Collagen type V promotes the malignant phenotype of pancreatic ductal adenocarcinoma

Cancer Letters 356 (2015) 721–732

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

Collagen type V promotes the malignant phenotype of pancreatic ductal adenocarcinoma Sonja Berchtold a, Barbara Grünwald b, Achim Krüger b, Anja Reithmeier a, Teresa Hähl a, Tao Cheng c, Annette Feuchtinger d, Diana Born e, Mert Erkan c,f, Jörg Kleeff c, Irene Esposito a,* a

Institute of Pathology, Technische Universität München, Munich, Germany Institute for Experimental Oncology and Therapy Research, Technische Universität München, Munich, Germany c Department of Surgery, Technische Universität München, Munich, Germany d Research Unit Analytical Pathology, Helmholtz Zentrum München-German Research Center for Environmental Health, Neuherberg, Germany e Pathology, Kantonspital Münsterlingen, Münsterlingen, Switzerland f Department of Surgery, Koc University School of Medicine, Istanbul, Turkey b

A R T I C L E

I N F O

Article history: Received 12 August 2014 Received in revised form 20 October 2014 Accepted 21 October 2014 Keywords: Collagen type V β1-integrin Pancreatic ductal adenocarcinoma Pancreatic stellate cells Epithelial–stromal interaction

A B S T R A C T

Excessive matrix production by pancreatic stellate cells promotes local growth and metastasis of pancreatic ductal adenocarcinoma and provides a barrier for drug delivery. Collagen type V is a fibrillar, regulatory collagen up-regulated in the stroma of different malignant tumors. Here we show that collagen type V is expressed by pancreatic stellate cells in the stroma of pancreatic ductal adenocarcinoma and affects the malignant phenotype of various pancreatic cancer cell lines by promoting adhesion, migration and viability, also after treatment with chemotherapeutic drugs. Pharmacological and antibodymediated inhibition of β1-integrin signaling abolishes collagen type V-induced effects on pancreatic cancer cells. Ablation of collagen type V secretion of pancreatic stellate cells by siRNA reduces invasion and proliferation of pancreatic cancer cells and tube formation of endothelial cells. Moreover, stable knockdown of collagen type V in pancreatic stellate cells reduces metastasis formation and angiogenesis in an orthotopic mouse model of ductal adenocarcinoma. In conclusion, paracrine loops involving cancer and stromal elements and mediated by collagen type V promote the malignant phenotype of pancreatic ductal adenocarcinoma and underline the relevance of epithelial–stromal interactions in the progression of this aggressive neoplasm. © 2014 Elsevier Ireland Ltd. All rights reserved.

Introduction Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. The very low five-year survival rate of 6% [1] could not be improved by decades of extensive research, indicating that a better and more detailed understanding of this type of cancer has to be achieved in order to design effective treatment strategies. One prominent feature of PDAC is the stromal reaction surrounding tumor cells and influencing their growth [2]. Tumor progression from precursor lesions to the invasive carcinoma is accompanied by the development of a stromal reaction. In fully developed PDAC the desmoplastic stroma accounts for up to 80% of the tumor mass [3]. Pancreatic stellate cells (PSCs) are the major fibrogenic cell type

Abbreviations: PSC, pancreatic stellate cells; PDAC, pancreatic ductal adenocarcinoma; Col V, collagen type V; αSMA, α-smooth muscle actin. * Corresponding author. Tel.: +49 89 4140 4166; fax: +49 89 4140 4865. E-mail address: [email protected] (I. Esposito). http://dx.doi.org/10.1016/j.canlet.2014.10.020 0304-3835/© 2014 Elsevier Ireland Ltd. All rights reserved.

within pancreatic desmoplasia. PSCs are myofibroblast-like cells that transdifferentiate from a quiescent to an activated state during tumor progression or inflammation [4,5]. Upon activation, PSCs produce excessive amounts of extracellular matrix (ECM) molecules, such as collagens, fibronectin, laminin and tenascin-C, and promote matrix remodeling via production of proteases and inhibitors, most prominently matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) [6–9]. The resulting desmoplastic microenvironment of PDAC is not a passive scaffold for the tumor cells but actively drives carcinogenesis [10]. These structural features of PDAC have been shown to directly impact tumor cell aggressiveness [6,11] and diminish treatment success, as the desmoplastic reaction acts as a barrier to drug delivery [12–14]; however, recent data point to a more complex and possibly controversial role of the stroma in pancreatic carcinogenesis [15,16]. In addition to the influence that PSCs and PSC-derived ECM proteins have on pancreatic cancer cells, accumulating evidence indicates that the epithelial and stromal compartments interact in a bidirectional manner to enhance the aggressive nature of PDAC. Stromal cells promote aggressiveness of cancer cells, while in turn

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pancreatic cancer cells are known to influence PSC migration, proliferation and secretion of ECM proteins [17]. The major components of the desmoplastic reaction are collagens [18], which under physiological conditions maintain tissue integrity. Collagen type V (Col V) is a regulatory fibril-forming collagen and in normal tissues comprises only 2–5% of the total collagens. During fibrillogenesis, Col V forms heterotypic fibrils with collagen type I and thereby regulates the diameter of the collagen type I fibrils [19]. In addition, Col V influences the total collagen content, as shown in Col V knockout mice, which present with a reduced number of fibrils with an abnormally large diameter [20]. Collagens are known to activate intracellular signaling pathways via binding to integrins [21]. The main receptor for Col V is α2β1-integrin [22,23]. In glomerular endothelial cells, β1-integrin was described to be essential for Col V-mediated signaling and subsequent downstream activation of focal adhesion kinase (FAK) and paxillin (PAX) [24]. This Col V-mediated activation of the β1-integrin signaling pathway promoted cell migration and motility [25]. Col V plays a functional role in some cancer entities, like breast cancer and colon cancer [26,27]. In breast cancer, Col V was found to be increased in the stromal reaction and produced by myofibroblasts rather than cancer cells [26]. In PDAC a protumorigenic role of collagen type I and collagen type III has been already described [28,29], whereas the function of Col V has not been addressed yet. Recently, Col V was found to be secreted by activated PSCs [30]. Here we show that Col V plays an important role in the interaction between pancreatic cancer cells and PSCs and affects the malignant phenotype of PDAC. Materials and methods Clinical samples Tissue samples were obtained from patients who underwent pancreatic resections for PDAC at the Technische Universität München, Munich, Germany. After surgical removal, tissue samples were fixed in 4% buffered formalin and embedded in paraffin after 24 hrs. The clinical diagnosis of PDAC was confirmed by histological analysis. Tissue arrays were obtained with a manual tissue arrayer (Beecher Instruments Inc., USA). The use of human material for the analysis was approved by the local ethics committee of the Technische Universität München, Munich, Germany, and written informed consent was obtained from all patients.

αSMA (1:100, clone HHF35, Dako, Hamburg, Germany) for the detection of activated PSCs and Col V. Immunofluorescence Cells were grown in a 24-well plate containing glass coverslips and fixed with methanol. Permeabilization was achieved with Triton X-100 (0.25% v/v in TBS) for 5 min followed by two washing steps and by blocking for 1 hr in blocking solution (TBS containing 10% goat serum and 0.1% Triton X-100). Then, incubation with antibodies anti-pPAX-Y118 (1:100, sc-101774, Santa Cruz Biotechnology, CA, USA), antipFAK-Y861 (1:200, sc-101679 Santa Cruz Biotechnology, CA, USA), anti-vinculin (1:200, V9131, Sigma-Aldrich, Inc., MO, USA) anti-Col V (1:100, C-5, Santa Cruz Biotechnology, CA, USA), anti-CD31 (1:100, ab28364, Abcam plc, UK) and anti-αSMA (1:100, ab5694, Abcam plc, UK) was performed overnight. Secondary antibodies Alexa Fluor 488 goat anti-mouse-IgG or Alexa Fluor 546 goat anti-rabbit-IgG (1:200, Life Technologies, CA, USA) were added 1:200 for 1 hr. Counterstaining was performed with Hoechst 33342 (0.2 mg/ml in water) for 5 min and coverslips were mounted on microscope slides. Protein extract preparation and immunoblotting Protein extracts for immunoblot analysis were prepared from attached and still non-attached cells, as well as from snap-frozen mouse tumor tissues. For the cell extracts, culture medium was removed and non-attached cells were collected by centrifugation. Both pelleted and still adherent cells as well as homogenized tumor tissues were lysed in cold lysis buffer (20 mM TRIS, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM EGTA pH 7.4) supplemented with protease and phosphatase inhibitor cocktails (Roche Diagnostics, Mannheim, Germany). Cell lysates were passed through a 26G needle and cellular debris was removed by centrifugation. Lysates were subjected to a 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were then incubated overnight at 4 °C with antibodies anti-pPAX-Y118 (1:1500, GTX24833, GeneTex, Inc., CA, USA), antipaxillin (1:1000, #2542, Cell Signaling Technology Inc., MA, USA), anti-pFAK-Y397 (1:750, #611722, BD Biosciences, CA, USA), anti-pFAK-Y576/577 (1:750, #3281, Cell Signaling Technology Inc., MA, USA), anti-FAK (1:500, #610087, BD Biosciences, CA, USA), anti-GAPDH (1:5000, sc-25778, Santa Cruz Biotechnology, CA), anti-Col V (1:500), anti-β1-integrin (1:1000, #610467, BD Biosciences, CA, USA) and anti-αSMA (1:2000). Col V coating Col V was purchased from Sigma Aldrich (C3657, Sigma Aldrich, Steinheim, Germany) and coating was performed to a final concentration of 10 μg/cm2. Col V was allowed to absorb overnight at 37 °C, before unbound protein was removed with PBS washing. Unspecific binding sites were blocked with 0.1% BSA (heat denatured at 85 °C, 10 min) at RT. Plates were washed afterwards twice and sterilized under UV light for 20 min. Uncoated plates, blocked with BSA, are referred in this manuscript as control plates.

Cell cultures and reagents

Adhesion assay

PSCs were isolated and characterized as previously described [31]. Briefly, the purity of the PSC preparations was assessed by morphology and staining for α-smoothmuscle actin (αSMA) (>95% of the cells), vimentin (100%), and desmin (20–40%). HUVEC were obtained by Promocell, Heidelberg, Germany. Fibroblasts from patients suffering from Ehlers–Danlos syndrome (F011 and F057) were provided by the Cell Line and DNA Biobank (Istituto G. Gaslini), member of the Telethon Network of Genetic Biobanks (project no. GTB12001). Human dermal fibroblasts (HDF) were kindly provided by Prof. Jürgen Schlegel, Institute of Pathology, Technische Universität München, Munich, Germany. All other cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). PSCs were maintained in DMEM/Ham’s F12 (1:1, vol/vol) supplemented with 20% fetal bovine serum (FBS, Gibco®, Darmstadt, Germany), 1% Penicillin–Streptomycin (Gibco®, Darmstadt, Germany) and 1% amphotericin (Biochrom AG, Berlin, Germany). HUVEC were cultured in endothelial growth medium (PromoCell, Heidelberg, Germany) supplemented with 1% Penicillin–Streptomycin. Cancer cells and fibroblasts (F011, F057 and HDF) were maintained in DMEM supplemented with 10% FBS and 1% Penicillin– Streptomycin.

Cells were starved for 24 hrs in medium with 1% FBS. 15,000 cells were then seeded into Col V coated 96-well plates and let adhered for 3 hrs. For experiments with the Src kinase inhibitor AZD0530, cells were preincubated with 2 μM of AZD for 15 min and for experiments with the blocking antibody P5D2 (2.5 μg/1 × 106 cells), cells were preincubated for 30 min. Non-adherent cells were removed with PBS washing. Adherent cells were then fixed with methanol, and stained with 0.5% toluidine blue in water. Cells were then lysed in 50 μl of 50 mM HCl diluted in 50% ethanol. Absorption was measured at 620 nm using a microplate reader. Experiments were performed with four technical and three biological replicates.

Immunohistochemistry Paraffin-embedded tissue sections were deparaffinized, rehydrated and rinsed in washing buffer (Dako, Hamburg, Germany). Heat-induced antigen retrieval was performed in citrate buffer for 10 min. The sections were then blocked with universal block, followed by blocking with normal goat serum. Primary antibodies against Col V (1:150, H-200, Santa Cruz Biotechnology, CA, USA) and β1-integrin (1:50, #610467, BD Biosciences, CA, USA) were added overnight at 4 °C. Biotinylated secondary antibody was added for 1 hr followed by incubation with streptavidin– horseradish peroxidase for 30 min at RT. Color reaction was performed with DAB as chromogen. Double immunohistochemistry was performed with antibodies against

Proliferation assay Cells were seeded into Col V coated 96-well plates (Nunc GmbH & Co. KG, Langenselbold, Germany) in cell numbers dependent on the cell line (AsPC-1: 7000; Capan-1: 8000; PANC-1: 5000; SU.86.86: 3000; F011, F057, HDF and PSC: 2000) in standard culture medium. For inhibition experiments and viability assay, cells were seeded and inhibitors or chemotherapeutics were added after the cells adhered. A dose of 1 mM 5-FU or 10 μM gemcitabine was chosen after dose–response assays had been performed (data not shown). Cells were then grown for 24 and 48 hrs before 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed as previously described [32]. Experiments were performed with four technical and three biological replicates. Migration assay Cells were plated onto Col V coated 24-well plates (Capan-1: 250,000; PANC1: 150,000; SU.86.86: 120,000, F011, F057, HDF and PSC: 100,000) for 24 hrs in standard culture medium. Then, the medium was changed to serum-free medium to exclude proliferation of the cells for another 24 hrs. The monolayer was then scraped

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with a 10 μl pipette tip. After three washes with PBS, cells were incubated at 37 °C, 5% CO2. Inhibitors were added directly after making the wound. Migration of cells into the open area was evaluated by taking images with an Axio Observer A.1 microscope (Carl Zeiss AG, Jena, Germany) and open areas were calculated using ImageJ. Experiments were performed with four technical and three biological replicates. Transfection with siRNA 50,000 PSC or 150,000 HUVEC were plated in a 6-well plate and let adhere overnight. The next day, cells were starved for 2 hrs in reduced serum media (OptiMEM®). Transfection was performed with X-tremeGENE siRNA Transfection Reagent (Roche Diagnostics, Mannheim, Germany) according to the user’s manual. Briefly, 1 μg siRNA (5′-UUACAGUCGAGGAUCAAGGTG-3′, Qiagen, Hilden, Germany) and transfection reagent (5 μl) were first diluted in Opti-MEM®, then mixed and incubated for 15 min at room temperature and added dropwise to the cells. Scrambled siRNA was used for control. After 5 hrs, the medium was replaced with a standard medium. Efficiency of transfection was checked by immunoblotting. Preparation of conditioned medium Conditioned media containing cell secretions were collected from the cell lines after 24 hrs of culture.

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Orthotopic mouse model Eight week-old athymic female mice (5 animals/group) (BALB/c nude, Charles River) were anesthetized and an incision was made in the left flank. The spleen and the tail of the pancreas were exteriorized and cells diluted in 50 μl matrigel were injected into the tail of the pancreas. The following cells were used: (1) SU.86.86: 0.5 × 106, (2) SU.86.86 + PSC_shNT: 0.5 × 106 each, (3) SU.86.86 + PSC_sh#10: 0.5 × 106 each, (4) SU.86.86 + PSC_sh#11: 0.5 × 106 each. Six weeks after surgery, mice were sacrificed, the pancreas removed and tumor size was measured according to an established formula (1/2 length × width 2 ). Tumors were further processed for immunoblotting and histological examination and livers were stained with 5-bromo4-chloro-3-indolyl-β-d-galactopyranoside to detect metastases as described [35]. All animal experiments were carried out in accordance with the German Animal Protection Law with permission from the responsible veterinary authority (reference number: 55.2-1-54–2532-42-13). Statistical analysis Data are presented as mean ± SEM (standard error of the mean) unless otherwise stated. Student’s t-test or one-way analysis of variance (ANOVA) was used for comparisons. Survival analysis was performed using Kaplan–Meier analysis.

Results Invasion Chemoinvasion assay was performed using 8 μm pore size polycarbonate membrane inserts coated with matrigel (BD BioCoat™, CA, USA). 500 μl of the cell suspension (25,000 cells/well) was laid onto the insert, and the lower well was filled with 750 μl of the chemoattractant (conditioned medium from transfected and not transfected PSCs). After 24 hrs incubation at standard conditions cells from the upper part of the insert were removed using a humidified cotton swab. Migrated cells were fixed with methanol for 30 min and stained with toluidine blue (2% in water). Experiments were performed with three technical and three biological replicates. Tube-formation assay Matrigel was thawed at 4 °C for around 2 hrs before use; pipette tips and a 96well plate were prechilled at −20 °C. 100 μl of matrigel was put in 96-well plates and polymerized at 37 °C for 30 min. HUVEC (25,000 cells) with and without a transient knock-down of Col V or in conditioned medium from PSC or pancreatic cancer cells without FBS were seeded on the matrigel layer. Pictures were taken 16 hrs after plating the cells, as described [33]. Genetic manipulation of stellate and tumor cells using lentiviral gene transfer Lentiviral particles were produced in HEK293T with the ViraPower™ Lentiviral Expression Systems (Life Technologies, CA, USA) according to the manufacturer’s instruction. PSCs were infected with lentiviral particles in the presence of polybrene (Sigma Aldrich, Munich, Germany) when they reached a confluence of 80–90%. Cells were incubated for 2 hrs at 37 °C before the virus was removed again. Lentiviral particles based on the respective empty vector were used as a control (shNT). Knockdown of Col V was achieved using plasmids purchased from Sigma Aldrich, Munich, Germany (TRCN0000082810, #10; TRCN0000082811, #11). RNA isolation, reverse transcription and quantitative RT-PCR RNA was isolated from cells using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription was performed with the High capacity cDNA Reverse Transcription Kit (Invitrogen, Carlsbad, CA, USA) using 1 μg of RNA, as previously described [34]. Primers and probes for Col5a1 were purchased from Roche (mRNA) or from Applied Biosystems® (internal 18S rRNA standard). Relative quantification of gene expression was performed using the Applied Biosystems ABI prism 7700 sequence detection system (TaqMan) as described previously [34].

Expression of Col V increases during PDAC progression In the normal pancreas, Col V shows a weak expression in the periductal connective tissue, whereas a stronger staining is observed in chronic pancreatitis of different etiologies, as previously described [36]. We investigated the amount and distribution of Col V during progression of PDAC by immunohistochemical staining of human PanIN and PDAC tissues. In precursor lesions, Col V was distributed in a basement membrane-like pattern, whereas in the cancer tissue a diffuse distribution of staining in stromal cells was observed (Fig. 1A). The number of cases positive for Col V increased during tumor progression, with 49.4% positive PanIN-1 cases, 88.8% PanIN-2, 92.3% PanIN-3 and 95.5% positive PDAC cases (Supplementary Fig. S1A and B). In order to verify if the expression of Col V in PDAC tissues affects prognosis, survival analysis was performed. The mean survival of patients with Col V-negative tumor tissues was 13 months, whereas patients with Col V-positive tumors survived 12 months (p = 0.82) (Supplementary Fig. S1C). Col V is expressed by PSC in PDAC tissues In order to identify the cellular source of Col V within the stromal compartment, we performed double immunohistochemistry using the PSC marker α-smooth muscle actin (αSMA) and Col V. αSMApositive PSCs were shown to express Col V within the desmoplastic stroma of PDAC (Fig. 1B). Immunocytochemical staining of freshly isolated PSCs confirmed expression of Col V with αSMA in this cell type (Fig. 1C). Contamination of the cell preparation with endothelial cells was excluded by CD31 staining (Fig. 1D). Col V expression in different cell types was further evaluated via immunoblotting. Strong expression of Col V was found in cell lysates of PSC. In all tested cell lysates of pancreatic cancer cell lines, weak or no Col V expression was detected (Fig. 1E). This indicates that PSCs are the main source of Col V in PDAC in vivo.

Indirect co-culture of PSC and HUVEC

Effects of endogenous and exogenous Col V on fibroblasts First, 50,000 PSCs were seeded in the 6-well plates (lower chamber) and let adhere. Thereafter, HUVEC were seeded in the same quantity into the cell culture inserts (upper chamber). Both cell types were afterwards co-cultured for 48 hrs. RNA was isolated and transcribed as described above. For PCR analysis of pro- and antiangiogenic factors the following primers were used: VEGF_Fwd: 5′-CTACCTCCACCATGCCAAGT-3′, VEGF_Rev: 5′-ATCTGCATGGTGATGTTGGA-3′; Angiogenin_Fwd: 5′CCTGGGCGTTTTGTTGTTGG-3′, Angiogenin_Rev: 5′-TGTGGCTCGGTACTGGCATG-3′, Angiostatin_Fwd: 5′-ACAGACCTAGATTCTCACCTGC-3′, Angiostatin_Rev: 5′CTTCACACTCAAGAATGTCGC-3′, TSP-1_Fwd: 5′-ACCGCATTCCAGAGTCTGGC-3′, TSP1_Rev: 5′-ATGGGGACGTCCAACTCAGC-3′, TSP-2_Fwd: 5′-CTGTGTCAACACTCAGCCTGGC3′, TSP-2_Rev: 5′-TCCTTCTCATCGGTCACACCG-3’.

Functional properties of PSC in the presence or absence of extracellular Col V were examined and compared with those of fibroblasts bearing a Col V mutation, obtained from patients suffering from the classical type Ehlers–Danlos syndrome, and of normal skin fibroblasts (HDF). Briefly, classical type Ehlers–Danlos syndrome is characterized by mutations in the genes of the alpha 1 (COL5A1) or alpha 2 chain (COL5A2) of Col V [37]. Most of them are nonsense or frameshift mutations resulting in a null-allele and

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Col V GAPDH Fig. 1. Col V expression increases during pancreatic cancer progression. (A) Immunohistochemical staining of Col V in precursor lesions and PDAC tissue shows an increasing expression along with tumor progression. PanIN lesions display a basement membrane-like pattern of expression, whereas a diffuse stromal expression is observed in PDAC. Scale bar: 50 μm. (B) Immunohistochemical double staining for Col V (red) and αSMA (brown) in human PDAC reveals co-expression in PSC (arrows). Scale bar: 100 μm. (C) Co-staining of Col V (green) and αSMA (red) is visible by immunofluorescence of freshly isolated human PSC. (D) No CD31 staining is observed, thereby confirming the purity of the PSC preparation. CD31 expression in HUVEC is shown for comparison. (E) Immunoblot analysis of Col V reveals a strong expression in PSCs. Cancer cells do not express Col V, with the exception of a weak expression in SU.86.86. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

causing haploinsufficiency, thereby leading to defective incorporation of Col V into the fibrils and to decrease in fibril size [38–40]. The expression levels of Col V are not drastically influenced by the presence of a mutation, as shown by immunoblotting and immunofluorescence (Supplementary Fig. S2A). When grown on a standard substrate, PSC showed a higher adhesion compared to Ehlers– Danlos fibroblasts and HDF (Supplementary Fig. S2B, left panel). PSCs and HDF showed a significantly higher proliferation (p < 0.001) and migration (p < 0.01) compared to Ehlers–Danlos fibroblasts, indi-

cating an important role of intact Col V in supporting these properties of stromal cells (Supplementary Fig. S2C and D, left panels). A Col V-enriched matrix significantly (p < 0.001) increased the adhesion of all three types of stromal cells (Supplementary Fig. S2B, central and right panels); in addition, it significantly promoted the proliferation (F011: p = 0.027; F057: p = 0.005; PSC: p = 0.036) and the migration (F011: p = 0.009; F057: p = 0.046; PSC: p = 0.022) of Ehlers– Danlos fibroblasts and PSC, but not of HDF (Supplementary Fig. S2C and D, central and right panels). However, the addition of

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Fig. 2. Expression of β1-integrin in pancreatic tissues and cell lines. (A) β1-integrin shows a membranous basolateral staining pattern in low-grade precursor lesions, whereas high-grade PanIN displays a membranous and cytoplasmic expression. Stromal cells around precursor lesions do not express β1-integrin. Scale bar: 50 μm. (B) In PDAC tissues, a restoration of membranous β1-integrin expression is observed. Stromal cells are also positive. Staining for Col V on consecutive sections is detected around β1-integrin positive tumor cells, indicating a potential interaction between the two proteins. (C) Expression of β1-integrin in PSC and pancreatic cancer cell lines is verified via immunoblotting. Arrow indicates the correct protein size. Scale bar: 50 μm. (D) A Col V-rich matrix leads to an increased expression of β1-integrin in the SU.86.86 cell line. Arrow indicates the correct protein size.

exogenous Col V could not completely rescue the effects of a mutation of endogenous Col V, since PSC still displayed significantly higher adhesion (p < 0.001), proliferation (p < 0.001) and migration (p < 0.001) and HDF significantly higher proliferation (p < 0.001) and migration (F011: p = 0.028; F057: p = 0.017) than Ehlers– Danlos fibroblasts (Supplementary Fig. S2B–D, central panels). To further assess the impact of endogenous Col V in PSC, transient siRNA knock-down of Col V was performed in these cells, resulting in a significant reduction of adhesion (p < 0.05), proliferation (p < 0.05) and migration (p < 0.01) of PSC compared to the controls (Supplementary Fig. S2E). Altogether, these data show that an intact endogenous Col V is relevant for adhesion, migration and proliferation of stromal cells and that exogenous Col V can further promote these properties, suggesting the possibility of autocrine loops involving PSCs in the stroma of PDAC. Col V activates the β1-integrin-signaling pathway α2β1-integrin is the major receptor for Col V [22] and mediates cell adhesion to Col V [24]. To check if the interaction between Col

V and α2β1-integrin is relevant in pancreatic tissues, the expression and localization of β1-integrin in precursor lesions (Fig. 2A) and PDAC (Fig. 2B) were analyzed by immunohistochemistry. Lowgrade precursor lesions showed membranous basolateral staining of β1-integrin, whereas in high-grade lesions the distribution of β1integrin appeared more disheveled, membranous and cytoplasmic. Stromal cells around PanIN lesions showed no expression of β1integrin. In PDAC the localization of β1-integrin was membranous in tumor cells and was also present in stromal cells. In consecutive sections of PDAC, Col V staining was observed surrounding β1integrin positive tumor cells, indicating a co-localization and thereby a potential interaction of both molecules during cancer progression (Fig. 2B). The expression of β1-integrin in PSC and in four pancreatic cancer cell lines of different sites of origin (primary tumor vs metastatic site) and degrees of differentiation was validated by immunoblotting (Fig. 2C). Next, we checked if Col V affects the expression of β1-integrin in the tumor cells. The only cell line that showed an up-regulation of β1-integrin when growing on a Col V substrate was SU.86.86 (Fig. 2D), which was already found to express Col V in a small amount. Further, to determine whether ECM receptors on pancreatic cancer cells mediate an ‘outside-in’ signal upon

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Fig. 3. Downstream signaling of Col V and its influence on pancreatic cancer cells in vitro. (A) Activation of FAK and PAX is found in AsPC-1, Capan-1 and SU.86.86, but not in PANC-1 grown on Col V-rich matrix via immunoblot analysis. (B) Col V induces morphological changes on pancreatic cancer cell lines with appearance of lamellipodia (arrows) 3 hrs after plating. (C) Immunofluorescence staining shows strong expression of pFAK and pPAX on the migration edges 3 hrs after plating the cells on Col V. Phosphorylation of FAK and PAX persisted over 24 hrs. Under the same conditions, vinculin is distributed evenly throughout the whole cell, thus indicating a specific activation of the β1-integrin pathway.

binding of Col V, we investigated the phosphorylation of two adaptor proteins of β1-integrin, paxillin (PAX) and focal adhesion kinase (FAK), as well as their intracellular localization in pancreatic cancer cells grown on Col V. FAK and PAX were found to be activated in AsPC1, Capan-1 and SU.86.86, but not in PANC-1 grown on Col V-rich matrix (Fig. 3A). Moreover, 3 hrs after plating, pancreatic cancer

cells displayed a change in morphology and showed lamellipodia (Fig. 3B) with strong expression of pFAK and pPAX (Fig. 3C). Phosphorylation of FAK and PAX persisted over 24 hrs. Under the same conditions, vinculin was distributed evenly throughout the whole cell (Fig. 3C), thus indicating a specific activation of the β1-integrin pathway.

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Fig. 4. Col V affects adhesion, migration, proliferation and viability of pancreatic cancer cells. (A) A Col V-rich matrix significantly increases the adhesion of AsPC-1, Capan-1 and SU 86.86 cell lines after 3 hrs. (B) A Col V-rich matrix significantly increases the migration of pancreatic cancer cell lines Capan-1 and SU.86.86 according to the results of the wound healing assay. (C) Proliferation of pancreatic cancer cell lines under the influence of Col V as growing substrate was found to be significantly increased in the AsPC-1, Capan-1 and SU.86.86 cell lines, according to the results of the MTT assay. (D) Viability rates of AsPC-1, Capan-1 and SU.86.86 cell lines grown on Col V after treatment with 5-FU (1 mM) and Gem (10 μM) were higher than those of control cells grown on BSA-coated plates, according to the results of the MTT assay. For all assays the results are presented as mean of three independent replicates and shown as relative values compared to controls. * p < 0.05.

Col V affects important biologic properties of pancreatic cancer cells Adhesion of pancreatic cancer cell lines to Col V-coated plates was significantly increased in three cell lines compared to control plates (AsPC-1, SU.86.86: p < 0.01; Capan-1: p = 0.035) (Fig. 4A). We next assessed the migratory activity of pancreatic cancer cells cultured on Col V coated plates (Fig. 4B). In accordance with the adhesion promoting effect of Col V, cell migration was enhanced by Col V for Capan-1 (p < 0.05) and SU.86.86 (p < 0.01) while no effect was seen for PANC-1 (Fig. 4A). AsPC-1 were excluded from this analysis since they do not build monolayers. Col V was found to influence

the proliferation of pancreatic cancer cells with statistically significant effects in AsPC-1 (p < 0.01), SU.86.86 (p < 0.001) and Capan-1 (p = 0.024) cell lines (Fig. 4C). Moreover, we found that Col V promoted the viability of cancer cells, and that this effect was present even upon treatment with two independent chemotherapeutics (1 mM 5-FU and 10 μM Gem). In both cases, cells cultured on Col V coated plates exhibited significantly increased survival rates compared to the respective controls (Fig. 4D). These differences were significant for AsPC-1 treated with Gem (p < 0.05) and for Capan-1 (5-FU: p < 0.05, Gem: p < 0.001) and SU.86.86 (5-FU: p < 0.05, Gem: p < 0.05) treated with both drugs.

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These findings indicate that Col V promotes the malignant phenotype of pancreatic cancer cells. Inhibition of the β1-integrin signaling pathway reduces Col V-induced adhesion and migration of pancreatic cancer cells To check if the effects of Col V on pancreatic cancer cell adhesion, migration, viability and proliferation were mediated through the integrin pathway, the above described experiments were repeated after treating cells with either AZD (an inhibitor of the Src kinase which subsequent blocks the phosphorylation of FAK) or P5D2 (a blocking antibody against β1-integrin which prevents the binding of Col V to its receptor). Treatment with both inhibitors reduced the phosphorylation of FAK and PAX (Supplementary Fig. S3A). As shown in Supplementary Fig. S3B–D, the reported effects were abolished by blocking the β1-integrin signaling pathway. In detail, administration of AZD as well as of P5D2 reduced the adhesion of pancreatic cancer cell lines to Col V coated plates for AsPC-1 (AZD: p < 0.001, P5D2: p = 0.046), Capan-1 (AZD: p = 0.019, P5D2: p < 0.001) and SU.86.86 (AZD: p < 0.01). A significant reduction in migration was seen for Capan-1 (AZD: p = 0.029, P5D2: p = 0.024) and SU.86.86 (AZD: p = 0.018, P5D2: p < 0.01) when grown on Col V. The effects on proliferation were diminished by the two inhibitors in AsPC-1 (AZD: p < 0.001, P5D2: p < 0.01) and Capan-1 (AZD: p < 0.001, P5D2: p = 0.025). The effects on viability of pancreatic cancer cell lines grown on Col V and treated with 5-FU were reversed by AZD (AsPC-1: p < 0.001, Capan-1: p < 0.001 and SU.86.86: p < 0.05) and P5D2 (AsPC-1: p = 0.026, Capan-1: p = 0.034 and SU.86.86: p < 0.001) (Supplementary Fig. S4, upper panels). Similarly, the effects on viability of cancer cell lines treated with gemcitabine were reversed in both approaches (5-FU: AsPC-1 p < 0.01, Capan-1 p < 0.001, SU.86.86: p < 0.001; P5D2: AsPC-1 p = 0.023, Capan-1 p < 0.01, SU.86.86: p < 0.001) (Supplementary Fig. S4, lower panels). Interactions between pancreatic stellate cells, cancer cells and endothelial cells To test the effects of Col V expression by PSC in the interaction with pancreatic cancer cells, transient knock-down of Col V in PSC was performed (Supplementary Fig. S5). PSCs were more spindle than stellate shaped after siRNA treatment, whilst the transdifferentiation to their myofibroblastic phenotype was not influenced (Fig. 5A and B). Moreover, pancreatic cancer cells treated with a conditioned medium obtained from PSCs after siRNA mediated knock-down of Col V displayed reduced invasive capacity and proliferation (Fig. 5C and D) in the Capan-1 and AsPC-1 cell lines, respectively. Interestingly, secretion of Col V by PSC was strongly increased upon treatment with the conditioned medium of pancreatic cancer cells, while the intracellular expression level was not influenced (Fig. 5E), indicating a paracrine effect of pancreatic cancer cells to modify the microenvironment. To check if angiogenesis was

affected by Col V, the expression of Col V and CD34 in the same tissue sections were quantified using software-based morphometric analysis, but no correlation was found (Supplementary Fig. S6A). However, HUVEC cells treated with the conditioned medium of PSCs after siRNA-mediated knock-down of Col V displayed reduced tube formation, whereas treatment with pancreatic cancer cell conditioned medium did not show any effects (Fig. 5F). Further, HUVEC transfected with siRNA against Col V had impaired abilities to form tubes, pointing to an important role of Col V in angiogenesis (Supplementary Fig. S6B). To investigate the effects of PSCs on tube formation, expression of different pro- and anti-angiogenic factors was checked. Here, we could show that HUVEC indirectly co-cultured with PSCs displayed a reduced expression of antiangiogenic factors, such as angiostatin, thrombospondin-1 and thrombospondin-2 (Supplementary Fig. S6C), while upon coculture with PSCs after Col V knock-down, a slight increase in VEGF expression was found (Supplementary Fig. S6D). siRNA treatment resulted in a reduced expression of angiogenic factors by PSCs themselves. On the other hand, co-culture of PSCs with HUVEC cells did not change the expression of angiogenic factors in PSC, with the exception for angiostatin, which was down-regulated (Supplementary Fig. S6E).

PSC-derived Col V promotes hepatic metastasis of pancreatic tumor cells To test the effects of PSC-derived Col V on tumor growth and metastasis in vivo, stable knock-down of Col V in PSCs was achieved by lentiviral transduction employing shRNA technology. In order to exclude off-target effects, two different constructs (shCol5a1#10, shCol5a1#11) were used. This resulted in a knock-down of 88% (shCol5a1#10) or 75% (shCol5a1#11) of Col V in PSCs on mRNA level as determined by qRT-PCR (Supplementary Fig. S7A) and of 29% or 61%, respectively on protein level in the PSCs (Supplementary Fig. S7B) as determined by immunoblotting. BALB/c nude mice received orthotopic injection of pancreatic cancer cells and PSCs and developed palpable tumors after six weeks. In order to allow detection of systemic metastasis at high resolution, we employed lacZtagging of cancer cells, as described previously [35]. Tumor growth and metastasis formation were increased in the presence of PSCs (Fig. 6A and B), thus confirming previously reported results [41]. Knock-down of Col V in PSCs did not affect primary tumor growth (Fig. 6A), although the knock-down of Col V was still detectable with 40.6% and 75.9% in the tumor tissue of the primary tumors (Supplementary Fig. S7C). However, metastasis formation to the liver was reduced (Fig. 6B). Neoangiogenesis, assessed by morphometric quantification of CD31 staining, revealed a lower vessel density in the tumors developed after Col V knock-down (Fig. 6C and D). Taken together, these data indicate that PSC-derived Col V influences formation of hepatic metastasis by pancreatic cancer cells at least partially by affecting angiogenesis in vivo.

Fig. 5. Col V influences the interaction of cancer cells with PSCs. (A) PSCs after transient knock-down of Col V (siCOL5) display a more spindle shaped than stellate shaped morphology compared to PSCs treated with scrambled siRNA (ctrl), as shown in the immunofluorescence picture (left) and in the bright field picture (right). Red fluorescence: αSMA. Green fluorescence: Col V. (B) The transdifferentiation status of PSCs after transient knock-down of Col V remains unchanged, as shown by the unmodified expression of αSMA. (C) Capan-1 cells show reduced invasive capacity in invasion assays performed using conditioned medium of transfected PSC as chemoattractant. Results are presented after normalization to the results obtained using the conditioned medium of untreated cells (mean of three independent replicates). (D) AsPC-1 cells show reduced proliferation in MTT assays performed using the conditioned medium of transfected PSCs. Results are presented after normalization to the results obtained using the conditioned medium of untreated cells (mean of three independent replicates). (E) Immunoblotting of Col V in the supernatant (SN) and cell lysate (CL) of PSCs after treatment with conditioned medium of pancreatic cancer cell lines. While the expression of Col V in the PSCs is not influenced, secretion of Col V is increased after treatment with the conditioned medium of cancer cells. (F) Tube-formation assay in HUVEC treated with standard medium of HUVEC (HUVEC-SM), with conditioned medium of PSCs before (PSC-CM) and after Col V knock-down (PSC-CMsiCol5) and of cancer cells (AsPC-1/Capan-1/SU.86.86-CM). HUVEC treated with the conditioned medium of PSC show well-formed vascular tubes, comparable to those obtained by HUVEC standard medium (HUVEC-SM). Exposure to conditioned medium of PSCs after Col V knockdown leads to poor tube formation. No tube formation is observed by exposure to the cancer cell conditioned medium. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Col V affects metastasis formation and angiogenesis in an orthotopic mouse model. (A) Primary tumor growth is increased in the presence of PSC, but no significant change in the tumor volume is observed using PSCs after stable Col V knock-down. (B) Liver metastasis formation is increased in the presence of PSC and stable knockdown of Col V in PSC affects metastasis formation in the liver, leading to a reduction in the number of metastatic foci. (C) Immunostaining for CD31 highlights the higher vascular density of tumors generated by injection of cancer cells and untreated PSC compared to tumors obtained after stable knock-down of Col V in PSC. (D) Morphometric quantification of the CD31 staining in tumor tissues discloses a lower vessel density in the tumors obtained using PSC after stable Col V knock-down.

Discussion The stromal reaction is important during PDAC progression and targeting the stroma represents one of the most recently developed therapeutic approaches against this tumor type [42], which might allow conventional therapeutics to reach cancer cells more easily, possibly also affecting intratumoral neoangiogenesis [13]. However, recent data suggest a possible protective role of the stromal reaction against the growth of malignant cells, indicating the existence of heterogeneous patterns of epithelial–stromal interactions in malignancies [15,16]. Collagens are the major components of the stromal reaction [2]. Col V has been recently described to be overexpressed in the microenvironment of PDAC [30] and it was found to be associated with tumor progression in the present study. In particular, in addition to an increase in the expression, a shift in the localization from a basement membrane-like pattern in the precursor lesions to a diffuse distribution in the stroma of invasive cancer

was observed. Expression of Col V by PSC, as described by Wehr et al. [30], could be verified here by double immunohistochemical staining of αSMA and Col V in PDAC tissues, as well as in freshly isolated PSCs by immunofluorescence. Col V expression in pancreatic cancer cells was either not present or weak, indicating PSCs as the main source of Col V in PDAC. Since the stromal response to a growing neoplasm in a specific organ is still probably less heterogeneous than the cancer cell component, the selective expression of Col V in the stroma of PDAC and of its precursor lesions could be used as a specific target for imaging of early PDAC [43]. Detailed understanding of the relevance of endogenous Col V in PSC was obtained investigating fibroblasts isolated from patients affected by the classical type of Ehlers–Danlos syndrome due to a Col V mutation. The higher rates of adhesion, migration and proliferation of PSCs and normal skin fibroblasts grown on a standard (i.e. not enriched with Col V) substrate compared to the Ehlers– Danlos fibroblasts, as well as the higher adhesion, proliferation and

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migration of PSCs on a Col V-rich matrix, underscore the functional relevance of this collagen type in stromal cells and support the concept of autocrine mechanisms involving Col V and promoting relevant functional properties of PSC. The significantly reduced adhesion, proliferation and migration of PSC after siRNA-mediated knock-down of Col V further confirm the relevance of this protein in stromal cell biology. Cancer cell signaling of Col V is mediated through α2β1-integrin [44] and its downstream targets FAK and PAX [24]. The receptor β1integrin has been previously described to play a role in adhesion and invasion of pancreatic cancer cells, as different cell lines show different invasive potential depending on the different constitutive activity of β1-integrin [45]. In the present study, a co-localization of β1-integrin and Col V in the same tissue areas was observed in PDAC and its precursor lesions by immunohistochemistry. Interestingly, β1-integrin expression changed from a membranous, basolateral staining in low-grade PanIN to a diffuse cytoplasmic staining in high-grade PanIN, possibly indicating a change in anchorage of epithelial cells to the basement membrane. In PDAC tissues, both cancer and stromal cells showed a restored membranous expression pattern of β1-integrin, arguing for a direct contact of the cancer cells to the PSCs after invasion through the basement membrane. This contact represents the basis for the following activation of the downstream signaling of Col V and for its effects on adhesion, proliferation, migration of pancreatic cancer cells and on tumor cell viability after treatment with chemotherapeutic drugs. In order to generate a comprehensive picture of the potential effects of Col V in PDAC progression, several pancreatic cancer cell lines with different genetic backgrounds were used for the in vitro experiments. The obtained data were mostly comparable, but not completely homogeneous among the used cell lines. This further underscores the known problem of tumor heterogeneity, which should be taken into account for the in vivo translation of data generated in vitro. For instance, PANC-1 cells seem to be quite resistant to the effects of exogenous Col V on their adhesion, proliferation, migration and viability upon treatment with chemotherapeutics. On the other hand, SU.86.86 is the only pancreatic cancer cell line among those tested in this study showing some levels of endogenous Col V expression and an up-regulation of β1-integrin when grown on a Col V-enriched matrix, possibly indicating a synergism of endogenous and exogenous Col V in achieving this effect. In addition, the presence of endogenous Col V in SU.86.86 can explain the less prominent effects of conditioned medium obtained from Col V-ablated PSCs on this cell line. The specificity of the effects of exogenous Col V on the pancreatic cancer cell lines was shown by blocking the β1-integrin receptor or its downstream molecule Src-kinase. These results underscore the biological relevance of the β1-integrin pathway in PDAC and support its pharmacological abrogation as a promising therapeutic target, as it has been previously suggested [46]. Due to its role as β1-integrin binding partner, Col V can exert pleiotropic effects within the tumor microenvironment, promoting cell proliferation, migration and survival via the Src/FAK/PAX signaling pathway [47]. We suggest that the Col V-FAK axis shapes the PDAC microenvironment in a tumor-promoting manner, substantiating the role of the tumor–stroma interaction in PDAC progression. The crosstalk between pancreatic cancer cells and PSCs is another field of investigation, which could be important for future treatment options in PDAC. Moderately differentiated pancreatic cancer cells showed a decrease in proliferation and invasion when treated with the supernatant of PSCs after siRNA-mediated knock-down of Col V. On the other side, PSCs treated with the supernatant of pancreatic cancer cells displayed a higher secretion of Col V, suggesting paracrine interactions between PSCs and pancreatic cancer cells. The in vitro effects of Col V on angiogenesis were confirmed by the in vivo experiments, since tumors orthotopically implanted after the

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knock-down of Col V in PSC showed a lower vessel density and a reduced number of liver metastases than tumors growing with unchanged expression of Col V. Since higher microvessel density is a known prerequisite for a higher metastatic potential of cancer [48], these data point to a pivotal role of Col V in affecting the metastatic process in PDAC in vivo. In conclusion, these results show for the first time that Col V plays an important role in PDAC progression. Col V progressively accumulates in the stroma of precursor lesions and – via activation of FAK signaling – affects relevant aspects of the malignant phenotype of the cancer cells, such as their adhesion to the surrounding matrix, their viability, proliferation and migration, until the formation of metastases. These data offer the rationale for targeting Col V or its downstream β1-integrin-mediated signaling pathway for diagnostic purposes (e.g. early detection, monitoring of metastatic disease), and as a possible new option in the context of multimodal therapy for PDAC. Authors’ contributions Conception and design: S. Berchtold, I. Esposito. Development of methodology: S. Berchtold, A. Reithmeier, B. Grünwald, A. Krüger, C. Tao, I. Esposito. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Erkan, J. Kleeff, I. Esposito. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Berchtold, A. Reithmeier, B. Grünwald, A. Krüger, I. Esposito. Writing, review, and/or revision of the manuscript: S. Berchtold, A. Krüger, M. Erkan, J. Kleeff, I. Esposito. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Erkan, J. Kleeff, I. Esposito. Study supervision: I. Esposito. Acknowledgements This study was supported by the EU-FP7 (project no. 256974) “Translational research on cancers with poor prognosis. EPC-TMNet (European Pancreatic Cancer-Tumour-Microenvironment Network)” and by the EU-FP7 Project NMP-2010-263307/SaveMe. We thank the Cell Line and DNA Biobank from Patients affected by Genetic Diseases (Istituto G. Gaslini), member of the Telethon Network of Genetic Biobanks (project no. GTB12001), for providing the F011 and F057 cell lines. We would like to thank Katrin Lindner for excellent technical assistance and Katja Steiger and Anna Melissa Schlitter for critically reviewing the manuscript. Conflict of interest The authors state no conflict of interests. Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2014.10.020. References [1] R. Siegel, D. Naishadham, A. Jemal, Cancer statistics, 2012, CA Cancer J. Clin. 62 (2012) 10–29. [2] M.V. Apte, S. Park, P.A. Phillips, N. Santucci, D. Goldstein, R.K. Kumar, et al., Desmoplastic reaction in pancreatic cancer: role of pancreatic stellate cells, Pancreas 29 (2004) 179–187.

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