- Research article
- Open Access
- Open Peer Review
FAP-overexpressing fibroblasts produce an extracellular matrix that enhances invasive velocity and directionality of pancreatic cancer cells
© Lee et al; licensee BioMed Central Ltd. 2011
- Received: 12 November 2010
- Accepted: 13 June 2011
- Published: 13 June 2011
Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions.
We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer.
We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses.
We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated.
Cancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and β1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking β1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions.
- Pancreatic Cancer Cell
- Human Pancreatic Cancer
- Fibroblast Activation Protein
- Pancreatic Stellate Cell
- Cancer Cell Behavior
Increasing evidence demonstrates the significance of the tumor microenvironment for tumor initiation and progression [1–6]. The tumor microenvironment is characterized by a heterogeneous complex of cellular and acellular components including tumor-associated fibroblasts, immune and endothelial cells, soluble cytokines, chemokines and proteases, as well as a characteristically remodeled ECM . These components act in a coordinated manner to regulate the growth and differentiation of adjacent cells, thus alterations in the stromal microenvironment towards a permissive environment provide important cues for tumor growth, invasion, and metastasis [7, 8]. In fact, the majority of proteases and stromal factors associated with malignant tumors are secreted by the host stroma rather than by the tumor cells themselves [9–11]. One of the most selective proteins for tumor stromal fibroblasts is the Fibroblast Activation Protein (FAP).
FAP is a serine protease that contains both dipeptidyl peptidase and gelatinase/collagenage activities in vitro . Because of its specific induction in tumor-associated fibroblasts in over 90% of epithelial tumors, including pancreas and breast among others, FAP was used as a platform for studying stromal specific effects on tumor behaviors [13–18]. Previously, we reported that FAP overexpression by tumor cells results in increased tumorigenicity and tumor growth  and its enzymatic activity played an important role in the promotion of tumor growth in mouse model . Although several studies have shown that FAP expression in human melanoma cell lines or hepatic stellate cells promotes an invasive phenotype through cell adhesion pathways [13–15], it is not clear how FAP expressing fibroblast-specific signals prepare a permissive stromal microenvironment and how modified ECMs influence cancer cell behavior in vitro.
Studies in several human cancer types describing alterations in stromal cells and their ECM compositions suggest that changes in the microenvironment and tissue architecture contribute to tumorigenesis . In vivo, the mesenchymal ECM is comprised of several proteins including collagens I, III and fibronectin, which assemble in an intricate fibrillar network and are engaged by transmembrane receptors. For example, integrins transmit biochemical and mechanical stimuli from the matrix to the cytoskeleton of the cell and back to the ECM, triggering distinct intracellular signaling pathways that control proliferation, survival, and migration . In fact, it has been shown that both desmoplastic fibronectin and collagen I fibers often align in parallel patterns in response to tumorigenesis [21, 22], and these fibers are engaged by integrin heterodimers in pancreatic and other cancers [6, 20], suggesting that cell behavior can be affected by the underlying stromal substrates via integrin engagement. Thus, alteration of the stromal ECM composition in cancers may be linked to cancer progression through tissue remodeling processes . Here, we show that FAP enzymatic activity locally modifies stromal ECM components thus facilitating the formation of a permissive microenvironment promoting tumor invasion on human pancreatic cancer.
Cell lines and murine xenograft model
All cell lines used in this study were originally purchased from ATCC or obtained from the Cell Culture Facility at Fox Chase Cancer Center. Three C.B17/Icr-scid mice were subcutaneously injected with 2 × 106 cells of each pancreatic cell line (Panc-1, Capan-1, AsPC-1, and HPAF-II). After 5 weeks inoculation, tumors were harvested for immunohistochemistry analysis.
Stable transfection of fap in NIH-3T3 fibroblasts
Mouse fap gene was cloned downstream of the Tet-response promoter in a tetracycline-inducible expression vector pTRE (a kind gift from Dr. Teresa Ramirez-Montagut, Memorial Sloan-Kettering Cancer Center, NY). This construct was co-transfected into NIH-3T3 cells with rtTA plasmid (pUHD172-1 neo) that encodes the pTRE promoter-binding transactivator, thus murine FAP expression was induced by adding Doxycycline (Dox, 2 μg/ml) into complete DMEM medium containing 10% Tet system approved Fetal Bovine Serum (Clontech, CA).
Isolation of human pancreatic stellate cells and matched pancreatic adenocarcinoma-associated fibroblasts
Fresh surgical tissue (decoded) samples from a pancreatic Whipple conducted at the Fox Chase Cancer Center was delivered with the assistance of the Protocol Laboratory and the Biosample Repository Facility following protocols approved by the Institutional Review Board. Tissue samples corresponding to pancreatic adenocarcinoma and distant (normal) pancreas were rinsed in cold PBS containing 100 μg/ml streptomycin and 100 U/ml penicillin. The two types of samples were carefully minced and incubated overnight with 0.2% collagenase at 37°C for digestive dissociation. The digested material was subjected to centrifugation at 1200 rpm for 5 min, thus precipitating a fibroblast-enriched fraction. This fraction was filtered through a series of 100 μm followed by 40 μm cell-strainer (BD Bioscience) before culturing in DMEM containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine at 37°C using a humidified atmosphere and 5% CO2 for a period of 4 hours before removing the non adherent cells. Cell homogeneity was confirmed by direct microscopic observations, while cells were designated as fibroblastic following confirmation of mesenchymal marker vimentin expression, as well as absence of epithelial marker cytokeratin 19 expression. In addition, normal fibroblasts were characterized as pancreatic stellate cells following confirmation of uptake and storage of vitamin A, by auto-fluorescence stemming from retinyl acetate (Sigma) containing droplets . The resultant (PaSCs) and pancreatic tumor-associated fibroblasts (which were confirmed to have lost the vitamin A droplets) were used for self-derived matrix characterization.
Western analysis of FAP induction in fibroblasts
FAP-transfected fibroblasts were harvested at different time points (0, 1, 2, 4, 6, 8, and 10 days) in the presence or absence of Dox. Total protein was extracted using M-Per reagent (Pierce, IL), resolved by 4-12% SDS-PAGE under reducing conditions (Invitrogen, CA), and blotted with rabbit monoclonal anti-mouse FAP antibody (0.08 μg/ml). Immuno positive bands were labeled using goat anti-rabbit-HRP (3000×, Amersham Bioscience, UK) and visualized by ECL reagent (Pierce, IL). Purified murine recombinant FAP protein (92 kD) and parental NIH-3T3 cell lysate were used as positive and negative controls, respectively.
Fibroblast-derived 3D matrix production
All fibroblasts were seeded at a concentration of 7 × 105 cells per sample onto 35 mm plates pre-coated with 0.2% gelatin. Confluent fibroblastic cultures were treated with media supplemented with 50 μg/ml ascorbic acid (and Dox when necessary) every other day for 8 days to obtain un-extracted 3D cultures. Alkaline detergent treatment (0.5% Triton X-100, 20 mM NH4OH in PBS) for 5 minutes at 37°C gave rise to cell-free in vivo-like 3D matrices [25, 26]. For control, FAP-transfected fibroblasts were used to make FAP- matrix in the absence of Dox. To make FAP+inhibitor matrix, FAP-specific small molecule inhibitor naphthalenecarboxy-Gly-boroPro (400 μM, provided by Dr. William Bachovchin, Tufts University, MA) was added to the media during matrix production.
Analysis of fibronectin fiber orientation
For indirect immunofluorescent labeling of matrix fibers, un-extracted 3D cultures were prepared on glass cover slips as described . Cells were stained with rabbit anti-mouse fibronectin antibody (25 μg/ml, Abcam, UK), a donkey anti-rabbit Cy5 conjugated antibody (15 μg/ml, Jackson ImmunoResearch, PA) and DAPI.
From two independent experiments with duplicate samples, minimum of 5 images per experimental sample were obtained using a z-stack function of the spinning disc microscope (PerkinElmer Life Sciences, PA). Each slice measured 0.5 μm and stacks were reconstituted as a maximum projection using the MetaMorph software as described in detail . Fiber distributions corresponding to each experimental setting were determined by the percentage of fibers that were oriented at 100 variation angles from the identified modes.
In-Cell Western analysis of un-extracted 3D cultures
FAP-transfected fibroblasts (1.5 × 104 cells/48-well plate) were cultured in the presence or absence of Dox during matrix production. Fibroblasts were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and stained with antibodies followed by manufacturer's directions (Li-Cor Bioscience, NE). The antibodies for this assay were tenascin C (50 μg/ml, Abcam, UK), α- SMA (5 μg/ml, Sigma, MO), collagen I (25 μg/ml, Chemicon International, CA), fibronectin (20 μg/ml, Abcam, UK), β-actin (1000×, Cell Signaling Technology, MA), and GAPDH (2 μg/ml, Chemicon International, CA). Fluorescence-labeled secondary antibodies IRDye 680 and IRDye800CW (Li-Cor Bioscience, NE) were used to scan by the Odyssey Infrared Imaging System. The protein levels were normalized by β-actin or GAPDH.
Western analysis of pancreatic cancer cells cultured within assorted matrices
Following 2 days culture on matrices, Panc-1 cells were lysed in extraction buffer (50 mM Tris-HCl, pH8.8, 1% SDS, 5 mM DTT, 13 mM iodoacetamide, 5 mM EDTA, 150 mM NaCl). Proteins were quantified by western blot analysis using ECL reagent (Pierce, IL) or Odyssey infrared imaging system following the manufacturer's directions (Li-Cor Bioscience, NE). The antibodies for this assay were β1-integrin (2500×, BD Transduction Laboratories, NJ), AKT (500×, BD Transduction Laboratories, NJ), pS473-AKT (1000×, Cell Signaling Technology, MA), FAK (1 μg/ml, Upstate, NY), pY397-FAK (1000×, Biosource, CA), and GAPDH (2 μg/ml, Chemicon International, CA). Specific activity of AKT and FAK were calculated as the ratio of the scanned optical density of phosphorylated protein/total protein. Then the ratios were normalized by GAPDH.
Motility assay within the assorted 3D matrices
As described in detail , tumor cells were re-plated onto the matrices and incubated overnight. Cell movements (10~15 cells), recording cells migrating both on and through the matrix, were recorded every 10 minutes during 12 hours using Nikon TE-2000U inverted microscope equipped with cool snap HQ camera. Individual cell dynamics were analyzed using the MetaMorph program following 4 distinctive factors: 1) the net path distance (D, μm) by calculating the number of μm/pixel. 2) The path trajectory of an individual cell during the recording period (T, μm). 3) Average velocity as the motility rate (AV, μm/hr). 4) Directionality calculated by the D/T ratio, which determines random (D/T = significantly smaller than 0.5) versus directional (D/T = close to 1) migration of individual cells [27–29]. Motility experiments using the assorted matrices were performed a minimum of 3 times using different batches of matrices and following a minimum of 10 individual cells.
In order to assess the effect on motility by integrin inhibitors, motility assays were performed in the presence of functional blocking β1 integrin antibody mAb13 (50 μg/ml, a kind gift from Dr. K. Yamada at NIH/NIDCR, Bethesda, MD) or the α5β1-integrin blocking peptide ATN-161 (Ac-PHSCN-NH2, 50 μg/ml, Attenuon, San Diego, CA). These experiments were performed at least 2 times using two alternative batches of matrices.
Data from 3D matrix fiber distribution were analyzed using "chi-squared test". The normalized western data were analyzed using linear regression. For the motility analysis, the average velocity, net path distance and the path trajectory were analyzed using Gamma regression, and the directionality was analyzed using linear regression.
In vivo tumor-dependent stromal FAP upregulation provides the rationale to study in vitroFAP fibroblastic over-expression effects
Some reports suggest that FAP expression can occur in both the stroma and epithelial compartments of cancers [30–33]. However, the selectivity of FAP for stromal fibroblasts, but not epithelial tumor cells, has been confirmed either by immunohistochemical studies in pancreas, colorectal and breast cancer patients [12, 34, 35], or by RT-PCR of pancreas, lung, and renal cell xenografts . To confirm the putative significance of studying FAP-expression effects in stromal matrices, we first examined FAP expression in the human pancreatic cancer patient tissues and mouse xenografts by immunohistochemistry. We observed that FAP is highly expressed on the stromal fibroblasts of human pancreatic cancer, while it is undetectable both in the epithelial cancer cells (Additional file 1, Fig. S1A) and normal tissue (not shown). In xenograft mouse models inoculating the human pancreatic cancer cell lines (HPAF-II, Capan-1, AsPC-1, and Panc-1), murine FAP expression was also found up-regulated specifically at the tumor stroma (Additional file 1, Fig. S1B). This observation confirmed the existence of a selectivity of FAP expression in tumor-associated fibroblasts and prompted us to believe that perhaps FAP is an attractive protein to study stromal effects imparted upon tumor behaviors.
Stable FAP expression on naive fibroblasts
In vivo, FAP is highly expressed on pancreatic tumor stromal fibroblasts, but its expression ex vivo on cultured primary cells is not maintained. To establish stable FAP-expressing fibroblasts, mouse fap gene was cloned under the Tet-inducible system. Induction of FAP expression was achieved as early as 24 hours following Dox treatment, and its expression was clearly maintained for at least 10 days (Figure 1A). Parental NIH-3T3 cells and FAP+ fibroblasts cultured in the absence of Dox showed no detectable FAP induction, allowing the use of these fibroblasts as negative controls.
FAP expression on fibroblasts during 3D matrix production induces tumor stromal-like parallel orientation of fibronectin and collagen I fibers
During the 8 days required to produce a fibroblast-derived 3D matrix, clear morphological differences on fibroblasts were observed between the parental NIH-3T3 and FAP- vs. FAP+ fibroblasts. FAP+ cells were elongated into an enhanced spindled shape, and they organized in a parallel pattern (data not shown). Since tumor-associated ECMs are organized in parallel patterns [21, 22, 37], we tested whether FAP expression affects the topography of fibroblasts-derived ECMs by indirect immunofluorescence using anti-fibronectin (Figure 1) or anti-collagen I (Additional file 2, Fig. S2) antibodies on un-extracted matrices containing their original matrix-synthesizing fibroblasts . Indeed, FAP+ fibroblasts produced ECM fibers oriented in parallel patterns when compared to FAP- matrices (Figure 1B). These patterns are reminiscent of previously observed tumor-associated patterns in vitro . In addition, the level of organization of the assorted fibroblast-produced fibronectin fibers was quantified by measuring the relative orientation angles of fibers . The average percent of parallel fibers that were oriented within ±100 of the mode angle was determined using the MetaMorph software. The measured percentages were 27% and 45% for FAP- and FAP+ 3D ECMs, respectively (p < 0.001) (Figure 1B). Furthermore, inhibition of FAP enzymatic activity by the FAP inhibitor during the matrix production effectively reversed the FAP-induced parallel matrix orientation (29%). Moreover, the observed architectural patterns were similar to the ones seen in desmoplastic reactions in human pancreatic cancers and in xenografted tumors formed in mice using human pancreatic cancer cells in vivo (see asterisks in Additional file 1, Fig. S1). Importantly, when assessing the architectural patterns of collagen I fibers using the FAP series of un-extracted matrices in vitro, our results showed that, although the collagen I fibrillogenesis levels during matrix production (8 days) were not as substantial as to allow the analysis of the fiber orientation, simple microscopy observations clearly demonstrated that FAP+ fibroblasts indeed affect collagen I fiber organization (Additional file 2, Fig. S2). These results suggest that the FAP enzymatic activity during matrix production is important for the topographical organization of the ECM fibers. Because of this topographical similarity to that of tumor permissive 3D ECMs [25, 37], the FAP+ 3D matrix system was used to study the mechanism of matrix-supported pancreatic cancer cell invasion.
Human pancreatic stellate cells- and adenocarcinoma-derived ECMs resemble FAP null and FAP+ECMs, respectively
Stromal FAP modulates the expression levels of assorted ECM proteins
FAP+3D matrices promote the invasiveness of pancreatic cancer cells
In order to question whether FAP+ matrix effects promoting cell motility of invasive pancreatic cancer cells (i.e., Panc-1) are specific for pancreatic cancers, we repeated this series of experiments using three well characterized breast cell lines. Additional file 3, Fig. S3 shows that compared to immortalized normal MCF-10A (AV = 7.59±1.3, D/T = 0.348±0.09) and tumorigenic MCF-7 (AV = 4.88±0.65, D/T = 0.17±0.042), highly invasive MDA-MB-231 cells moved faster (AV = 12.0±1.0, p = 0.04, p < 0.001, respectively) and more direct (D/T = 0.68±0.04, p = 0.001, p < 0.001, respectively) on FAP+ matrix. The results demonstrate that FAP+ matrix effects may be important in neoplasias other than just pancreatic cancers.
In summary, FAP+ 3D matrices represent a permissive environment for pancreatic (and breast cancer) invasion, and perhaps these matrices play an equally important role along with the epithelial cell component. Given their matrix-induced velocity and persistent directionality, Panc-1 (and in some instances MDA-MB-231) cells were selected for the further studies.
FAP+3D matrices induce enhanced invasion behaviors upon Panc-1
Matrix-mediated invasive phenotypes are regulated by β1-integrins
Given the enhanced expression and spatial orientation of fibronectin in FAP+ matrix, the importance of the fibronectin receptor α5β1-integrin was also tested. Inhibition of α5β1-integrin using ATN-161 showed significant decrease in the FAP+ matrix-induced invasive behavior of Panc-1 cells (Figure 6). Compared with controls, inhibition of α5β1-integrin decreased both velocity (AV = 7.73 ± 0.84, 43% inhibition, p = 0.002) and directionality (D = 0.34 ± 0.03, 53% inhibition, p < 0.001) of cells on FAP+ matrix, although not to the same extent as that of more general integrin inhibitors. Given the incomplete abrogation of invasive behavior of Panc-1 cells in the presence of ATN-161, it is possible that additional β1-integrins could also contribute to pancreatic cancer invasion facilitated by the FAP+ 3D matrices. In summary we concluded that, since the enhanced motility of Panc-1 cells on FAP+ 3D matrices can be significantly reversed by blocking the function of β1-integrins, a potential mechanism of matrix mediated tumor invasion is an attractive possibility.
FAP+-dependent enhanced invasion behavior of Panc-1 is regulated by the β1-integrin/FAK signaling pathway
In this work, we provide evidence that FAP is important for remodeling a permissive stromal ECM that supports pancreatic (and perhaps also breast) cancer invasion in vitro. The experimental approach used to study the role of FAP in tumor invasion utilized an in vivo-like 3D matrix system that has been shown to effectively recapitulate stromal ECMs from various murine and human tissues [21, 28, 43]. Fibroblasts-derived 3D matrix system can avoid the flat surface of a tissue culture dish which impose an artificially rigid environment onto the cell . On recent studies analyzing the direct effects of tumor stromal ECMs on cancer cell behavior, we demonstrated (among other things) that different breast cancer cell lines present distinct cell motility phenotypes when cultured within tumor-associated ECMs . Here we also performed assays using the same breast cancer lines on our FAP+ matrices and found that FAP+ matrices effectively recapitulate many aspects of tumor-associated ECMs (data shown in supplemental material). Thus FAP+ matrices were utilized as a stromal landscape to study matrix-induced pancreatic (and in some instances breast) cancer cell behaviors.
In this study, we observed that FAP+ fibroblast-derived matrices presented higher organization levels of fibers when compared to FAP- matrices. Importantly, we showed that FAP+ matrices contain parallel fiber organization features that are reminiscent of tumor-associated ECMs of pancreatic desmoplastic tissues associated with pancreatic adenocarcinoma looking at human normal and tumor ECMs both in vivo and in vitro. The observed enhanced directionality and velocity of cancer cells invading through FAP+ 3D matrices was effectively reversed in matrices produced from FAP+ fibroblasts in the presence of FAP enzymatic inhibitor. By determining alterations in collagen at the vicinity of tumors in vivo, it has been suggested that a strong relationship exists between both collagen density and matrix architectural organization and observed increased breast tumorigenesis . Likewise, tumor-associated collagen features similar to the above-mentioned were observed by depleting FAP in vivo . Interestingly, our human pancreatic adenocarcinoma-associated fibroblast ECMs, as well as our FAP+ 3D matrices in vitro, also presented the parallel organized patterns highlighted by the two studies above. These facts reinforce the notion that our in vitro models may be regarded as being highly relevant.
Our FAP+inhibitor matrices resembled FAP- matrices with lower ECM levels of organization and supported relatively impeded tumor cell behaviors. We also report that inhibition of β1-integrins abrogates the FAP+ ECM facilitated invasive capabilities of pancreatic and breast tumor cells, suggesting that a cell-matrix β1-integrin engagement is important for this FAP+ matrix-dependent process. We elucidated a potential molecular mechanism underlying the enhanced pancreatic cancer cell motility mediated by FAP+ matrix. We found that FAP remodels the extracellular matrices through alterations of ECM proteins levels, as well as through increased fibronectin fiber patterned orientation. FAP expression regulates tenascin C, collagen I, fibronectin and α-SMA expressions, all known to play important roles in pancreatic tumorigenesis [7, 46]. FAP activity seems to be important for the positive collagen I and the negative tenascin C regulation, yet absence of FAP activity seems to significantly block collagen I while enhancing fibronectin and α-SMA levels of expression. Our results suggest that FAP enzymatic activity plays a role not only in regulating fiber orientation but also in regulating expression levels of collagen I. Moreover, we were able to distinguish among FAP expression and/or activity dependent functions in regulating stromal markers. When measuring the levels of collagen I expression, we observed that, while FAP activity is important to both fibronectin and collagen architectural fiber organization, this activity is mostly important for assessing collagen I as opposed to fibronectin (or α-SMA) levels of expression. In summary, we observed that both FAP expression and/or activity dependent functions can regulate ECMs compositions.
Interestingly, we learned that the velocity of the migrating cancer cells is dependent on FAP expression regardless of its activity, whereas the latter is crucial for directionality. The observed change in architecture and the levels of components of the ECM leads to enhanced ECM permissiveness, which facilitates pancreatic cancer invasiveness via β1-integrin engagement. Interestingly, our results using MDA-MB-231 cells were comparable to the ones observed with Panc-1, and thus we concluded that fibroblastic FAP-dependent matrix alterations and the importance of β1-integrin in the regulation of cancer cell motility are effects that are not necessarily restricted to pancreatic cancers and that additional cancers such as breast, where stromal FAP expression levels have been shown to be increased , show similar behaviors. Furthermore, FAP+ matrix-induced regulatory molecules in cancer cells revealed that it is associated with an increased activation of FAK that is independent of AKT activity. Because β1-integrins are major receptors responsible for ECM assembly, these results suggest that FAP remodels ECM fibers to provide directional paths for pancreatic (and other cancer, i.e., breast) cells to engage β1-integrin/FAK for invasion. As a response to altered signals from the tumor stroma, cancer cell behavior seems to be influenced through patterning of the underlying matrix, resulting in track-dependent cellular migration. Conversely, the inhibition of α5β1-integrin function only partially affected FAP+ matrix-induced Panc-1 invasion. This specific integrin inhibition did not significantly alter the constitutive FAK activation seen in Panc-1 cells cultured into FAP+ matrices, suggesting that additional members of the β1-integrin family are also responsible for the matrix-induced effects. Importantly, the changes in the ECM produced by FAP+ fibroblasts effectively recapitulate many aspects of stromal ECMs, including increased tumorigenic behaviors , enhance ability to metastasize in vivo , promote epithelial cancer cells to move along insoluble tumor-associated ECM fibers leading them towards the intravasation sites during metastasis , and recapitulate pancreatic stromal characteristics, such as increased collagen I expression and up-regulation of desmoplastic marker α-SMA .
Our data suggests that cancer cell invasiveness can be affected by alterations in the tumor microenvironment. We provide evidence that FAP is important for remodeling a permissive stromal ECM to produce directional paths for pancreatic (and breast) cells that engage β1-integrin/FAK for invasion. Therefore, our observations imply that a better understanding of the changes in stromal fibroblasts and their influence on epithelial tumor cell behavior can lead to novel strategies for the prevention and treatment of cancer. The identification of fibroblastic 3D-dependent signaling pathways may be important to block the transition to a permissive microenvironment and even reverse stromal fibroblast-dependent neoplasia . This study provides the pre-clinical rationale that inhibition of FAP proteolytic activity and selected β1-integrin family members in combination may abrogate the invasive capabilities of pancreatic tumors by interfering with the architectural organization of tumor-associated ECMs and disrupting the tumor-stromal interactions.
Acknowledgements and Funding
We thank Dr. Wen-Tien Chen, Dr. Teresa Ramirez-Montagut, Dr. William Bachovchin, and Dr. Kenneth Yamada for providing reagents. We also thank Dr. Fang Zhu for statistical analysis assistance. This work was supported by grants from the National Institutes of Health CA090468 (JDC), CA122301 (JDC), CA113451 (EC) and CA06927, by an Appropriation from the Commonwealth of Pennsylvania, as well as by Fox Chase Cancer Center's Internal Director's Fund and the Ewing Trust for Pancreatic Cancer research.
- Kiaris H, Chatzistamou I, Kalofoutis C, Koutselini H, Piperi C, Kalofoutis A: Tumour-stroma interactions in carcinogenesis: basic aspects and perspectives. Mol Cell Biochem. 2004, 261 (1-2): 117-122.View ArticlePubMedGoogle Scholar
- Nakagawa H, Liyanarachchi S, Davuluri RV, Auer H, Martin EW, de la Chapelle A, Frankel WL: Role of cancer-associated stromal fibroblasts in metastatic colon cancer to the liver and their expression profiles. Oncogene. 2004, 23 (44): 7366-7377. 10.1038/sj.onc.1208013.View ArticlePubMedGoogle Scholar
- Ruiter D, Bogenrieder T, Elder D, Herlyn M: Melanoma-stroma interactions: structural and functional aspects. Lancet Oncol. 2002, 3 (1): 35-43. 10.1016/S1470-2045(01)00620-9.View ArticlePubMedGoogle Scholar
- Sato N, Maehara N, Goggins M: Gene expression profiling of tumor-stromal interactions between pancreatic cancer cells and stromal fibroblasts. Cancer Res. 2004, 64 (19): 6950-6956. 10.1158/0008-5472.CAN-04-0677.View ArticlePubMedGoogle Scholar
- Ostman A, Augsten M: Cancer-associated fibroblasts and tumor growth--bystanders turning into key players. Curr Opin Genet Dev. 2009, 19 (1): 67-73. 10.1016/j.gde.2009.01.003.View ArticlePubMedGoogle Scholar
- Li H, Fan X, Houghton J: Tumor microenvironment: the role of the tumor stroma in cancer. J Cell Biochem. 2007, 101 (4): 805-815. 10.1002/jcb.21159.View ArticlePubMedGoogle Scholar
- Mahadevan D, Von Hoff DD: Tumor-stroma interactions in pancreatic ductal adenocarcinoma. Mol Cancer Ther. 2007, 6 (4): 1186-1197. 10.1158/1535-7163.MCT-06-0686.View ArticlePubMedGoogle Scholar
- Mueller MM, Fusenig NE: Tumor-stroma interactions directing phenotype and progression of epithelial skin tumor cells. Differentiation. 2002, 70 (9-10): 486-497. 10.1046/j.1432-0436.2002.700903.x.View ArticlePubMedGoogle Scholar
- Edwards DR, Murphy G: Cancer. Proteases--invasion and more. Nature. 1998, 394 (6693): 527-528. 10.1038/28961.View ArticlePubMedGoogle Scholar
- Johnsen M, Lund LR, Romer J, Almholt K, Dano K: Cancer invasion and tissue remodeling: common themes in proteolytic matrix degradation. Curr Opin Cell Biol. 1998, 10 (5): 667-671. 10.1016/S0955-0674(98)80044-6.View ArticlePubMedGoogle Scholar
- Kalluri R, Zeisberg M: Fibroblasts in cancer. Nat Rev Cancer. 2006, 6 (5): 392-401. 10.1038/nrc1877.View ArticlePubMedGoogle Scholar
- Park JE, Lenter MC, Zimmermann RN, Garin-Chesa P, Old LJ, Rettig WJ: Fibroblast activation protein, a dual specificity serine protease expressed in reactive human tumor stromal fibroblasts. J Biol Chem. 1999, 274 (51): 36505-36512. 10.1074/jbc.274.51.36505.View ArticlePubMedGoogle Scholar
- Monsky WL, Lin CY, Aoyama A, Kelly T, Akiyama SK, Mueller SC, Chen WT: A potential marker protease of invasiveness, seprase, is localized on invadopodia of human malignant melanoma cells. Cancer Res. 1994, 54 (21): 5702-5710.PubMedGoogle Scholar
- Mueller SC, Ghersi G, Akiyama SK, Sang QX, Howard L, Pineiro-Sanchez M, Nakahara H, Yeh Y, Chen WT: A novel protease-docking function of integrin at invadopodia. J Biol Chem. 1999, 274 (35): 24947-24952. 10.1074/jbc.274.35.24947.View ArticlePubMedGoogle Scholar
- Wang XM, Yu DM, McCaughan GW, Gorrell MD: Fibroblast activation protein increases apoptosis, cell adhesion, and migration by the LX-2 human stellate cell line. Hepatology. 2005, 42 (4): 935-945. 10.1002/hep.20853.View ArticlePubMedGoogle Scholar
- Cheng JD, Dunbrack RL, Valianou M, Rogatko A, Alpaugh RK, Weiner LM: Promotion of tumor growth by murine fibroblast activation protein, a serine protease, in an animal model. Cancer Res. 2002, 62 (16): 4767-4772.PubMedGoogle Scholar
- Cheng JD, Valianou M, Canutescu AA, Jaffe EK, Lee HO, Wang H, Lai JH, Bachovchin WW, Weiner LM: Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth. Mol Cancer Ther. 2005, 4 (3): 351-360.PubMedGoogle Scholar
- Cohen SJ, Alpaugh RK, Palazzo I, Meropol NJ, Rogatko A, Xu Z, Hoffman JP, Weiner LM, Cheng JD: Fibroblast activation protein and its relationship to clinical outcome in pancreatic adenocarcinoma. Pancreas. 2008, 37 (2): 154-158. 10.1097/MPA.0b013e31816618ce.View ArticlePubMedGoogle Scholar
- Bissell MJ, Rizki A, Mian IS: Tissue architecture: the ultimate regulator of breast epithelial function. Curr Opin Cell Biol. 2003, 15 (6): 753-762. 10.1016/j.ceb.2003.10.016.View ArticlePubMedPubMed CentralGoogle Scholar
- Grzesiak JJ, Ho JC, Moossa AR, Bouvet M: The integrin-extracellular matrix axis in pancreatic cancer. Pancreas. 2007, 35 (4): 293-301. 10.1097/mpa.0b013e31811f4526.View ArticlePubMedGoogle Scholar
- Amatangelo MD, Bassi DE, Klein-Szanto AJ, Cukierman E: Stroma-derived three-dimensional matrices are necessary and sufficient to promote desmoplastic differentiation of normal fibroblasts. Am J Pathol. 2005, 167 (2): 475-488. 10.1016/S0002-9440(10)62991-4.View ArticlePubMedPubMed CentralGoogle Scholar
- Provenzano PP, Eliceiri KW, Campbell JM, Inman DR, White JG, Keely PJ: Collagen reorganization at the tumor-stromal interface facilitates local invasion. BMC Med. 2006, 4 (1): 38-10.1186/1741-7015-4-38.View ArticlePubMedPubMed CentralGoogle Scholar
- Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, Fong SF, Csiszar K, Giaccia A, Weninger W, et al: Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell. 2009, 139 (5): 891-906. 10.1016/j.cell.2009.10.027.View ArticlePubMedPubMed CentralGoogle Scholar
- Miura N, Kanayama Y, Nagai W, Hasegawa T, Seko Y, Kaji T, Naganuma A: Characterization of an immortalized hepatic stellate cell line established from metallothionein-null mice. J Toxicol Sci. 2006, 31 (4): 391-398. 10.2131/jts.31.391.View ArticlePubMedGoogle Scholar
- Castello-Cros R, Cukierman E: Stromagenesis during tumorigenesis: characterization of tumor-associated fibroblasts and stroma-derived 3D matrices. Methods Mol Biol. 2009, 522: 275-305. 10.1007/978-1-59745-413-1_19.View ArticlePubMedPubMed CentralGoogle Scholar
- Cukierman E: A visual-quantitative analysis of fibroblastic stromagenesis in breast cancer progression. J Mammary Gland Biol Neoplasia. 2004, 9 (4): 311-324. 10.1007/s10911-004-1403-y.View ArticlePubMedGoogle Scholar
- Cukierman E: Cell migration analyses within fibroblast-derived 3-D matrices. Cell migration: Developmental methods and protocols. Edited by: Guan J. 2005, Totowa NJ: Humana Press, 294: 79-93.View ArticleGoogle Scholar
- Cukierman E, Pankov R, Stevens DR, Yamada KM: Taking cell-matrix adhesions to the third dimension. Science. 2001, 294 (5547): 1708-1712. 10.1126/science.1064829.View ArticlePubMedGoogle Scholar
- Pankov R, Endo Y, Even-Ram S, Araki M, Clark K, Cukierman E, Matsumoto K, Yamada KM: A Rac switch regulates random versus directionally persistent cell migration. J Cell Biol. 2005, 170 (5): 793-802. 10.1083/jcb.200503152.View ArticlePubMedPubMed CentralGoogle Scholar
- Goodman JD, Rozypal TL, Kelly T: Seprase, a membrane-bound protease, alleviates the serum growth requirement of human breast cancer cells. Clin Exp Metastasis. 2003, 20 (5): 459-470. 10.1023/A:1025493605850.View ArticlePubMedGoogle Scholar
- Jin X, Iwasa S, Okada K, Mitsumata M, Ooi A: Expression patterns of seprase, a membrane serine protease, in cervical carcinoma and cervical intraepithelial neoplasm. Anticancer Res. 2003, 23 (4): 3195-3198.PubMedGoogle Scholar
- Iwasa S, Jin X, Okada K, Mitsumata M, Ooi A: Increased expression of seprase, a membrane-type serine protease, is associated with lymph node metastasis in human colorectal cancer. Cancer Lett. 2003, 199 (1): 91-98. 10.1016/S0304-3835(03)00315-X.View ArticlePubMedGoogle Scholar
- Ariga N, Sato E, Ohuchi N, Nagura H, Ohtani H: Stromal expression of fibroblast activation protein/seprase, a cell membrane serine proteinase and gelatinase, is associated with longer survival in patients with invasive ductal carcinoma of breast. Int J Cancer. 2001, 95 (1): 67-72. 10.1002/1097-0215(20010120)95:1<67::AID-IJC1012>3.0.CO;2-U.View ArticlePubMedGoogle Scholar
- Scanlan MJ, Raj BK, Calvo B, Garin-Chesa P, Sanz-Moncasi MP, Healey JH, Old LJ, Rettig WJ: Molecular cloning of fibroblast activation protein alpha, a member of the serine protease family selectively expressed in stromal fibroblasts of epithelial cancers. Proc Natl Acad Sci USA. 1994, 91 (12): 5657-5661. 10.1073/pnas.91.12.5657.View ArticlePubMedPubMed CentralGoogle Scholar
- Henry LR, Lee HO, Lee JS, Klein-Szanto A, Watts P, Ross EA, Chen WT, Cheng JD: Clinical implications of fibroblast activation protein in patients with colon cancer. Clin Cancer Res. 2007, 13 (6): 1736-1741. 10.1158/1078-0432.CCR-06-1746.View ArticlePubMedGoogle Scholar
- Niedermeyer J, Scanlan MJ, Garin-Chesa P, Daiber C, Fiebig HH, Old LJ, Rettig WJ, Schnapp A: Mouse fibroblast activation protein: molecular cloning, alternative splicing and expression in the reactive stroma of epithelial cancers. Int J Cancer. 1997, 71 (3): 383-389. 10.1002/(SICI)1097-0215(19970502)71:3<383::AID-IJC14>3.0.CO;2-H.View ArticlePubMedGoogle Scholar
- Castello-Cros R, Khan DR, Simons J, Valianou M, Cukierman E: Staged stromal extracellular 3D matrices differentially regulate breast cancer cell responses through PI3K and beta1-integrins. BMC Cancer. 2009, 9: 94-10.1186/1471-2407-9-94.View ArticlePubMedPubMed CentralGoogle Scholar
- Levy MT, McCaughan GW, Abbott CA, Park JE, Cunningham AM, Muller E, Rettig WJ, Gorrell MD: Fibroblast activation protein: a cell surface dipeptidyl peptidase and gelatinase expressed by stellate cells at the tissue remodelling interface in human cirrhosis. Hepatology. 1999, 29 (6): 1768-1778. 10.1002/hep.510290631.View ArticlePubMedGoogle Scholar
- Omary MB, Lugea A, Lowe AW, Pandol SJ: The pancreatic stellate cell: a star on the rise in pancreatic diseases. J Clin Invest. 2007, 117 (1): 50-59. 10.1172/JCI30082.View ArticlePubMedPubMed CentralGoogle Scholar
- Armstrong T, Packham G, Murphy LB, Bateman AC, Conti JA, Fine DR, Johnson CD, Benyon RC, Iredale JP: Type I collagen promotes the malignant phenotype of pancreatic ductal adenocarcinoma. Clin Cancer Res. 2004, 10 (21): 7427-7437. 10.1158/1078-0432.CCR-03-0825.View ArticlePubMedGoogle Scholar
- Loukopoulos P, Kanetaka K, Takamura M, Shibata T, Sakamoto M, Hirohashi S: Orthotopic transplantation models of pancreatic adenocarcinoma derived from cell lines and primary tumors and displaying varying metastatic activity. Pancreas. 2004, 29 (3): 193-203. 10.1097/00006676-200410000-00004.View ArticlePubMedGoogle Scholar
- Hsia DA, Mitra SK, Hauck CR, Streblow DN, Nelson JA, Ilic D, Huang S, Li E, Nemerow GR, Leng J, et al: Differential regulation of cell motility and invasion by FAK. J Cell Biol. 2003, 160 (5): 753-767. 10.1083/jcb.200212114.View ArticlePubMedPubMed CentralGoogle Scholar
- Quiros RM, Valianou M, Kwon Y, Brown KM, Godwin AK, Cukierman E: Ovarian normal and tumor-associated fibroblasts retain in vivo stromal characteristics in a 3-D matrix-dependent manner. Gynecol Oncol. 2008, 110 (1): 99-109. 10.1016/j.ygyno.2008.03.006.View ArticlePubMedPubMed CentralGoogle Scholar
- Beacham DA, Cukierman E: Stromagenesis: the changing face of fibroblastic microenvironments during tumor progression. Semin Cancer Biol. 2005, 15 (5): 329-341. 10.1016/j.semcancer.2005.05.003.View ArticlePubMedGoogle Scholar
- Santos AlM, Jung J, Aziz N, Kissil JL, Puré E: Targeting fibroblast activation protein inhibits tumor stromagenesis and growth in mice. The Journal of Clinical Investigation. 2009, 119 (12): 3613-3625. 10.1172/JCI38988.View ArticlePubMedPubMed CentralGoogle Scholar
- Farrow B, Rowley D, Dang T, Berger DH: Characterization of Tumor-Derived Pancreatic Stellate Cells. J Surg Res. 2009, 157 (1): 96-102.View ArticlePubMedGoogle Scholar
- Mersmann M, Schmidt A, Rippmann JF, Wuest T, Brocks B, Rettig WJ, Garin-Chesa P, Pfizenmaier K, Moosmayer D: Human antibody derivatives against the fibroblast activation protein for tumor stroma targeting of carcinomas. Int J Cancer. 2001, 92 (2): 240-248. 10.1002/1097-0215(200102)9999:9999<::AID-IJC1170>3.0.CO;2-U.View ArticlePubMedGoogle Scholar
- Condeelis J, Segall JE: Intravital imaging of cell movement in tumours. Nat Rev Cancer. 2003, 3 (12): 921-930. 10.1038/nrc1231.View ArticlePubMedGoogle Scholar
- Binkley CE, Zhang L, Greenson JK, Giordano TJ, Kuick R, Misek D, Hanash S, Logsdon CD, Simeone DM: The molecular basis of pancreatic fibrosis: common stromal gene expression in chronic pancreatitis and pancreatic adenocarcinoma. Pancreas. 2004, 29 (4): 254-263. 10.1097/00006676-200411000-00003.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/245/prepub
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