Proteome Profiling of Cancer-Associated Fibroblasts Identifies Novel Proinflammatory Signatures and Prognostic Markers for Colorectal Cancer

Published OnlineFirst September 11, 2013; DOI: 10.1158/1078-0432.CCR-13-1130

Clinical Cancer Imaging, Diagnosis, Prognosis Research

Proteome Profiling of Cancer-Associated Fibroblasts Identifies Novel Proinflammatory Signatures and Prognostic Markers for Colorectal Cancer

Soﬁa Torres1, Ruben A. Bartolome1, Marta Mendes1, Rodrigo Barderas1, M. Jesus Fernandez-Acenero~ 3, Alberto Pelaez-García 1, Cristina Pena~ 4, María Lopez-Lucendo2, Roi Villar-Vazquez 1, Antonio García de Herreros5, Felix Bonilla4, and J. Ignacio Casal1

Abstract Purpose: Cancer-associated fibroblasts (CAF) are essential components of the stroma that play a critical role in cancer progression. This study aimed to identify novel CAFs markers that might contribute to the invasion and the prognosis of colorectal cancer. Experimental Design: The azoxymethane/dextran sodium sulfate mouse model of sporadic colon cancer represents an adequate source for the isolation of CAFs and normal fibroblasts. By using the explants technique, we purified CAFs and normal fibroblasts from colon tissues. Whole-cell extracts and supernatants were subjected to in-depth quantitative proteomic analysis by tandem mass spectrometry. Further valida- tions of upregulated proteins in CAFs were carried out by chemokine microarray and immunohistochemical analyses of mouse and human tissues. Results: Using a fold-change of 1.4 or more, we found 132 and 125 differentially expressed proteins in whole-cell extracts and supernatants, respectively. We found CAFs-associated proinflammatory and desmoplastic signatures. The proinflammatory signature was composed of several cytokines. Among them, CCL2 and CCL8 caused an increase in migration and invasion of colorectal cancer KM12 cells. The desmoplastic signature was composed of 30 secreted proteins. In mouse and human samples, expression of LTBP2, CDH11, OLFML3, and, particularly, FSTL1 was significantly increased in the tumoral stroma, without significant expression in the cancer epithelial cells. The combination of CALU and CDH11 stromal expression showed a significant association with disease-free survival and poor prognosis. Conclusion: We have identified LTBP2, CDH11, OLFML3, and FSTL1 as selective biomarkers of cancer stroma, and CALU and CDH11 as candidate stromal biomarkers of prognostic significance in colon cancer. Clin Cancer Res; 19(21); 6006–19. 2013 AACR.

Introduction although it also contains infiltrating immune cells and The tumor stroma comprises most of the cancer mass and pericytes (1). Stroma nurtures cancer cells and facilitates is mainly composed of fibroblasts and endothelial cells, tumor development and invasion. Clinical and experimental data support the hypothesis that tumor stroma promotes invasion and cancer metastasis (2). Within stroma, fibroblasts are key components for cancer progression. Stromal fi 1 Authors' Af liations: Department of Cellular and Molecular Medicine; fibroblasts are called activated fibroblasts, myofibroblasts, 2Proteomics Core Facility, Centro de Investigaciones Biologicas (CIB- CSIC); 3Department of Pathology, Fundacion Jimenez Díaz; 4Department or cancer-associated fibroblasts (CAF), and they acquire a of Oncology, Hospital Puerta de Hierro Majadahonda, Madrid; and 5IMIM- particular phenotype similar to fibroblasts present in skin Hospital del Mar, Barcelona, Spain wounds. Carcinoma progression is associated with an Note: Supplementary data for this article are available at Clinical Cancer increase in the production of fibrosis, known as desmopla- Research Online (http://clincancerres.aacrjournals.org/). sia, similar to that present in wound healing (3). CAFs S. Torres and R.A. Bartolome contributed equally to this work. respond to profibrotic and promigratory factors, such as TGF-b, platelet-derived growth factor (PDGF), hepatocyte Current address for R. Barderas: Department of Biochemistry and Molec- ular Biology, I. Universidad Complutense, Madrid, Spain. growth factor (HGF), or fibroblast growth factor 2 (FGF2), thereby promoting cancer progression. They are character- Corresponding Author: J. Ignacio Casal, Department of Cellular and Molecular Medicine, Centro de Investigaciones Biologicas (CIB-CSIC), ized by increased expression of myofibroblastic markers Ramiro de Maeztu, 9, 28040 Madrid, Spain. Phone: 34-918373112; Fax: such as a-smooth muscle actin (a-SMA), FSP1, or prolyl-4- 34-915360432; E-mail: [email protected] hydroxilase (4, 5). Cancer fibroblasts proliferate more than doi: 10.1158/1078-0432.CCR-13-1130 their normal counterparts and secrete more constituents 2013 American Association for Cancer Research. of the extracellular matrix (ECM) and ECM-degrading

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Molecular Signatures of Colon Cancer–Associated Fibroblasts

immortalization (13). We propose the use of direct intes- Translational Relevance tinal explants in culture for fibroblast isolation and Our knowledge of cancer-associated stroma is rather proteomic characterization (14). This strategy strongly limited in comparison with that of cancer epithelial cells. reduces sample complexity and heterogeneity. The use Stroma constitutes the cancer microenvironment that of an inbred mouse model reduces biologic variation due nurtures cancer cells and facilitates invasion and metas- to genetic and environmental heterogeneity. Because tasis. Moreover, stromal myofibroblasts have been primary fibroblasts duplicate only a few times before associated with recurrence and survival. Novel biomar- entering senescence, we discarded metabolic labeling for kers are required for the isolation and characterization quantification. Therefore, iTRAQ (isobaric tag for relative of colorectal cancer–associated fibroblasts (CAF) to and absolute quantification) was preferred because of its improve overall survival and recurrence prediction in reproducibility and reliability (15). colorectal cancer. We, here, describe a strategy based on Here, we compared the protein component of the an in-depth proteome profiling of CAFs obtained from a whole-cell extracts and conditioned medium of primary mouse model of human sporadic colorectal cancer. The CAFs and normal fibroblasts (NF) isolated from AOM/ use of this model facilitates the isolation of pure popula- DSS–induced sporadic colorectal cancer mice or controls, tions of CAFs, avoiding other cell contaminants. The respectively. We identified 132 and 125 proteins deregu- validity of this strategy is supported by the identification lated in whole-cell extracts and conditioned medium, of multiple stromal biomarkers previously described in respectively, of CAFs versus normal fibroblasts. In silico humans. In addition, we report a collection of novel studies demonstrated a predominant association of upre- stromal biomarkers as well as comprehensive proinflam- gulated proteins to deposition of ECM, wound healing, matory and desmoplastic CAFs signatures for colorectal and inflammation. Proinflammatory and desmoplastic cancer. The value and relevance of these novel stromal signatures, specific for colon cancer, were defined for biomarkers for colon cancer prognosis and survival were CAFs-associated proteins. A number of proteins were validated in human samples. identified as promising tumor-associated stromal prognostic markers. Among these proteins, high expression of CALUandCDH11wasassociatedtopoorsurvivalin human cancer samples. proteases (6). In addition, CAFs can mediate inflammation and angiogenesis, as reported in skin cancer (7). Despite the Materials and Methods significant number of markers and secreted proteins already AOM/DSS–induced colon cancer model associated with activated fibroblasts, the study of their All mouse studies were performed under the approval of contribution to tumor growth and invasion would benefit the Animal Ethics Committee of the Spanish Research of additional markers for cell selection, prognosis, and Council (CSIC, Madrid, Spain). Animals were housed invasion prediction (8). under pathogen-free conditions, and were given autoclaved With regard to human colon adenocarcinomas, CAFs food and water ad libitum. For the AOM/DSS model, FVB/N synthesize ECM components such as fibronectin, tenascin, mice (4- to 6-week-old) were weighed and given a single collagens types I, III, IV, V, and XII, and proteoglycans intraperitoneal injection of azoxymethane (10 mg/kg) or (biglycan, fibromodulin, perlecan, and versican; refs. 9, vehicle (PBS). Five days later, animals received either 2.5% 10). In addition, they contribute to the formation of base- DSS or normal drinking water. Chronic colitis-derived ment membranes by secreting collagen type IV and laminin. colon cancer was induced after three cycles of DSS treat- For a more comprehensive molecular analysis, we propose ment, which consisted of 5 days with 2.5% DSS followed by the use of sensitive proteomic techniques combined with a 16 days with normal water. At the end of the protocol, mouse model of colon cancer, cell isolation, and mass animals were sacrificed and distal colons were longitudi- spectrometry (MS). We have selected the well-established nally cut, rinsed twice with ice-cold PBS, and cut into small murine model of colitis-associated cancer (CAC) based on pieces. Pieces of intestine were either cultured to isolate the use of azoxymethane (AOM) and dextrane sodium fibroblasts or fixed in 10% buffered formalin overnight for sulfate (DSS). Although the model does not progress to immunohistochemical analysis. metastasis (11), it mimics quite well many steps in cancer progression of sporadic colorectal cancer. In fact, this model Cell line culture has been very useful for the elucidation of the role of TNF-a, KM12C and KM12SM human colon cancer cells were interleukin (IL)-6, NF-kB, and other molecules in the ini- obtained from the laboratory of I. Fidler (MD Anderson tiation and progression of inflammation-associated cancer Cancer Center, Houston, TX). A large batch of working (see ref. 12 for a review). aliquots of KM12 cells was stored in liquid nitrogen. For Fibroblast characterization has been largely complicated each experiment, cells were thawed and kept in culture for by the necessity to isolate primary fibroblasts from the a maximum of 10 passages in Dulbecco’s modified Eagle colonic tissue. In other publications, the characterization medium (DMEM; Gibco-Life Technologies) containing of CAFs was carried out by coculturing normal fibroblasts 10% fetal calf serum (FCS) and antibiotics at 37C. Cells and colon cancer cell lines (10), cell sorting (7), or cell were tested for Mycoplasma but not authenticated, because

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these lines were originally isolated in Dr Fidler’s labora- fraction, and desalting was performed with Zip-Tip. Pep- tory. CT26 murine colon cancer cell line was obtained tide mixtures were vacuum-dried and reconstituted with 6 directly from the American Type Culture Collection mL 0.1% formic acid. Then, peptides were trapped onto a (which authenticated the cell line by short-tandem repeat precolumn C18-A1 ASY-Column (2 cm, ID100 mm, 5mm; profiling), and passaged for fewer than 6 months after Thermo Scientific) and run with a linear gradient of 2% receipt or resuscitation according to the manufacturer’s to 35% ACN in 0.1% aqueous solution of formic acid. instructions. The gradient was performed over 180 minutes using an Easy-nLC (Proxeon) at a flow-rate of 300 nL/min onto a Fibroblast isolation and culture Biosphere C18 column (75 mm, 16 cm, and 3 mm; CAFs and normal fibroblasts cultures were established NanoSeparations). Then, peptides were scanned and frag- from colonic tissues from AOM/DSS–treated mice (n ¼ 16) mented with a linear ion trap-Orbitrap Velos (Thermo and vehicle-treated (n ¼ 16) by the explant technique Scientific). The Orbitrap Velos was operated in a data- (14, 16). Tissues were cut into 2 to 3 mm fragments and dependent mode to automatically switch between MS and planted in fibroblast growth medium-2 (Lonza) containing tandem mass spectrometry (MS–MS). Survey full-scan MS 2% FCS and 1% penicillin/streptomycin/amphotericin B spectra were acquired from m/z 400 to 1,600 after accu- 6 (Invitrogen) at 37 C in a 5% CO2-humidified atmosphere. mulation to a target value of 10 in the Orbitrap at a Normal fibroblasts and CAFs grew around the explants and resolution of 60,000 at m/z 400. For internal mass cali- were cultured for approximately 3 weeks. Then, tissue frag- bration, we used the 445.120025 ion for lock mass. ments were removed, and cells were trypsinized and seeded Charge-state screening was enabled, and precursors with at 70% to 90% confluence and cultured in complete medi- charge state unknown or 1 were excluded. After the survey um. When cells reached 95% to 100% confluence (1.7– scan, the 10 most intense precursor ions were selected 2.0 104 cells/cm2), they were washed with PBS, incubated for collision-induced dissociation—higher energy colli- with serum-free medium for 1 hour, washed again, and sion dissociation (CID-HCD) MS–MS fragmentation. incubated for 24 hours in serum-free medium. Then, cells Peptide identification was performed in both CID and and conditioned medium were collected, pooled in two HCD spectra and quantification of iTRAQ reporter ions different batches for normal fibroblasts and CAFs, and was performed in HCD. HCD was carried out with excess mixed for performing the different experiments. We recov- of collision energy for effectively maximizing abundance ered 7.0 and 13.5 106 cells from normal fibroblasts and of the reporter ions. For CID fragmentation, the target CAFs cultures, respectively. value was set to 10,000 and normalized collision energy to 35%. For HCD, target value was set to 50,000 and Sample preparation and iTRAQ labeling collision energy was set to 55%. Dynamic exclusion was For a detailed description of sample preparation, see applied during 30 seconds. All MS data were analyzed and Supplementary Methods. iTRAQ labeling was carried out quantified with Proteome Discoverer (version 1.3.0.339; using iTRAQ Reagent 4-Plex kit (AB SCIEX), according to Thermo Scientific) using standardized workflows (see the manufacturer’s instructions but with minor modifica- Supplementary Materials and Methods for a more tions. Briefly, 100 mg of protein from whole-cell lysate or 50 detailed description). mg of protein from conditioned medium from normal fibroblasts and CAFs were digested with 5 mg trypsin (Pro- Mouse cytokine array mega) overnight at 37C. Peptide mixtures were labeled Conditioned medium from CAFs and normal fibroblasts with iTRAQ reagents: 114 and 116 for normal fibroblasts were collected after 24 hours in serum-free medium and and 115 and 117 for CAFs as illustrated in Supplementary incubated with Mouse Cytokine Antibody Array C6 for Fig. S1. To remove interfering substances, strong cation semiquantitative analysis of 97 mouse cytokines (RayBio- exchange (SCX) chromatography was carried out using a tech) according to the manufacturer’s instructions. Then, Resource S column (GE Healthcare). To adjust the pH membranes were scanned and analyzed using Redfin, a two- between 2.5 and 3.3, the sample mixture was diluted with dimensional gel image analysis software (Ludesi). Relative 15 volume of 10 mmol/L KH2PO4 pH 3.0 containing 25% semiquantitative cytokine intensities were normalized in acetonitrile (ACN). Peptides were eluted in 1 mL of elution comparison with control spots on the same membrane. buffer (25% ACN, 350 mmol/L KCl, 10 mmol/L KH2PO4, Expression ratios were calculated comparing the signal pH 3.0). Then, peptides were desalted with Sep-Pak C18 intensities for each spot of the different cytokines. The limit cartridges (Waters), dried, and reconstituted in OFFGEL of detection, sensitivity, and the dynamic range of the solution (Agilent Technologies). All chemicals were obt- measured cytokines were more than 1 pg/mL, where most ained from Sigma-Aldrich. of cytokines can be detected.

Peptide fractionation and MS analysis Cell proliferation, adhesion, migration, and invasion Peptides were recovered in 12 fractions by isoelectric assays focusing using a low-resolution strip (pH 3–10) in a 3100 KM12C and KM12SM human colon cancer cells (17) OFF-GEL fractionator (Agilent Technologies). Five micro- were cultured in DMEM (Life Technologies) containing liters of 25% triﬂuoroacetic acid (TFA) were added to each 10% FCS and antibiotics at 37 C in a 5% CO2-humidiﬁed

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atmosphere. Invasion, migration, adhesion, and cell pro- S100A4, and Snail, considered markers of activated fibro- liferation assays were carried out by following established blasts in desmoplastic tumors (Fig. 1B). To establish CAFs procedures (18), with the addition of the indicated cyto- and normal fibroblasts in cultures, distal colons from 16 kines: CCL2 (10 ng/mL), CCL8 (50 ng/mL), and IL-9 AOM/DSS–treated and vehicle-treated mice were cut into (2 ng/mL). See Supplementary Materials and Methods for small fragments and placed in culture. Fibroblasts migrated a detailed description. outside the explants and expanded (Fig. 1C). Cells at 95% to 100% confluence (2 104 cells/cm2) were collected, Immunohistochemical analysis divided into two fractions, and mixed for iTRAQ experi- Pieces of intestine from AOM/DSS–treated or control ments. On Western blot analysis, it was confirmed that mice were fixed in 10% buffered formalin overnight to AOM/DSS–isolated fibroblasts had higher expression of perform hematoxylin and eosin (H&E) and immunohis- markers corresponding to activated fibroblasts such as tochemical staining. For the prognostic studies in humans, a-SMA, S100A4, and Snail1 (Fig. 1D). The expression was a total of 80 patients, diagnosed and treated of colorectal equivalent to the immunohistochemical staining in the adenocarcinoma between 2001 and 2006 in Fundacion original tumor (Fig. 1B). As expected, purified fibroblasts Jimenez Daz (Madrid, Spain) and followed in the long expressed vimentin, a mesenchymal marker, but not the term, were included for the study. Human samples were epithelial marker Epcam. prepared as described previously (18). All human biopsies were obtained with the patient’s consent and approval of Protein identification, iTRAQ quantification, Gene the Ethical Committee of Hospital Fundacion Jimenez Ontology, and functional networks alterations Daz in accordance with the official Spanish regulations. Briefly, 100 (cell lysates) and 50 mg (conditioned We reviewed the clinical records of the patients to deter- media) of proteins from cultures of CAFs and normal mine tumor stage at the time of diagnosis and outcome fibroblasts were trypsin-digested. Peptides were labeled (18). Each sample was deparaffinized for antigen retrieval with iTRAQ for relative quantification and fractionated using the PT Link Module (Dako) at high pH for 20 with OFFGEL, pH 3–10. To avoid biases in peptide minutes, rehydrated, and then incubated with the primary labeling that could affect the final quantification, we antibody against a-SMA, Snail1, S100A4, CALU, CDH11, performed two replicate analyses using the 114 and LTBP2, FSTL1, or OLFML3 (Supplementary Table S1). The 116 iTRAQ tags for normal fibroblasts peptides and reaction was revealed using 3,30-diaminobenzidine (DAB) 115 and 117 for CAFs peptides (Supplementary Fig. as the chromogen and hematoxylin for counterstaining, S1). In total, 3,846 and 734 peptides corresponding to and observed in an Olympus microscope. ImageJ software 1,353 and 295 proteins were identified in the whole-cell was used to quantify the DAB staining of immunohisto- extract and conditioned medium, respectively. For quan- chemical images. tification, peptide ratios were calculated by comparing the intensity of the reporter ions in the MS–MS spectra, Bioinformatics and normalized either using the ratios corresponding to Ingenuity Pathway Analysis (IPA; Ingenuity systems, seven different housekeeping proteins in the whole-cell Inc.) was used to analyze the predicted biologic functions extract or the median in the secretome (Supplementary of the proteins deregulated in CAFs and to determine Fig. S2). A total of 1,102 and 250 proteins were quan- protein interactions as well as for network analysis. Bio- tified in the whole-cell extract and conditioned medium, GPS software was used to determine the top-5 tissues with respectively (Supplementary Tables S2 and S3). We used highest expression of each altered protein compartment several criteria for protein selection, including: (i) a fold- to determine the desmoplasticsignature(10).FatiGo change of 1.4 or more relative to control samples; (ii) was used to get further insight in deregulated functions proteins present in both replicates; (iii) expression ratios using a cutoff value of less than 0.1 for the adjusted P following a similar trend in both replicates; and (iv) value (19). proteins identified with a single unique peptide or var- iability higher than 50% were manually inspected to Results verify that the peptide only corresponded to a single Isolation and characterization of fibroblasts from protein. We selected fold-change of 1.4 as a minimum AOM/DSS colon cancer tumors value to include all relevant proteins that showed sys- After AOM/DSS treatment, development of adenocar- tematic change and to compensate for the compression of cinomas in the distal colon of mice was visible by visual iTRAQ ratios, which leads to underestimation of fold- inspection and histologic staining (Fig. 1A). Mouse ade- changes (20). Using a fold-change of 1.4 or more, we nocarcinomas were equivalent to human colorectal ade- found 132 proteins deregulated in complete cell lysates, nocarcinomas, corresponding to a well-differentiated infil- 109 upregulated, and 23 downregulated (Supplementary trating enteroid adenocarcinoma, with the presence of Table S4), as well as 125 proteins in the secretome, with large numbers of flat and polypoid malignant tumors. The 72 upregulated and 53 downregulated (Supplementary mucosa from control mice was normal, without dysplastic Table S5). changes (Fig. 1A). On immunohistochemical analysis, Gene Ontology analysis of whole-cell extracts results AOM/DSS tumors showed high expression level of a-SMA, from CAFs showed a clear upregulation of ECM

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α-SMA Snail1 S100A4 A H&E B Control Control AOM/DSS AOM/DSS

Ratio* 4.8 6.2 2.6

C DAY 1 DAY 7 DAY 15 DAY 25 D

NFs CAFs Ratio*

S100-A4

NFs 1.0 2.0 2.0 α-SMA Control 1.0 5.0 5.0 Snail1

1.0 26.0 26.0 Vim CAFs 1.0 1.0 1.0 Epcam AOM/DSS 0.0 0.0 – α-Tub

Figure 1. Isolation and culture of fibroblast from colorectal tissue from AOM/DSS–treated mice. A, mice treated with AOM/DSS showed engrossed intestine with polypoid tumors, whereas mice treated with vehicle displayed a normal colon (left). H&E staining of tumoral and normal colon (right). B, immunohistochemical analysis of cancer and normal tissue using antibodies against S100A4, Snail1, and a-SMA. C, fibroblast isolation from colonic tissues by the explants technique between days 1 and 25. Tissues were cut into 2- to 3-mm fragments and placed in culture medium for 3 weeks to allow fibroblast migration. Top, control explants; bottom, AOM/DSS explants. Explants are indicated with white arrows. D, Western blot analysis of isolated fibroblasts using antibodies against S100A4, Snail1, and a-SMA. Vimentin was used as positive fibroblast marker and EpCAM as negative control. B and D, ratio of AOM-DSS/control was calculated for Snail1, a-SMA, and S100A4 using ImageJ software or QuantityOne 1-D Analysis Software, respectively.

constituents (collagen a1 types I, V, III, and XII, collagen secretome, we found "Cancer" to be the most represented a2 types I and V, and fibulin 2), proteins for matrix biologic function altered (n ¼ 97 proteins and P values assembly (decorin, prolyl 4-hydroxylase a2, and leprecan ranging from 2.82E14 to 1.35E04), and "Organismal 1), proteins related to TGF-b signaling, such as LTBP2 Injuries and Abnormalities" as the top biologic function (latent TGF-b–binding protein 2), cadherin 11, fibrillin 1, altered (n ¼ 54 proteins and P values ranging from and insulin-like growth factor (IGF) 2 receptor (IGF2R; 2.55E14 to 1.54E04). With regard to altered network Supplementary Table S6). In the secretome, 26 of 125 function "Dermatological Diseases and Conditions, Cellu- proteins were related to ECM. In addition, we identified lar Assembly and Organization, Cellular Function and differentially expressed chemokines, such as CCL11, Maintenance" with 31 proteins emerged as identified in CCL8, CXCL5, or CCL2, IGF-related proteins such as cell extracts (Fig. 2A), and "Cell morphology, Cellular IGFBP7, or IGF1, cell adhesion, and wound response Assembly and Organization, Cellular Function and Main- (Supplementary Table S7). Collectively, these data sug- tenance" was significantly altered in the secretome, with 28 gest that activated fibroblasts participate in ECM remo- proteins identified (Fig. 2B). deling, wound healing, and epithelial differentiation, and With FatiGo, we found that oxidative phosphorylation are important regulators of inflammation. and molecular functions related to redox processes were Altered biologic functions, networks, and pathways were altered in whole-cell extract. Transport of proteins and analyzed using IPA (Supplementary Table S8) and FatiGO their cellular localization emerged as the top altered (Supplementary Table S9). Using IPA in whole-cell extract, biologic processes. In the secretome, the ECM and focal "Connective Tissue disorders," related with ECM remodel- adhesion were the most altered processes. In addition, we ing, was the top altered function (n ¼ 21 proteins and observed a focus toward modulation of molecular func- P values ranging from 1.59E11 to 1.34E02). In the tions through receptor binding, whereas the most altered

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Figure 2. Network functions affected in CAFs. Protein networks were identified by IPA using the 132 and 125 differentially expressed proteins identified in whole-cell extract and secretome, respectively. A, "Dermatological Diseases and Conditions, Cellular Assembly and Organization, Cellular Function and Dermatological Diseases and Conditions, Cellular Maintenance" network was identified in whole- Assembly and Organization, Cellular Function and cell extracts with a score of 47. The network Maintenance consisted of 26 upregulated and five downregulated proteins from 34 direct- B interacting proteins. B, "Cell Morphology, Hematological System Development and Function, Inflammatory Response" was significantly altered in the secretome with a score of 44. The network consisted of 27 upregulated and one downregulated proteins from 35 direct-interacting proteins.

Cell morphology, Cellular Assembly and Organization, Cellular Function and Maintenance

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biologic processes were the response to wounding, exter- whereas CSF1 was downregulated in CAFs (Supplementary nal stimulus, and inflammatory response. Collectively, Table S7). To investigate other additional changes in cyto- these protein networks indicate the association of CAF kines, we used a mouse chemokine microarray for CAF/NF proteins with desmoplastic and proinflammatory signa- supernatants (Fig. 4A). CCL11, CCL8, SAA3, and CXCL5 tures, respectively. antibodies were not present in the arrays. Microarray semiquantitative data confirmed the increase in CCL2 expres- Validation and fibroblast specificity of selected sion, although not as relevant as in iTRAQ, and showed an biomarkers increase in other proinflammatory cytokines such as IL-6, After previous network analyses and data-mining using CXCL2, CCL20, and osteopontin (OPN; Fig. 4B). Overall, the GeneCards database, we selected top upregulated our chemokine profile was similar to the one described for proteins that were not previously associated to colon cancer skin fibroblasts that showed NF-kB–dependent can- cancer and pertained to major networks (Fig. 2 and cer-promoting activity (7). Supplementary Tables S8 and S9). Therefore, 11 differ- To be activated by cytokines, cancer cells should express entially expressed proteins (five from whole-cell extract the corresponding receptors. To detect CCL2 and CCL8 and six from secretome) were validated using Western blot receptors(CCR1,2,3,and5)aswellasthereceptorforIL- analysis. We selected a relatively large number of proteins 9 (IL-9R), we performed RT-PCR assays with KM12C and from both locations to make a more comprehensive SM cells. These assays showed that both cell lines express analysis, as they would represent different family proteins IL-9R as well as CCR1 and CCR3, whereas CCR2 and and functionalities. Results of Western blot analysis were CCR5 were detected only in KM12C and KM12SM, consistent with the iTRAQ quantification data (Fig. 3A respectively (Fig. 4C). Then, we investigated the effect of and B). Molecules such as CRABP1, LTBP2, FDPS, and CCL2, CCL8, and IL-9 on cell proliferation, adhesion, CDH11 were upregulated in whole-cell extracts (Fig. 3A), migration, and invasion of colon cancer cell lines (Fig. whereasOLFML3,CALU,AEBP1,LMOD1,SPON2,and 4D). A more than 2-fold increase was observed at the FSTL1 were upregulated in CAFs supernatants (Fig. 3B). adhesion, migration, and invasion level in KM12 cells, To confirm that purified fibroblasts were equivalent to after addition of CCL2 and CCL8, but there was no effect those present in the tumor, we validated a subset of these for IL-9. In contrast, IL-9 induced a significant increase in markers in the original tissues of the AOM/DSS model by the proliferation of KM12 cell lines. Collectively, these quantitative real-time PCR (RT-PCR) and Western blot data suggest that CCL2 and CCL8 play an important role analysis. Protein and mRNA expression values were sim- in tumorigenesis and the acquisition of invasive capacity ilar between purified fibroblasts and original tissues (Sup- of cancer cells. plementary Fig. S3). To confirm fibroblast specificity of stromal biomarkers, Desmoplastic signature in AOM/DSS colon fibroblasts wecomparedthepresenceoftheseproteinsinCAFswith and prognostic value regard to CT26—a colon adenocarcinoma murine cell Because activated fibroblasts are characterized by the line—by Western blot analysis. Expression of LTBP2, acquisition of a myofibroblast phenotype consisting of FDPS, CDH11, and OLFML3 was specific or clearly upre- different smooth–muscle-like proteins, such as a-SMA, gulated in fibroblasts with regard to the epithelial CT26 one aim of this work was to define a desmoplastic cells(Fig.3C).Whenwetestedthesupernatants,SPON2 signature for colon cancer. We searched for desmoplastic and FSTL1 were also specific for fibroblasts. Finally, we markers on the differentially expressed proteins using the investigated the capacity of CDH11 for cell sorting of Bio-GPS database. In the secretome, we found 30 secreted fibroblasts. PDGF receptor a (PDGFRa)wasusedasa proteins (24% of the total) preferentially expressed in positive control (7). CDH11 antibodies were able to myofibroblasts (Table 1), including seven collagens, col- isolate fibroblast cell lines, such as mouse NIH-3T3 or lagen metabolism-related proteins (LOX and PCOLCE), human BJ-hTERT, but did not work with epithelial cell fibronectin, proteoglycans (perlecan and byglican), cyto- lines such as CT26 or RKO (Fig. 3D). These results sug- kines (CCL11, CCL8, and CCL2), and calcium metabo- gested that all these six proteins were stromal biomarkers lism proteins [calumenin (CALU)], IGF-binding proteins specific for colon cancer. (IGFBP6 and -7), and FSTL1. The cell lysate showed a similar profile. Cytokines and growth factors deregulated in the Then, we studied the value of the proteins in this desmo- secretome of activated fibroblasts are plastic signature as prognostic biomarkers in human proinflammatory and protumorigenic on colon patients. Given the large number of candidates, we carried cancer cells out a meta-analysis using databases from the "cBio Cancer CAFs secrete chemokines that control critical steps of the Genomics Portal" (23), which contains mRNA expression adhesion–invasion–metastasis cascade and affect recruit- datasets from 274 samples of colon cancer. Using Kaplan– ment of inflammatory cells and enhancement of angiogen- Meier for overall survival analysis, we found some combi- esis (21, 22). By MS, we observed a significant increase in the nations of proteins with prognostic value from whole-cell expression of cytokines such as CCL11 (Eotaxin-1), CCL8 extract (Supplementary Fig. S4A) and the secretome (Sup- (MCP-2), CCL2 (MCP-1), SAA3, and CXCL5 (ENA-78), plementary Fig. S4B). The most significant combination

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Ratio Ratio

A ) B

NFs CAFs NFs CAFs ) ^3 CAFs/NFs CAFs/NFs 300 ^3 200 iTRAQ WB iTRAQ WB 200 Crabp1 3.93 3.6 Olfml3 100 15.06 8.5 Intensity 100 Intensity [counts](10 [counts](10 114 115 116 117 0 m/z 114 115 116 117 m/z ) ^3

15 ) 250 ^3 Ltbp2 10 2.42 2.2 Calu 150 11.88 8.1

Intensity 5 50 [counts](10 Intensity

114 115 116 117 [counts](10 m/z 114 115 116 117 m/z ) ) ^3 120 140 ^3

Fdps 60 2.07 2.1 Aebp1 80 11.75 7.9 Intensity

20 Intensity 20 [counts](10 [counts](10 114 115 116 117 114 115 116 117 m/z m/z ) ) ^3

^3 20 600 Cdh11 400 2.03 4.9 Lmod1 10 11.03 3.7 Intensity

Intensity 200 [counts](10 [counts](10 114 115 116 117 114 115 116 117 m/z m/z )

^3 100 ) ^3 600 50 Thy1 400 0.29 0.1 Spon2 10.33 3.8 Intensity Intensity

200 [counts](10 10

[counts](10 114 115 116 117 114 115 116 117 m/z m/z )

^3 35 ) 2.5 ^3 25 α-Tub 1.5 1.19 1.2 Fstl1 15 3.08 2.4 Intensity 5

Intensity 0.5 [counts](10

[counts](10 114 115 116 117 114 115 116 117 m/z m/z CDH11 PDGFRα CD1.34 1.44 Cell Iysates Supernatants NIH-3T3 CT26 CAFs CT26 CAFs kDa kDa Crabp1 Calu 10 48 0.63 0.59 Ltbp2 180 Aebp1 75 BJ-hTERT

48 Fdps Lmod1 63 130 0.88 0.79 100 Cdh11 Spon2 35 75 CCD18

35 Cell number Olfml3 35 Fstl1 0.32 0.31 α-Tub 63 35 RhoGDI CT26

0.31 2.01 RKO

00100 101 102 103 100 101 102 103 Fiuorescence intensity

Figure 3. Western blotting validation of iTRAQ data and biomarker expression on stromal cells. Protein samples from whole-cell extract (A) or concentrated conditioned media (B) of CAFs and normal fibroblasts were separated on SDS–PAGE gels, transferred to nitrocellulose membranes, and probed with the indicated antibodies. Tubulin was used as a control. Protein abundance was quantified by densitometry, and CAF/NF ratios were compared with the iTRAQ ratios. C, Western blot analysis of the expression of selected stromal biomarkers in the murine adenocarcinoma cell line CT26 and CAFs using complete cell extracts and concentrated supernatants, respectively. D, flow cytometric analysis for testing CDH11 and PDGFRa specificity (gray areas) on fibroblasts and cancer cell lines. An irrelevant antibody was used as a control (white areas). www.aacrjournals.org Clin Cancer Res; 19(21) November 1, 2013 6013

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A Galectin-1 NFs Eotaxin IL-6 CCL-20 Galectin-1 CAFs Eotaxin IL-6 CCL-20

CCL-5 CCL-2 CXCL-2 CCL-5 CCL-2 CXCL-2

B 50,000

45,000 NFs 40,000 CAFs 35,000 30,000 25,000 20,000 15,000 Arbitrary units 10,000 5,000 0

CCR1 CCR2 CCR3 CCR5 IL−9R GAPDH

D KM12C KM12SM KM12C KM12SM Proliferation Adhesion 250 300

0.5 0.8 2 *** *** * *** *** *** *** 0.7 200 250 0.4 * 0.6 * 200 0.3 *** 0.5 150 0.4 150 0.2 100 0.3 100 0.2 0.1 50 50 0.1 Adhered cells/mm

Absorbance (560 nm) 0 0 0 0

Migration Invasion 120 350 m/h) 4 *** 10 *** *** μ *** *** *** 300 *** *** 100 3 8 *** 250 *** 80 200 6 60 2 150 4 40 100

1 Invasive cells 2 20 50 0 0 0 0

Cell migration speed ( μ

6014 Clin Cancer Res; 19(21) November 1, 2013 Clinical Cancer Research

Molecular Signatures of Colon Cancer–Associated Fibroblasts

included RPN2, IVL, RP1, and CALU (Supplementary Fig. expression displayed a significant association with disease- S4C), which displayed a highly significant association to free survival (log-rank test P ¼ 0.015) and overall survival overall survival (log-rank test P ¼ 0.000095). In contrast, (log-rank test P ¼ 0.009) in human cancer samples (Fig. the association with survival in ovarian or lung adenocar- 5D). The rest of the biomarkers did not reach significant cinoma did not deliver significant P values (data not correlation with metastasis or survival (Supplementary Fig. shown). S5A–K).

Verification of stromal markers and prognostic Discussion association in human cancer samples The use of the AOM/DSS murine model of sporadic Immunohistochemical staining was performed to vali- colon cancer for the isolation of purified and homoge- date the expression of the five selected candidate stromal neous populations of CAFs, together with in-depth prote- biomarkers CALU, CDH11, FSTL1, OLFML3, and LTBP2 in omic analysis, allowed the identification of an elevated AOM/DSS–induced tumor samples. SPON2 was not tested number of proteins deregulated after fibroblast activation because of the lack of suitable antibodies. The selected in cancer. The identification of many of the currently markers were strongly expressed in the stroma of AOM/ known stromal biomarkers confirmed the validity of DSS–induced murine adenocarcinoma, with little or no our experimental approach. As an example, we found an expression in normal colon mucosa (Fig. 5A). The expres- increase in collagens type III and XII, which are mainly sion of these markers was more intense in the leading edge implicated in desmoplasia and colorectal cancer metas- of tumors, which could be associated with the desmoplastic tasis (10). At least six identified proteins: LTBP2, CDH11, invasion front. FDPS, OLFML3, SPON2, and FSTL1 were novel candidate Finally, to investigate the relevance of these markers in stromal biomarkers, as no expression was observed in the human colon cancer samples, we used a tissue micro- murine adenocarcinoma cell line CT26. In addition, array. Clinical information, together with the stromal CDH11 was useful to isolate colon cancer fibroblasts. expression of the selected CAF markers, is summarized The relevance of these biomarkers for colorectal cancer in Supplementary Table S10. We observed an increased prognosis was confirmed by studies with human tissue expression for CALU, CDH11, FSTL1, OLFML3, and microarrays. The expression of CDH11, FSTL1, OLFML3, LTBP2 in the tumoral respect to normal stroma (Fig. and LTBP2, together with CALU, was increased in the 5B).Allofthemshowedstrongerexpressioninthe tumoral stroma of patients with colorectal cancer. More- leading edge of the tumors, where the tumor infiltrates over, we observed an association of CALU and CDH11 the surrounding areas. Tumoral epithelial cells showed an with poor survival and prognosis in human patients with increased expression in more than 70% of the tumors for cancer. Together, these data support the value of the CALU,CDH11,andOLFML3,butnosignificantexpres- AOM/DSS murine model for the discovery of stromal sion was detected for FSTL1 (Fig. 5B). In contrast, in the biomarkers applicable in human patients. stroma of normal tissue, the staining of these markers was Despite this, the azoxymethane model presents some mainly observed to be negative or weak in contrast to limitations. The most relevant is its inability to develop tumoral stroma where the markers were highly overex- metastasis. Recently, an adenomatous polyposis coli (APC) pressed (Fig. 5B). mutant mouse model of sporadic colon cancer was used to Then, we determined if the expression of these proteins in analyze the proximal fluid proteome of whole tumor tis- the stroma was associated with survival in human cancer sues, without isolation of pure cell populations (24). Look- using the tissue microarray data for patients followed on the ing at the candidate protein biomarkers identified in that long term (>5 years). The series was retrospectively selected model, we have observed several coincidences in ECM (Supplementary Table S10). High stromal expression of fibroblast-related proteins: biglycan, cingulin, collagens, CALU correlated significantly with lymph node involve- decorin, or lamin A/C, confirming the relative similarity of ment at the moment of diagnosis (P ¼ 0.034; Fig. 5C) and both models. with poor prognosis (P ¼ 0.010; Fig. 5D). In addition, CAFs exhibited specific proinflammatory and desmo- stromal expression of CDH11 correlated with disease-free plastic protein signatures for colon cancer. Several reports survival (P ¼ 0.051; Fig. 5E), but not with prognosis (P ¼ support the role of the desmoplastic microenvironment 0.105). In addition, the combination of CALU with CDH11 in tumor promotion (2). For instance, Tsujino and

Figure 4. Chemokine expression in conditioned medium from CAFs and normal fibroblasts. Functional studies with selected chemokines. A, a representative image of a chemokine microarray after screening of conditioned medium from CAFs and normal fibroblasts. White arrows indicate the most deregulated cytokines in CAFs and normal fibroblasts. B, most abundant chemokines and cytokines differentially expressed in CAFs versus normal fibroblasts. Bar graphs were calculated in arbitrary units with the mean value of the duplicates present in the microarrays. C, RT-PCR analysis of the indicated cytokine receptors in KM12C and KM12SM cell lines. D, proliferation, adhesion, migration, and invasion of colon cancer KM12 cells in presence or absence of the indicated chemokines. Proliferation was determined by MTT assays after 24 hours of culture. Cell adhesion to Matrigel was performed after 2 minutes of incubation with the indicated chemokines and 4 minutes of adhesion. Migration speed was calculated as the distance covered by the cells in 24 hours. Cell invasion was done across Matrigel of the KM12 cell lines treated as indicated. Experiments were performed three times. Error bars, SD (, P < 0.05; , P < 0.005).

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Table 1. Myoﬁbroblastic signature associated with proteins in the CAF secretomea

Accession Average number Description Name fold-change SD P48298 Eotaxin Ccl11 25.37 25.83 Q61581 IGFBP7 Igfbp7 11.06 5.21 Q9Z121 C–C motif chemokine 8 Ccl8 11.00 10.87 Q61554 Fibrillin-1 Fbn1 7.81 5.60 Q60847 Collagen a-1(XII) chain Col12a1 6.12 9.61 O35887 Calumenin Calu 6.11 2.59 P11087 Collagen a-1(I) chain Col1a1 5.51 2.09 P08121 Collagen a-1(III) chain Col3a1 5.48 0.38 D3YYT0 Cadherin-2 Cdh2 4.46 1.41 P28301 Protein-lysine 6-oxidase Lox 4.31 1.98 G5E8M2 Fibronectin 1, isoform CRA_d Fn1 4.27 2.17 P10148 C–C motif chemokine 2 Ccl2 4.11 1.42 Q01149 Collagen a-2(I) chain Col1a2 3.73 1.02 Q61398 Procollagen C-endopeptidase enhancer 1 Pcolce 3.35 0.63 P98063 Bone morphogenetic protein 1 Bmp1 3.28 1.06 P28653 Biglycan Bgn 3.21 0.56 P08122 Collagen a-2(IV) chain Col4a2 3.17 1.55 E9QL02 Heparan sulfate proteoglycan 2 Hspg2 3.13 0.49 Q91X24 Igfbp6 protein Igfbp6 3.12 0.95 Q9WVJ9 EGF-containing ﬁbulin-like ECM protein 2 Efemp2 3.04 1.41 P10493 Nidogen-1 Nid1 2.14 0.26 P35441 Thrombospondin-1 Thbs1 2.08 0.36 Q3U962 Collagen a-2(V) chain Col5a2 2.05 0.30 P07214 SPARC Sparc 1.81 0.21 Q8BND5 Sulfhydryl oxidase 1 Qsox1 1.80 0.38 P28862 Stromelysin-1 Mmp3 1.80 0.67 O88207 Collagen a-1(V) chain Col5a1 1.71 0.38 P12032 Metalloproteinase inhibitor 1 Timp1 1.69 0.45 Q62356 Follistatin-related protein 1 Fstl1 1.59 0.16 P10639 Thioredoxin Txn 0.19 0.06

aDifferentially expressed proteins were identiﬁed as preferentially expressed in smooth muscle if they were among the top ﬁve tissues with highest expression using the Bio-GPS database.

colleagues (25) reported a strong association between the survival, alone or in combination with other proteins such presence of abundant stromal myofibroblasts, liver as CDH11. Cadherin-11 is a mesenchymal cadherin, upre- metastasis, and shorter disease-free survival. In addition, gulated by TGF-b, which has been identified as a novel target it has been reported that tumor-derived ECM is stiffer for pulmonary fibrosis and associated with invasion in than normal ECM. Moreover, increased matrix stiffness squamous cell carcinoma (32, 33). The combination of and ECM remodeling was observed in premalignant tis- CALU with CDH11 significantly improved the prognostic sue. This increase was shown to contribute to malignant value of disease-free survival. transformation in the breast (26). In colorectal cancer, We found several other TGF-b–related deregulated pro- previous studies showed a correlation between the pres- teins as stromal biomarkers. OLFML3 belongs to the olfac- ence of myofibroblasts (25), vimentin (27), or expression tomedin domain-containing proteins. They are known of fibroblast activation protein (FAP; ref. 28) with prog- BMP antagonists (34), and also showed proangiogenic nosis and recurrence. activity in cancer (35). FSTL1 expression was the most Here, we identified several new prognostic markers asso- specific for the human stromal compartment. FSTL1 is ciated with fibroblasts. Calumenin is a calcium-binding a TGF-b–inducible gene, SPARC-related, that enhances protein that regulates the activity of coagulation factors inflammatory cytokine/chemokine expression (36) and (29), and was reported as being overexpressed in colon seems to be implicated in angiogenesis and revasculariza- cancer (30, 31). Although calumenin expression is not tion (37). Another TGF-b signaling-related protein was restricted to the stroma, we have found a good association LTBP2, which colocalizes with fibronectin and collagen with stromal expression, lymph node invasion, and lower type I for ECM regulation and remodeling (38, 39). LTBP2

6016 Clin Cancer Res; 19(21) November 1, 2013 Clinical Cancer Research

Molecular Signatures of Colon Cancer–Associated Fibroblasts

Control AOM/DSS Human Human A mouse mouse B normal mucosa cancer mucosa CALU CDH11 FSTL1 OLFML3 LTBP2

Negative 1.0 Negative 1.0 CDH11 C D CALU Positive Positive 0.8 0.8

0.6 0.6 CALU staining Negative 0.4 0.4 Cum survival Moderate 0.2 0.2 Intense Disease-free survival P = 0.010 P = 0.051 0.0 0.0 100 P = 0.034 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 80 Time (mo) Time (mo) 60 1.0 CALU Negative 1.0 CALU Negative Positive Positive 40 CDH11 CDH11 0.8 0.8 % patients 20 0.6 0 0.6 NO YES 0.4 0.4 Cum survival Lymph node 0.2 0.2 involvement Disease-free survival P = 0.009 P 0.0 0.0 = 0.015 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 Time (mo) Time (mo)

Figure 5. Validation of stromal biomarkers and prognostic value of CALU and CDH11 in patients with colorectal cancer. A, immunohistochemical staining of AOM/DSS cancer and normal mouse tissues using antibodies against CALU, CDH11, FSTL1, OLFML3, and LTBP2. B, immunohistochemical analysis of CALU, CDH11, FSTL1, OLFML3, and LTBP2 expression in human tissue microarrays. White arrows indicate the leading edge of the tumor. Pictures were taken at 200 magniﬁcation. C, percentage of patients with invasion of regional lymph nodes at time of diagnosis in function of CALU stromal expression; P values were calculated with x2 test. D, Kaplan–Meier analyses of overall and disease-free survival of patients according to the stromal expression of CALU or CDH11 and their combination, respectively. For statistical analysis of signiﬁcance, we used the log-rank test. www.aacrjournals.org Clin Cancer Res; 19(21) November 1, 2013 6017

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is overexpressed in pancreatic cancer, being mainly located markers in colon cancer was identified. Among them, the in the cancer stroma (40). TGF-b activates CAFs by changing combination of CDH11 with CALU gave a significant the secretion profile of chemokines to assist colorectal prognostic value. In addition, verification of the results cancer cells in the progression of the disease. An inflam- with human samples confirmed the value of the model matory microenvironment can promote malignant progres- and the strategy for identification of novel candidate sion and participate in the recruitment and retention of prognostic biomarkers with clinical potential. In summa- infiltrating leukocytes to inhibit the resolution of inflam- ry, this study defines a new set of stromal biomarkers with mation (41, 42). good prognostic value that might constitute an alternative Evidence was provided of a significant increase in to current markers such as a-SMA or the presence of proinflammatory chemokines (CCL2, CCL11, CCL8, myofibroblasts in colon cancer. CCL20, CXCL5, and IL-9) in the conditioned media of mouse CAFs, either by MS or chemokine microarrays. In Disclosure of Potential Conflicts of Interest addition to function as leukocyte chemoattractants and No potential conflicts of interest were disclosed. proangiogenic properties, human CCL2, CCL8, and IL-9 promoted tumorigenesis in two human colorectal cancer Authors' Contributions cell lines. CCL8—also called MCP2—a pluripotent che- Conception and design: M.J. Fernandez-Acenero,~ A. Garca de Herreros, I. Casal mokine, attracts monocytes (43) and lymphocytes (44). Development of methodology: S. Torres, M. Mendes, M.J. Fernandez- From our results, CCL2 and CCL8 regulated the prolif- Acenero,~ C. Pena~ eration, adhesion, migration, and invasion of two colo- Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Torres, R.A. Bartolome, R. Barderas, M.J. rectal cancer cell lines, whereas IL-9 was only efficient in Fernandez-Acenero,~ A. Pelaez-Garca, M. Lopez-Lucendo, R. Villar-Vazquez, tumor growth proliferation. Because CCL2 and CCL8 A. Garca de Herreros, F. Bonilla share at least three receptors (CCR2, CCR3, and CCR5; Analysis and interpretation of data (e.g., statistical analysis, biosta- tistics, computational analysis): S. Torres, R.A. Bartolome, M. Mendes, R. ref. 45), this could explain the similar effect induced by Barderas, M.J. Fernandez-Acenero,~ R. Villar-Vazquez, I. Casal both chemokines in colorectal cancer. In fact, several Writing, review, and/or revision of the manuscript: S. Torres, M. Mendes, M.J. Fernandez-Acenero,~ F. Bonilla, I. Casal solid cancers, including colorectal cancer, showed expres- Administrative, technical, or material support (i.e., reporting or orga- sion of functional chemokine receptors on tumor cells, nizing data, constructing databases): S. Torres, C. Pena,~ M. Lopez- able to promote proliferation and metastasis (46). There- Lucendo, R. Villar-Vazquez Study supervision: I. Casal fore, CCL8 and IL-9 are new colon cancer–associated chemokines that deserve further investigation. Stromal chemokines might work through either paracrine or Grant Support This work was financially supported by a grant to established research autocrine pathways. CAFs and myofibroblasts could dif- groups of the Asociacion Espanola~ Contra el Cancer (AECC); to R.A. ferentiate from resident tissue fibroblasts under various Bartolome, and by the Spanish Ministry of Science and Innovation (grant no. BIO2012-31023 and the Comunidad de Madrid (grant no. S2010/BMD- paracrine stimuli (47). In this regard, resident monocytes 2344/Colomics2). S. Torres is the recipient of a Juan de la Cierva programme or other immune cells might secrete chemokines that fellowship. M. Mendes was the recipient of a ProteoRed contract. R. Barderas activate the proinflammatory signature in CAFs by para- was a recipient of a JAE-DOC/FSE Contract (CSIC), and currently has a fellowship of the Ramon y Cajal programme. A. Pelaez has a Formacion del crine stimulation. In addition, carcinoma cells may pro- Personal Investigador (FPI) fellowship from the MINECO. R. Villar-Vazquez duce chemokines that affect CAFs. In turn, CAFs secrete has a Formacion del Personal Universitario (FPU) fellowship from the cytokines that can modulate tumor cells or other fibro- Ministry of Education and Culture (MEC). The costs of publication of this article were defrayed in part by the blasts in different ways. payment of page charges. This article must therefore be hereby marked Our data provide a comprehensive view of the many advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate molecules secreted by CAFs that favor the progression and this fact. invasion of tumors, from angiogenesis to desmoplasia Received April 30, 2013; revised August 9, 2013; accepted August 24, 2013; and profibrotic mechanisms. A panel of new stromal published OnlineFirst September 11, 2013.

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www.aacrjournals.org Clin Cancer Res; 19(21) November 1, 2013 6019

Proteome Profiling of Cancer-Associated Fibroblasts Identifies Novel Proinflammatory Signatures and Prognostic Markers for Colorectal Cancer

Sofia Torres, Rubén A. Bartolomé, Marta Mendes, et al.

Clin Cancer Res 2013;19:6006-6019. Published OnlineFirst September 11, 2013.

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