Molecular Signatures of Colon Cancer-Associated Fibroblasts

Author Manuscript Published OnlineFirst on September 11, 2013; DOI: 10.1158/1078-0432.CCR-13-1130 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Molecular signatures of colon cancer-associated fibroblasts

PROTEOME PROFILING OF CANCER-ASSOCIATED FIBROBLASTS IDENTIFIES NOVEL PRO-INFLAMMATORY SIGNATURES AND PROGNOSTIC MARKERS FOR COLORECTAL CANCER

Sofia Torres1*, Rubén A. Bartolomé1*, Marta Mendes1, Rodrigo Barderas1†, Mª Jesús Fernandez-Aceñero2, Alberto Peláez-García1, Cristina Peña3, María Lopez-Lucendo4, Roi Villar-Vázquez1, Antonio Garcia de Herreros5, Felix Bonilla3 and J. Ignacio Casal1*

1. Department of Cellular and Molecular Medicine. Centro de Investigaciones Biológicas (CIB-CSIC). 28040 Madrid, Spain. 2. Pathology Department. Fundación Jiménez Díaz. Madrid. Spain 3. Oncology Department. Hospital Puerta de Hierro Majadahonda. Madrid. Spain 4. Proteomics core facility. CIB-CSIC. Madrid. Spain 5. IMIM-Hospital del Mar. Barcelona. Spain

*. Equal authorship

†. Present address. Department of Biochemistry and Molecular Biology I. Universidad Complutense. Madrid. Spain

RUNNING TITLE: Molecular signatures of colon cancer-associated fibroblasts

KEYWORDS: Cancer-associated fibroblasts; prognostic biomarkers, chemokines, colon cancer, proteomics

Words count: 5254 Figures: 5 Tables: 1

CONFLICT OF INTEREST: The authors declare no conflict of interest.

GRANT SUPPORT: This research was supported by a grant to established research groups of the “Asociación Española Contra el Cancer (AECC), grant BIO2012-31023 from the Spanish Ministry of Science and Innovation and grant S2010/BMD-2344/ Colomics2 from Comunidad de Madrid.

*Corresponding author: J. Ignacio Casal Department of Cellular and Molecular Medicine Centro de Investigaciones Biológicas (CIB-CSIC) Ramiro de Maeztu, 9 28040 Madrid. Spain Phone: +34 918373112 Fax: +34 91 5360432 e-mail: [email protected]

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 11, 2013; DOI: 10.1158/1078-0432.CCR-13-1130 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Molecular signatures of colon cancer-associated fibroblasts

Translational relevance

Our knowledge of cancer-associated stroma is rather limited in comparison with cancer

epithelial cells. Stroma constitutes the cancer microenvironment that nurtures cancer

cells and facilitates invasion and metastasis. Moreover, stromal myofibroblasts have

been associated to recurrence and survival. Novel biomarkers are required for the

isolation and characterization of colorectal cancer (CRC)-associated fibroblasts (CAFs)

in order to improve overall survival and recurrence prediction in CRC. We here describe

a strategy based on an in-depth proteome profiling of CAFs obtained from a mouse

model of human sporadic CRC. The use of this model facilitates the isolation of pure

populations of CAFs, avoiding other cell contaminants. The validity of this strategy is

supported by the identification of multiple stromal biomarkers previously described in

humans. In addition, we report a collection of novel stromal biomarkers as well as

comprehensive pro-inflammatory and desmoplastic CAFs signatures for CRC. The

value and relevance of these novel stromal biomarkers for colon cancer prognosis and

survival were validated in human samples.

Molecular signatures of colon cancer-associated fibroblasts

ABSTRACT

Purpose: Cancer-associated fibroblasts (CAFs) 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 (NFs). By using the explants technique we purified CAFs and NFs

from colon tissues. Whole cell extracts and supernatants were subjected to in-depth

quantitative proteomic analysis by tandem mass spectrometry. Further validations of up-

regulated proteins in CAFs were carried out by chemokine microarray analysis and

immunohistochemistry of mouse and human tissues.

Results: Using a fold-change ≥1.4, we found 132 and 125 differentially-expressed

proteins in whole cell extracts and supernatants, respectively. We found CAFs-

associated pro-inflammatory and desmoplastic signatures. The pro-inflammatory

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 to disease-free survival and poor prognosis.

Molecular signatures of colon cancer-associated fibroblasts

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.

Molecular signatures of colon cancer-associated fibroblasts

INTRODUCTION

The tumor stroma comprises most of the cancer mass and is mainly composed of

fibroblasts and endothelial cells, although also contains infiltrating immune cells and

pericytes (1). Stroma nurtures cancer cells and facilitates 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 fibroblasts are called activated fibroblasts,

myofibroblasts or cancer-associated fibroblasts (CAFs) and they acquire a particular

phenotype similar to fibroblasts present in skin wounds. Carcinoma progression is

associated with an increase in the production of fibrosis, known as desmoplasia, similar

to that present in wound healing (3). CAFs respond to profibrotic and promigratory

factors, such as TGFβ, PDGF, HGF or FGF2, promoting cancer progression. They are

characterized by increased expression of myofibroblastic markers like α-smooth muscle

actin (α-SMA), FSP1 or prolyl-4-hydroxilase (4, 5). Cancer fibroblasts proliferate more

than their normal counterparts and secrete more extracellular matrix (ECM) constituents

and ECM-degrading proteases (6). In addition, CAFs can mediate inflammation and

angiogenesis, as reported in skin cancer (7). Despite the significant number of markers

and secreted proteins already associated to activated fibroblasts, the study of their

contribution to tumor growth and invasion would benefit of additional markers for cell

selection, prognosis and invasion prediction (8).

Regarding human colon adenocarcinomas, CAFs synthesize ECM components

such as fibronectin, tenascin, collagens types I, III, IV, V and XII and proteoglycans

(biglycan, fibromodulin, perlecan and versican) (9, 10). They also contribute to the

formation of basement membranes by secreting collagen type IV and laminin. For a

more comprehensive molecular analysis, we propose the use of sensitive proteomic

Molecular signatures of colon cancer-associated fibroblasts

techniques combined with a mouse model of colon cancer, cell isolation and mass

spectrometry. We have chosen the well-established murine model of colitis-associated

cancer (CAC) based on the use of azoxymethane (AOM) and dextrane sodium sulphate

(DSS). Although the model does not progress to metastasis (11), it mimics quite well

many steps in cancer progression of sporadic colorectal cancer. In fact, this model has

been very useful for the elucidation of the role of TNF-α, IL-6, NFκB and other

molecules in the initiation and progression of inflammation-associated cancer (see (12)

for a review).

Fibroblast characterization has been largely complicated by the necessity to

isolate primary fibroblasts from the colonic tissue. In other publications, the

characterization of CAFs was carried out by co-culturing normal fibroblasts and colon

cancer cell lines (10), cell sorting (7) or cell immortalization (13). We propose the use

of direct intestinal explants in culture for fibroblast isolation and proteomic

characterization (14). This strategy strongly reduces sample complexity and

heterogeneity. The use of inbred mouse model reduces biologic variation due to genetic

and environmental heterogeneity. Since primary fibroblasts duplicate only a few times

before entering senescence, we discarded metabolic labeling for quantification.

Therefore, iTRAQ (isobaric tag for relative and absolute quantification) was preferred

because of its reproducibility and reliability (15).

Here, we compared the protein component of the whole cell extracts and

conditioned medium of primary CAFs and normal fibroblasts (NFs) isolated from

AOM/DSS-induced sporadic colorectal cancer mice or controls, respectively. We

identified 132 and 125 proteins deregulated in whole cell extracts and conditioned

medium, respectively, of CAFs versus NFs. In silico studies demonstrated a

predominant association of up-regulated proteins to deposition of extracellular matrix,

Molecular signatures of colon cancer-associated fibroblasts

wound healing and inflammation. Pro-inflammatory and desmoplastic signatures,

specific for colon cancer, were defined for CAFs-associated proteins. A number of

proteins were identified as promising tumor-associated stromal prognostic markers.

Among these proteins, high expression of CALU and CDH11 was associated to poor

survival in human cancer samples.

Molecular signatures of colon cancer-associated fibroblasts

MATERIAL AND METHODS

AOM/DSS-induced colon cancer model

All mouse studies were performed under the approval of the Animal Ethics Committee

of the Spanish Research Council (CSIC. Madrid. Spain). Animals were housed under

pathogen-free conditions and were given autoclaved food and water ad libitum. For the

AOM/DSS model, FVB/N mice (4-6 weeks-old) were weighed and given a single

intraperitoneal injection of azoxymethane (AOM; 10 mg/kg) or vehicle (PBS). Five

days later, animals received either 2.5% DSS or normal drinking water. Chronic colitis-

derived colon cancer was induced after three cycles of DSS treatment, which consisted

of 5 days with 2.5% DSS followed by 16 days with normal water. At the end of the

protocol, animals were sacrificed and distal colons were longitudinally cut, rinsed twice

with ice-cold PBS and cut in small pieces. Intestine pieces were either cultured to

isolate fibroblasts or fixed in 10% buffered formalin overnight for

immunohistochemistry analysis.

Cell line culture

KM12C and KM12SM human colon cancer cells were obtained from I. Fidler´s

laboratory (MD Anderson Cancer Center. USA). A large batch of working aliquots of

KM12 cells was stored in liquid nitrogen. For each experiment, cells were thawed and

kept in culture for a maximum of 10 passages in DMEM (Gibco-Life Technologies)

containing 10% fetal calf serum and antibiotics at 37ºC. Cells were tested for

mycoplasma but not authenticated, as Dr Fidler’s laboratory is where these lines were

originally isolated. CT26 murine colon cancer cell line was obtained directly from the

ATCC (which authenticated the cell line by short tandem repeat profiling), and

Molecular signatures of colon cancer-associated fibroblasts

passaged for fewer than 6 months after receipt or resuscitation according to provider

instructions.

Fibroblast isolation and culture

CAFs and NFs cultures were established from colonic tissues from AOM/DSS-treated

mice (n=16) and vehicle-treated (n=16) by the explant technique (14, 16). Tissues were

cut into 2-3 mm fragments and planted in Fibroblast Growth Medium-2 (Lonza)

containing 2% fetal calf serum, 1% penicillin/streptomycin/amphotericin B (Invitrogen)

at 37ºC in a 5% CO2-humidified atmosphere. NFs and CAFs grew around the explants

and were cultured approximately for 3 weeks. Then, tissue fragments were removed and

cells were trypsinized and seeded at a 70-90% confluence and cultured in complete

medium. When cells reached 95-100% confluence (approximately 1.7-2.0x104

cells/cm2) were washed with PBS, incubated with serum-free medium for 1 h, washed

again and incubated for 24 h in serum-free medium. Then, cells and conditioned

medium were collected, pooled in two different batches for NFs and CAFs and mixed

for performing the different experiments. We recovered 7.0 and 13.5 x 106 cells from

NFs and CAFs cultures, respectively.

Sample preparation and iTRAQ labeling

For a detailed description of sample preparation see Supplementary Methods. iTRAQ

labeling was carried out using iTRAQ Reagent 4-Plex kit (AB SCIEX), following the

manufacturer´s protocol with minor modifications. Briefly, 100 µg of protein from

whole cell lysate or 50 µg of protein from conditioned medium from NFs and CAFs

were digested with 5 µg trypsin (Promega) overnight at 37ºC. Peptide mixtures were

labeled with iTRAQ reagents, 114, 116 for NFs and 115, 117 for CAFs as illustrated in

Molecular signatures of colon cancer-associated fibroblasts

Supplementary Fig. S1. To remove interfering substances, SCX chromatography was

carried out using a Resource S column (GE healthcare). To adjust the pH between 2.5

and 3.3 the sample mixture was diluted with 15 vol of 10mM KH2PO4 pH 3.0

containing 25% acetonitrile (ACN). Peptides were eluted in 1ml of elution buffer (25%

ACN, 350mM KCl, 10mM KH2PO4, pH 3.0). Then, peptides were desalted with Sep-

Pak C18 cartridges (Waters), dried and reconstituted in OFFGel solution (Agilent

Technologies). All chemicals were obtained from Sigma-Aldrich.

Peptide Fractionation and Mass Spectrometry Analysis

Peptides were recovered in 12 fractions by isoelectric focusing using a low resolution

strip (pH 3-10) in a 3100 OFF-GEL fractionator (Agilent Technologies). Five µl of 25%

trifluoroacetic acid (TFA) were added to each fraction and desalting was performed

with Zip-Tip. Peptide mixtures were vacuum-dried and reconstituted with 6 µl 0.1%

formic acid. Then, peptides were trapped onto a precolumn C18-A1 ASY-Column (2

cm, ID100 µm, 5µm) (Thermo Scientific) and run with a linear gradient of 2–35% ACN

in 0.1% aqueous solution of formic acid. The gradient was performed over 180 min

using an Easy-nLC (Proxeon) at a flow-rate of 300 nL/min onto a Biosphere C18

column (75 µm, 16 cm, 3 µm) (NanoSeparations). Then, peptides were scanned and

fragmented with a linear ion trap-Orbitrap Velos (Thermo Scientific). The Orbitrap

Velos was operated in data-dependent mode to automatically switch between MS and

MS/MS. Survey full-scan MS spectra were acquired from m/z 400 to 1600 after

accumulation to a target value of 106 in the Orbitrap at a resolution of 60,000 at m/z

400. For internal mass calibration, we used the 445.120025 ion for lock mass. Charge

state screening was enabled and precursors with charge state unknown or 1 were

excluded. After the survey scan, the 10 most intense precursor ions were selected for

Molecular signatures of colon cancer-associated fibroblasts

CID-HCD MS/MS fragmentation. Peptide identification was performed in both CID

and HCD spectra and quantification of iTRAQ reporter ions was performed in HCD.

HCD was carried out with excess of collision energy for effectively maximizing

abundance of the reporter ions. For CID fragmentation the target value was set to

10,000 and normalized collision energy to 35%. For HCD, target value was set to

50,000 and collision energy was set to 55 %. Dynamic exclusion was applied during

30s. All MS data were analyzed and quantified with Proteome Discoverer (version

1.3.0.339) (Thermo) using standardized workflows (see supplementary Methods for a

more detailed description).

Mouse cytokine array

Conditioned medium from CAFs and NFs were collected after 24h in serum-free

medium and incubated with Mouse Cytokine Antibody Array C6 for semi-quantitative

analysis of 97 mouse cytokines (RayBiotech) according to manufacturer´s instructions.

Then, membranes were scanned and analyzed using Redfin, a 2D-gel image analysis

software (Ludesi). Relative semi-quantitative cytokine intensities were normalized in

comparison to control spots on the same membrane. Expression ratios were calculated

comparing the signal intensities for each spot of the different cytokines. The limit of

detection, sensitivity and the dynamic range of the measured cytokines were above 1

pg/mL, where most of cytokines can be detected.

Cell proliferation, adhesion, migration and invasion assays

KM12C and KM12SM human colon cancer cells (17) were cultured in Dulbecco´s

modified Eagle medium (Life Technologies) containing 10% fetal calf serum and

antibiotics at 37ºC in a 5% CO2-humidified atmosphere. Invasion, migration, adhesion

Molecular signatures of colon cancer-associated fibroblasts

and cell proliferation assays were carried out following established procedures (18),

with the addition of the indicated cytokines: CCL2 (10 ng/ml), CCL8 (50 ng/mL) and

IL-9 (2 ng/ml). See Supplementary Methods for a detailed description.

Immunohistochemistry analysis

Intestine pieces from AOM/DSS treated or control mice were fixed in 10% buffered

formalin overnight to perform hematoxylin/eosin and immunohistochemical staining.

For the prognostic studies in humans, a total of 80 patients diagnosed and treated of

colorectal adenocarcinoma between 2001 and 2006 in Fundación Jiménez Díaz

(Madrid) and followed in the long term were used for the study. Human samples were

prepared as described in (18). All human biopsies were obtained with the patient´s

consent and the approval of the Ethical Committee of Hospital Fundación Jiménez Díaz

according to Spanish official regulations. We reviewed the clinical records of the

patients to determine tumor stage at the time of diagnosis and outcome (18). Each

sample was deparaffinized for antigen retrieval using the PT Link Module (Dako) at

high pH for 20 min, rehydrated and then incubated with the primary antibody against α-

SMA, Snail1, S100A4, CALU, CDH11, LTBP2, FSTL1 or OLFML3 (Supplementary

Table S1). The reaction was revealed using DAB as chromogen and hematoxylin for

counterstaining, and observed in an Olympus microscope. ImageJ was used to quantify

the DAB staining of immunohistochemistry images.

Bioinformatics

Ingenuity Pathway Analysis (IPA) (Ingenuity systems, Inc) was used to analyze the

predicted biological functions of the proteins deregulated in CAFs and to determine

protein interactions and network analysis. Bio-GPS software was used to determine the

Molecular signatures of colon cancer-associated fibroblasts

top-5 tissues with highest expression of each altered protein compartment in order to

determine the desmoplastic signature (10). FatiGo was used to get further insight in

deregulated functions using a cut-off <0.1 for the adjusted p value (19).

Molecular signatures of colon cancer-associated fibroblasts

RESULTS

Isolation and characterization of fibroblasts from AOM/DSS colon cancer tumors

After AOM/DSS treatment, development of adenocarcinomas in the distal colon of

mice was visible by visual inspection and histological staining (Fig. 1A). Mouse

adenocarcinomas were equivalent to human colorectal adenocarcinomas, corresponding

to a well-differentiated infiltrating enteroid adenocarcinoma, with presence of large

numbers of flat and polypoid malignant tumors. The mucosa from control mice was

normal, without dysplastic changes (Fig. 1A). By immunohistochemical analysis,

AOM/DSS tumors showed high expression level of α-SMA, S100A4 y Snail,

considered markers of activated fibroblasts in desmoplastic tumors (Fig. 1B). To

establish CAFs and NFs cultures, distal colons from 16 AOM/DSS-treated and vehicle-

treated mice were cut in small fragments and placed in culture. Fibroblasts migrated

outside the explants and expanded (Fig. 1C). Cells at 95-100% confluence

(approximately 2x104 cells/cm2) were collected, divided in two fractions and mixed for

iTRAQ experiments. By western blot, AOM/DSS-isolated fibroblasts confirmed the

higher expression of markers corresponding to activated fibroblast like α-SMA, S100A4

and Snail1 (Fig. 1D). Expression was equivalent to the immunohistochemical staining

in the original tumor (Fig. 1B). As expected, purified fibroblasts expressed vimentin, a

mesenchymal marker, but not the epithelial marker Epcam.

Protein identification, iTRAQ quantification, gene ontology and functional

networks alterations

Briefly, 100 µg (cell lysates) and 50 µg (conditioned media) of proteins from CAFs and

NFs cultures were trypsin-digested. Peptides were labeled with iTRAQ for relative

quantification and fractionated with OFFGEL, pH 3-10. To avoid biases in peptide

Molecular signatures of colon cancer-associated fibroblasts

labeling that could affect the final quantification, we performed two replicate analyses

using the 114 and 116 iTRAQ tags for NFs peptides and 115 and 117 for CAFs peptides

(Supplementary Fig. S1). In total, 3,846 and 734 peptides corresponding to 1,353 and

295 proteins were identified in the whole cellular extract and conditioned medium,

respectively. For quantification, peptide ratios were calculated by comparing the

intensity of the reporter ions in the MS/MS spectra and normalized either using the

ratios corresponding to seven different house-keeping proteins in the whole cell extract

or the median in the secretome (Supplementary Fig. S2). A total of 1,102 and 250

proteins were quantified in the whole cell extract and conditioned medium, respectively

(Supplementary Tables S2 and S3). We used several criteria for protein selection, 1)

a fold-change ≥1.4 relative to control samples, 2) proteins present in both replicates, 3)

expression ratios following a similar trend in both replicates and 4) proteins identified

with a single unique peptide or variability higher than 50% were manually inspected to

verify that the peptide only corresponded to a single protein. We selected 1.4 fold-

change as a compromise value to include all relevant proteins that showed systematic

change and to compensate the compression of iTRAQ ratios that leads to

underestimation of fold changes (20). Using a fold-change ≥ 1.4, we found 132 proteins

deregulated in complete cell lysates, 109 up-regulated and 23 down-regulated

(Supplementary Table S4), and 125 proteins in the secretome, with 72 up-regulated

and 53 down-regulated (Supplementary Table S5).

Gene ontology analysis of whole cellular extracts results from CAFs showed a

clear up-regulation of ECM constituents (collagen α1 types I, V, III and XII, collagen

α2 types I and V, fibulin 2), proteins for matrix assembly (decorin, prolyl 4-hydroxylase

α2 and leprecan 1), proteins related to TGFβ signaling, such as LTBP2 (Latent

transforming growth factor beta binding protein 2), cadherin 11, Fibrillin 1 and IGF2R

Molecular signatures of colon cancer-associated fibroblasts

(insulin-like growth factor 2 receptor) (Supplementary Table S6). In the secretome,

26 out of 125 proteins were related to ECM. In addition, we identified differentially-

expressed chemokines such as CCL11, CCL8, CXCL5 or CCL2, insulin growth factor-

related proteins such as IGFBP7, or IGF1, cell adhesion and wound response

(Supplementary Table S7). Collectively, these data suggest that activated fibroblasts

participate in extracellular matrix remodeling, wound healing, epithelial differentiation

and are important regulators of inflammation.

Altered biological functions, networks and pathways were analyzed using IPA

(Supplementary Table S8) and FatiGO (Supplementary Table S9). Using IPA in

whole cellular extract, “Connective Tissue disorders”, related with ECM remodeling,

was the top altered function (n= 21 proteins and p values ranging from1.59E-11 to

1.34E-02). In secretome, we found “Cancer” as the most represented biological function

altered (n= 97 proteins and p values ranging from 2.82E-14 to 1.35E-04), and

“Organismal Injuries and Abnormalities” as the top biological function altered (n= 54

proteins and p values ranging from 2.55E-14 to 1.54E-04). Regarding altered network

function “Dermatological Diseases and Conditions, Cellular Assembly and

Organization, Cellular Function and Maintenance” with 31 proteins emerging as

identified in cell extracts (Fig. 2A) and “Cell morphology, Cellular Assembly and

Organization, Cellular Function and Maintenance” was significantly altered in the

secretome with 28 proteins identified (Fig. 2B).

With FatiGo we found that oxidative phosphorylation and molecular functions

related to redox processes were altered in whole cellular extract. Transport of proteins

and their cellular localization emerged as the top altered biological processes. In the

secretome, the extracellular matrix and focal adhesion were the most altered processes.

In addition, we observed a focus towards modulations of molecular functions through

Molecular signatures of colon cancer-associated fibroblasts

receptor binding, whereas the most altered biological processes were the response to

wounding, external stimulus and inflammatory response. Collectively, these protein

networks are indicating the association of CAFs proteins with desmoplastic and pro-

inflammatory signatures, respectively.

Validation and fibroblast specificity of selected biomarkers

After previous network analyses and data-mining using GeneCards database, we

selected top up-regulated proteins that were not previously associated to colon cancer

and pertained to major networks (Fig. 2, Supplementary Tables S8, S9). So, eleven

differentially-expressed proteins (5 from whole cellular extract and 6 from secretome)

were validated using Western Blot. We selected a relatively large number of proteins

from both locations to make a more comprehensive analysis, as they would represent

different family proteins and functionalities. Western blot results were consistent with

the iTRAQ quantification data (Fig. 3A, B). Molecules like CRABP1, LTBP2, FDPS

and CDH11 were up-regulated in whole cellular extracts (Fig. 3A), whereas OLFML3,

CALU, AEBP1, LMOD1, SPON2 and FSTL1 were up-regulated in CAFs supernatants

(Fig. 3B). In order to confirm that purified fibroblasts were equivalent to those present

in the tumor, we validated a subset of these markers in the original tissues of the

AOM/DSS model by quantitative RT-PCR and western blot. Protein and mRNA

expression values were similar between purified fibroblasts and original tissues

(Supplementary Fig. S3).

To confirm fibroblast specificity of stromal biomarkers, we compared the

presence of these proteins in CAFs respect to CT26, a colon adenocarcinoma murine

cell line, by western blot. LTBP2, FDPS, CDH11 and OLFML3 expression was specific

or clearly up-regulated in fibroblasts respect to the epithelial CT26 cells (Fig. 3C).

Molecular signatures of colon cancer-associated fibroblasts

When we tested the supernatants, SPON2 and FSTL1 were also specific of fibroblasts.

Finally, we investigated the capacity of CDH11 for cell sorting of fibroblasts. PDGFRα

was used as a positive control (7). CDH11 antibodies were able to isolate fibroblast cell

lines, like mouse NIH-3T3 or human BJ-hTERT, but did not work with epithelial cell

lines like CT26 or RKO (Fig. 3D). These results suggested that all these six proteins

were specific colon cancer stromal biomarkers.

Cytokines and growth factors deregulated in the secretome of activated fibroblasts

are pro-inflammatory and pro-tumorigenic on colon cancer cells

CAFs secrete chemokines that control critical steps of the adhesion-invasion-metastasis

cascade and affect recruitment of inflammatory cells and enhancement of angiogenesis

(21, 22). By mass spectrometry, we observed a significant increase in the expression of

cytokines like CCL11 (Eotaxin-1), CCL8 (MCP-2), CCL2 (MCP-1), SAA3 and CXCL5

(ENA-78), whereas CSF1 was down-regulated in CAFs (Supplementary Table S7).

To investigate other additional changes in cytokines, we used a mouse chemokine

microarray for CAFs/NFs supernatants (Fig. 4A). CCL11, CCL8, SAA3 and CXCL5

antibodies were not present in the arrays. Microarray semi-quantitative data confirmed

the increase in CCL2 expression, although not as relevant as in iTRAQ, and showed an

increase in other pro-inflammatory cytokines like IL-6, CXCL2, CCL20 and

osteopontin (OPN) (Fig. 4B). Overall, our chemokine profile was similar to the one

described for cancer skin fibroblasts that showed cancer-promoting activity, NF-κB

dependent (7).

To be activated by cytokines, cancer cells should express the corresponding

receptors. In order to detect CCL2 and CCL8 receptors (CCR1, 2, 3 and 5) as well as

the receptor for IL-9 (IL9R), we performed RT-PCR assays with KM12C and SM cells.

Molecular signatures of colon cancer-associated fibroblasts

These assays showed that both cell lines express IL9R and CCR1 and CCR3, whereas

CCR2 and CCR5 were detected only in KM12C and KM12SM, respectively (Fig. 4C).

Then, we investigated the effect of CCL2, CCL8 and IL-9 on cell proliferation,

adhesion, migration and invasion of colon cancer cell lines (Fig. 4D). A >2-fold

increase was observed at the adhesion, migration and invasion level in KM12 cells, after

CCL2 and CCL8 addition, but no effect for IL-9. In contrast, IL-9 induced a significant

increase in the proliferation of KM12 cell lines. Collectively, these data suggest that

CCL2 and CCL8 play an important role in tumorigenesis and the acquisition of invasive

capacity of cancer cells.

Desmoplastic signature in AOM/DSS colon fibroblasts and prognostic value

Since activated fibroblasts are characterized by the acquisition of a myofibroblast

phenotype consisting of different smooth-muscle-like proteins, like α-SMA, one aim of

this work was to define a desmoplastic signature for colon cancer. We searched for

desmoplastic markers on the differentially-expressed proteins using the Bio-GPS

database. In the secretome, we found 30 secreted proteins (24% of the total)

preferentially expressed in myofibroblasts (Table 1), including seven collagens,

collagen metabolism-related proteins (LOX, PCOLCE), fibronectin, proteoglycans

(perlecan and byglican), cytokines (CCL11, CCL8 and CCL2) and calcium metabolism

proteins (calumenin (CALU)), insulin growth factor binding proteins (IGFBP6, 7) and

FSTL1. The cell lysate showed a similar profile.

Then, we studied the value of the proteins in this desmoplastic signature as

prognostic biomarkers in human patients. Given the large number of candidates, we

carried out a meta-analysis using databases from the “cBio Cancer Genomics Portal”

(23), which contains mRNA expression data sets from 274 samples of colon cancer.

Molecular signatures of colon cancer-associated fibroblasts

Using Kaplan-Meier for overall survival analysis, we found some combinations of

proteins with prognostic value from whole cellular extract (Supplementary Fig S4A)

and the secretome (Supplementary Fig S4B). The most significant combination

included RPN2, IVL, RP1 and CALU (Supplementary Fig S4C), which displayed a

highly significant association to overall survival (log-rank test p value = 0.000095). In

contrast, the association to survival in ovarian or lung adenocarcinoma did not deliver

significant p values (data not shown).

Verification of stromal markers and prognostic association in human cancer

samples

Immunohistochemical staining was performed to validate the expression of the five

selected candidate stromal biomarkers CALU, CDH11, FSTL1, OLFML3 and LTBP2

in AOM/DSS-induced tumor samples. SPON2 was not tested due to the lack of suitable

antibodies. The selected markers were strongly expressed in the stroma of AOM/DSS-

induced murine adenocarcinoma with little or no expression in normal colon mucosa

(Fig 5A). The expression of these markers was more intense in the leading edge of

tumors, which could be associated to desmoplastic invasion front.

Finally, to investigate the relevance of these markers in human colon cancer

samples we used a tissue microarray. Clinical information together with the stromal

expression of the selected CAF markers is summarized in Supplementary Table S10.

We observed an increased expression for CALU, CDH11, FSTL1, OLFML3 and

LTBP2 in the tumoral respect to normal stroma (Fig. 5B). All of them showed stronger

expression in the leading edge of the tumors, where the tumor infiltrates the surrounding

areas. Tumoral epithelial cells showed an increased expression in >70% of the tumors

for CALU, CDH11and OLFML3, but not significant expression was detected for

Molecular signatures of colon cancer-associated fibroblasts

FSTL1 (Fig. 5B). In contrast, in the stroma of normal tissue the staining of these

markers was mainly observed to be negative or weak in contrast to tumoral stroma

where the markers were highly over-expressed (Fig. 5B).

Then, we determined if the expression of these proteins in the stroma was

associated to survival in human cancer using the tissue microarray data for patients

followed on the long term (more than five years). The series was retrospectively

selected (Supplementary Table S10). High stromal expression of CALU correlated

significantly with lymph node involvement at the moment of diagnosis (p=0.034) (Fig.

5C) and with poor prognosis (p = 0.010) (Fig. 5D). Also stromal expression of CDH11

correlated with disease-free survival (p=0.051, Fig. 5E), but no with prognosis

(p=0.105). In addition, the combination of CALU with CDH11 expression displayed a

significant association to disease-free survival (log-rank test p value = 0.015) and

overall survival (log-rank test p value = 0.009) in human cancer samples (Fig. 5D). The

rest of biomarkers did not reach significant correlation with metastasis or survival

(Supplementary Fig. S5A-K).

Molecular signatures of colon cancer-associated fibroblasts

DISCUSSION

The use of the AOM/DSS murine model of sporadic colon cancer for the isolation of

purified and homogeneous populations of CAFs, together with in-depth proteomic

analysis, allowed the identification of an elevated number of proteins deregulated after

fibroblast activation in cancer. The identification of many of the currently known

stromal biomarkers confirmed the validity of our experimental approach. As an

example, we found an increase in collagens Type III and Type XII, which are mainly

implicated in desmoplasia and colorectal cancer metastasis (10). At least six identified

proteins: LTBP2, CDH11, FDPS, OLFML3, SPON2 and FSTL1 were novel candidate

stromal biomarkers, as no expression was observed in the murine adenocarcinoma cell

line CT26. In addition, CDH11 was useful to isolate colon cancer fibroblasts. The

relevance of these biomarkers for CRC prognosis was confirmed by studies with human

tissue microarrays. The expression of CDH11, FSTL1, OLFML3, and LTBP2 together

with CALU, was increased in the tumoral stroma of patients with CRC. Moreover, we

observed an association of CALU and CDH11 with poor survival and prognosis in

human cancer patients. Together, these data support the value of the AOM/DSS murine

model for the discovery of stromal biomarkers applicable in human patients.

Despite this, the AOM model presents some limitations. The most relevant is its

inability to develop metastasis. Recently, an APC mutant mouse model of sporadic

colon cancer was used to analyze the proximal fluid proteome of whole tumor tissues,

without isolation of pure cell populations (24). Looking at the candidate protein

biomarkers identified in that model, we have observed several coincidences in ECM

fibroblast-related proteins: biglycan, cingulin, collagens, decorin or lamin A/C,

confirming the relative similarity of both models.

Molecular signatures of colon cancer-associated fibroblasts

CAFs exhibited specific proinflammatory and desmoplastic protein signatures

for colon cancer. Several reports support the role of the desmoplastic microenvironment

in tumor promotion (2). For instance, Tsujino et al (25) reported a strong association

between presence of abundant stromal myofibroblasts, liver metastasis and shorter

disease-free survival. In addition, it has been reported that tumor-derived ECM is stiffer

than normal ECM. Moreover, increased matrix stiffness and ECM remodeling was

observed in pre-malignant tissue. This increase was shown to contribute to malignant

transformation in the breast (26). In colorectal cancer, previous studies showed a

correlation between the presence of myofibroblasts (25), vimentin (27) or fibroblast

activation protein (FAP) expression (28) with prognosis and recurrence.

Here, we identified several new prognostic markers associated to fibroblasts.

Calumenin is a calcium-binding protein that regulates the activity of coagulation factors

(29) and was reported as overexpressed in colon cancer (30, 31). Although calumenin

expression is not restricted to the stroma, we have found a good association with

stromal expression, lymph node invasion and lower survival, alone or in combination

with other proteins like CDH11. Cadherin-11 is a mesenchymal cadherin, up-regulated

by TGFβ, which has been identified as a novel target for pulmonary fibrosis and

associated with invasion in squamous cell carcinoma (32, 33). The combination of

CALU with CDH11 improved significantly the disease-free survival prognostic value.

We found several other TGFβ-related deregulated proteins as stromal

biomarkers. OLFML3 belongs to the olfactomedin domain-containing proteins. They

are known BMP antagonists (34) and showed proangiogenic activity in cancer (35).

FSTL1 expression was the most specific for human stromal compartment. FSTL1 is a

TGFβ-inducible gene, SPARC-related, that enhances inflammatory cytokine/chemokine

expression (36) and seems to be implicated in angiogenesis and revascularization (37).

Molecular signatures of colon cancer-associated fibroblasts

Another TGFβ signaling-related protein was LTBP2, which co-localizes with

fibronectin and collagen type 1 for ECM regulation and remodeling (38, 39). LTBP2 is

overexpressed in pancreatic cancer, being mainly located in the cancer stroma (40).

TGFβ activates CAFs by changing the secretion profile of chemokines to assist CRC

cells in the progression of the disease. An inflammatory microenvironment can promote

malignant progression and participate in the recruitment and retention of infiltrating

leukocytes to inhibit the resolution of inflammation (41, 42).

Evidence was provided of a significant increase in pro-inflammatory

chemokines (CCL2, CCL11, CCL8, CCL20, CXCL5, IL-9) in the conditioned media of

mouse CAFs either by mass spectrometry or chemokine microarrays. In addition to

function as leukocyte chemoattractants and pro-angiogenic properties, human CCL2,

CCL8 and IL-9 promoted tumorigenesis in two human colorectal cancer cell lines.

CCL8, also called MCP2, a pluripotent chemokine, attracts monocytes (43) and

lymphocytes (44). From our results, CCL2 and CCL8 regulated the proliferation,

adhesion, migration and invasion of two colorectal cancer cell lines, whereas IL-9 was

only efficient in tumor growth proliferation. Since CCL2 and CCL8 share, at least, 3

receptors (CCR2, CCR3 and CCR5) (45), this could explain the similar effect induced

by both chemokines in colorectal cancer. In fact, several solid cancers, including CRC,

showed expression of functional chemokine receptors on tumor cells, able to promote

proliferation and metastasis (46). Then, CCL8 and IL-9 are new colon cancer-

associated chemokines that deserve further investigation. Stromal chemokines might

work either in paracrine or autocrine ways. CAFs and myofibroblasts could differentiate

from resident tissue fibroblasts under various paracrine stimuli (47). In this regard,

resident monocytes or other immune cells might secrete chemokines that activate the

pro-inflammatory signature in CAFs by paracrine stimulation. Also, carcinoma cells

Molecular signatures of colon cancer-associated fibroblasts

may produce chemokines that affect CAFs. In turn, CAFs secrete cytokines that can

modulate tumor cells or other fibroblasts in different ways.

Our data provide a comprehensive view of the many molecules secreted by

CAFs that favor the progression and invasion of tumors, from angiogenesis to

desmoplasia and pro-fibrotic mechanisms. A panel of new stromal markers in colon

cancer was identified. Among them, the combination of CDH11 with CALU gave a

significant prognostic value. In addition, verification of the results with human samples

confirmed the value of the model and the strategy for identification of novel candidate

prognostic biomarkers with clinical potential. In summary, this study defines a new set

of stromal biomarkers with good prognostic value that might constitute an alternative to

current markers like α-SMA or the presence of myofibroblasts in colon cancer.

Molecular signatures of colon cancer-associated fibroblasts

ACKNOWLEDGEMENTS

S. Torres was a recipient of a Juan de la Cierva programme. R.A. Bartolomé was

supported by a grant to established research groups of the Asociación Española Contra

el Cáncer (AECC). Marta Mendes was a recipient of a ProteoRed contract. R. Barderas

was a recipient of a JAE-DOC/FSE Contract (CSIC) and is currently a fellow of the

Ramón y Cajal programme. A. Peláez was a FPI fellow from the MINECO. R. Villar-

Vazquez was an FPU fellow from the MEC.

Molecular signatures of colon cancer-associated fibroblasts

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Molecular signatures of colon cancer-associated fibroblasts

Table 1. Myofibroblastic signature associated to proteins1 in the CAFs secretome

Acession Average Fold- Std Description Name Number change Dev P48298 Eotaxin Ccl11 25.37 25.83 Insulin-like growth factor-binding protein Q61581 Igfbp7 11.06 5.21 7 Q9Z121 C-C motif chemokine 8 Ccl8 11.00 10.87 Q61554 Fibrillin-1 Fbn1 7.81 5.60 Q60847 Collagen alpha-1(XII) chain Col12a1 6.12 9.61 O35887 Calumenin Calu 6.11 2.59 P11087 Collagen alpha-1(I) chain Col1a1 5.51 2.09 P08121 Collagen alpha-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 alpha-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 alpha-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 EGF-containing fibulin-like extracellular Q9WVJ9 Efemp2 3.04 1.41 matrix protein 2 P10493 Nidogen-1 Nid1 2.14 0.26 P35441 Thrombospondin-1 Thbs1 2.08 0.36 Q3U962 Collagen alpha-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 alpha-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

1. Differentially-expressed proteins were identified as preferentially expressed in smooth muscle if they were among the top-5 tissues with highest expression using the Bio-GPS database.

Molecular signatures of colon cancer-associated fibroblasts

Legend to Figures

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).

Hematoxylin/eosin staining of tumoral and normal colon (right). B,

Inmunohistochemical analysis of cancer and normal tissue using antibodies against

S100A4, Snail1 and α-SMA. C, Fibroblast isolation from colonic tissues by the explants

technique between days 1 and 25. Tissues were cut into 2-3 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 α-

SMA. Vimentin was used as positive fibroblast marker and EpCAM as negative control.

B, D, Ratio AOM-DSS/Control was calculated for Snail1, α-SMA and S100A4 using

ImageJ or QuantityOne 1-D Analysis Software, respectively.

Figure 2. Network functions affected in CAFs. Protein networks were identified by

IPA using the 132 and 125 differentially-expressed proteins identified in whole cellular

extract and secretome, respectively. A, “Dermatological Diseases and Conditions,

Cellular Assembly and Organization, Cellular Function and Maintenance” network was

identified in whole cellular extracts with a score of 47. The network consisted of 26 up-

regulated and 5 down-regulated proteins from 34 direct-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 up-regulated and 1 down-regulated proteins from 35 direct-interacting

proteins.

Molecular signatures of colon cancer-associated fibroblasts

Figure 3. Western Blot validation of iTRAQ data and biomarker expression on

stromal cells. Protein samples from A, whole cellular extract or B, concentrated

conditioned media of CAFs and NFs 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 CAFs/NFs 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

cytometry analysis for testing CDH11 and PDGFRα specificity (grey areas) on

fibroblasts and cancer cell lines. An irrelevant antibody was used as a control (white

areas).

Figure 4. Chemokine expression in conditioned medium from CAFs and NFs.

Functional studies with selected chemokines. A, A representative image of a

chemokine microarray after screening of conditioned medium from CAFs and NFs.

White arrows indicate the most deregulated cytokines in CAFs and NFs. B, Most

abundant chemokines and cytokines differentially-expressed in CAFs versus NFs. 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 h of culture. Cell adhesion to Matrigel was

performed after 2 min of incubation with the indicated chemokines and 4 min of

adhesion. Migration speed was calculated as the distance covered by the cells in 24h.

Cell invasion was done across Matrigel of the KM12 cell lines treated as indicated.

Molecular signatures of colon cancer-associated fibroblasts

Experiments were performed three times. Error bars: Standard Deviation (* p < 0.05, **

p < 0.01, *** p < 0.005).

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 x200 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 Chi-square 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 employed the log-rank test.

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2013 American Association for Cancer Research. α-sma Snail1 S100A4 ABH&E Downloaded from Author ManuscriptPublishedOnlineFirstonSeptember11,2013;DOI:10.1158/1078-0432.CCR-13-1130 Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. ol ntrol r r o o C Cont clincancerres.aacrjournals.org S S AOM/DS AOM/DSS on September 27, 2021. © 2013American Association for Cancer

Research. 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 CAF 1.0 1.0 1.0 s s M/DSS O O Epcam A 0.0 0.0 - α-Tub Figure 1 Author Manuscript Published OnlineFirst on September 11, 2013; DOI: 10.1158/1078-0432.CCR-13-1130 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Dermatological Diseases and Conditions, Cellular Assembly and Organization, Cellular Function and Maintenance B

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

Figure 2

CDH11 PDGFRα 1.34 1.44 C Cell lysates Supernatants D CT26 CAFs CT26 CAFs NIH-3T3 kDa kDa Crabp1 Calu 10 48 0.63 0.59 Ltbp2 180 Aebp1 75 BJ-hTERT

48 Fdps Lmod1 63 130 er 0880.88 0790.79 100 Cdh11 35 75 Spon2 CCD18

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

0.31 2.01 RKO

0 100 101 102 1030 100 101 102 103 Figure 3 Fluorescence intensity

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 11, 2013; DOI: 10.1158/1078-0432.CCR-13-1130 A Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 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 50000 45000 NFs 40000 CAFs 35000 30000 rary units rary t 25000 20000 Arbi 15000 10000 5000 0

CCR1 CCR2 CCR3 CCR5 IL9R GAPDH D

KM12C KM12SM KM12C KM12SM PROLIFERATION ADHESION 250 300 0.5 0.8 2 *** * *** *** *** 0.7 *** 250 *** 0.4 * 200 0.6 200 (560nm)

* ells/mm 0.3 *** 0.5 150 c c e 0.4 150 0.2 100 0.3 100 0.2 0.1 50 0.1 Adhered 50 Absorbanc 0 0 0 0

MIGRATION INVASION 350 4 10 120 *** *** 300 *** *** *** *** *** 100 8 *** 3 *** 250 *** 80 6 200 2 60 150 4 40 100 nvasive cells ion speed (µm/h) I t 1 2 20 50 0 0 0 0 Cell migra

Downloaded from clincancerres.aacrjournals.org on SeptemberFigure 27, 42021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on September 11, 2013; DOI: 10.1158/1078-0432.CCR-13-1130 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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

101.0 Negative 101.0 CDH11 Negative D CALU Positive Positive 0.8 0.8

C 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 (months) Time (months) 60 1.0 CALU Negative 1.0 CALU Negative Positive Positive 40 CDH11 CDH11 0.8 0.8 % patients 20 0.6 0.6

0 survival ree survival f

NO YES m 0.4 0.4 Cu Lymph node 0.2 0.2 involvement Disease- P= 0.009 0.0 0.0 P= 0.015 0 2040 60 80 100 120 140 0 2040 60 80 100 120 140 Figure 5 Time (months) Time (months)

PROTEOME PROFILING OF CANCER-ASSOCIATED FIBROBLASTS IDENTIFIES NOVEL PRO-INFLAMMATORY SIGNATURES AND PROGNOSTIC MARKERS FOR COLORECTAL CANCER

Sofia Torres, Ruben A Bartolome, Marta Mendes, et al.

Clin Cancer Res Published OnlineFirst September 11, 2013.

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