairs of prostate groups (1). Chemokines exert their cellular effect by activation of cancer and normal prostate were performed to further compare cell surface receptors belonging to the G protein-coupled-receptor CXCL14 expression in normal and tumor stroma. These analyses family. Approximately 20 chemokine receptors have been identi- revealed a statistically significant (P Ͻ 0.05) increase in stromal fied (1, 2). Many receptors bind multiple ligands, whereas others are expression of CXCL14 in 15/27 (56%) cancer samples (Fig. 1B). highly specific such as CXCR4 and CXCR6, which bind only Variable CXCL14 expression in the epithelial compartment of CXCL12 and CXCL16, respectively. For some of the chemokines, normal and cancer tissue was also observed. Double immunoflu- such as CXCL14, the receptor has not yet been identified. orescent staining with antibodies against CXCL14 and the PDGF Recent studies have suggested important functions of chemo- ␤-receptor, used as a fibroblast marker, confirmed that fibroblasts kines in various aspects of tumor growth (2). Chemokines of human prostate cancer produced CXCL14 (Fig. S1). Stromal and contribute to leukocyte infiltration in tumors, and some, such as epithelial CXCL14 status were not significantly associated with IL-8, CXCL1–3, and CXCL5, also have direct proangiogenic Gleason grade. effects (3, 4). Concerning chemokines acting on malignant cells, most attention has been paid to CXCL12, which stimulates These analyses demonstrate that up-regulation of CXCL14 in tumor cell proliferation and migration through CXCR4 and CAFs is a common feature of human prostate cancer. CXCR7 (5, 6). CXCL12 also contributes to tumor growth by recruitment of bone marrow–derived endothelial precursor cells Author contributions: M.A., C.H., and A.O. designed research; M.A., C.H., E.O., C.S., P.T., (7). Additionally, mesenchymal stem cell–derived CCL5/ T.L., and Å.B. performed research; T.L., M.J.F., and L.E. contributed reagents/analytic tools; RANTES was recently shown to confer prometastatic effects on M.A., C.H., E.O., C.S., P.T., Å.B., P.M., L.E., and A.O. analyzed data; and M.A., C.H., P.M., and breast cancer cells (8). Most recently, CCL3 was implicated as a A.O. wrote the paper. prometastatic agent acting through multiple mechanisms includ- The authors declare no conflict of interest. ing stimulation of fibroblasts (9). 1M.A. and C.H. contributed equally to this work. Within the tumor microenvironment, multiple cell types have 2Present address: Department of Medicine (Cancer Research), West German Cancer Center, been identified as sources of chemokine production, including the University Hospital Essen, Essen, Germany. malignant cells and inflammatory leukocytes. Recent characteriza- 3To whom correspondence should be addressed. E-mail: [email protected]. tion of the expression profiles of cancer-associated fibroblasts This article contains supporting information online at www.pnas.org/cgi/content/full/ (CAFs) has also identified this cell type as an important producer 0813144106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813144106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 28, 2021 A 400 the fibroblast markers puromycin (present in the pBABE vector of NIH-ctr and NIH-CXCL14 cells) and vimentin indicated no signif- icant differences, although a tendency toward an increased fibro- blast content in LNCaP/NIH-CXCL14 tumors was observed (Fig. S3). LNCaP/NIH-CXCL14 tumors displayed a significantly higher 200 proliferation rate, as compared to control tumors (Fig. 2D). Be- cause no major differences in epithelia-stroma ratio were observed, expression (AU) it was concluded that that the increased proliferation was occurring in both these compartments. CXCL14 These analyses indicate protumorigenic effects of NIH-CXCL14 cells, which involve increased cell proliferation in tumors, occurring 0 in the absence of major alterations of the epithelia-stroma ratio. sample 12345678 LNCaP/NIH-CXCL14 Tumors Are Characterized by Increased Macro- B Normal Tumor phage Infiltration and Increased Angiogenesis. Analysis of angiogen- esis in the 2 tumor types, based on CD31 staining of tumor sections, revealed an increased vessel content in NIH-CXCL14 tumors (Fig. 2E). qRT-PCR analyses indicated an Ϸ2-fold increase in CD31- postive cells along with reduced ␣SMA levels in the LNCaP/NIH- CXCL14 tumors (Fig. S3). Moreover, a highly significant associa- Patient A tion (P ϭ 0.0039) was noted between CXCL14 and CD31 levels (Fig. 2F). Analyses of sections double-stained with fluorescent labeled CD31 and ␣SMA antibodies demonstrated a reduced relative content of pericytes in the vasculature of LNCaP/NIH- CXCL14 tumors (Fig. S4). Macrophage staining using a CD68 antibody revealed a locally enriched staining pattern, which was found more frequent in the Patient B LNCaP/NIH-CXCL14 tumors (Fig. 2G). qRT-PCR analyses with another macrophage-marker, CSF-1R, confirmed higher macro- phage content in LNCaP/NIH-CXCL14 tumors (Fig. S3). Also, the levels of CXCL14 and CSF1R mRNA displayed a highly significant correlation (P ϭ 0.0008) (Fig. 2H). However, analysis of NK-cell content (NK1.1) did not indicate any difference between the 2 tumor types (Fig. S3). Taken together, the data show that LNCaP/NIH-CXCL14 tu- Patient C mors are characterized by a higher density of immature vessels and macrophages. Fig. 1. CXCL14 is up-regulated in fibroblast-enriched prostate cancer stroma. CXCL14 Stimulates Growth and Migration of Fibroblasts. To obtain a (A) Stroma from normal prostate (open bars) and prostate cancer tissue (filled mechanistic understanding of the observed tumor phenotypes, bars) was microdissected from 8 different patients and the expression of several in vitro studies were performed. When cultured in 1% FCS, CXCL14 was analyzed with qRT-PCR. (B) Examples of CXCL14 immunohisto- NIH-CXCL14 cells grew significantly faster than control cells (Fig. chemistry analyses of paired tissues with up-regulation of