Oncolytic adenovirus expressing relaxin (YDC002) enhances therapeutic efficacy of gemcitabine against pancreatic cancer

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Cancer Letters xxx (2017) 1e12

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Original Article

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Oncolytic adenovirus expressing relaxin (YDC002) enhances therapeutic efficacy of gemcitabine against pancreatic cancer Kyung Hee Jung a, 1, Il-Kyu Choi b, 1, Hee-Seung Lee a, Hong Hua Yan a, Mi Kwon Son a, Hyo Min Ahn b, JinWoo Hong b, Chae-Ok Yun b, **, Soon-Sun Hong a, * a b

Department of Biomedical Sciences, College of Medicine, Inha University, 3-ga, Sinheung-dong, Jung-gu, Incheon, 400-712, Republic of Korea Department of Bioengineering, College of Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, 133-791, Seoul, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 February 2017 Received in revised form 7 March 2017 Accepted 7 March 2017

Pancreatic cancer is a highly lethal disease for which limited therapeutic options are available. Pancreatic cancer exhibits a pronounced collagen-rich stromal reaction, which induces chemoresistance by inhibiting drug diffusion into the tumor. Complementary treatment with oncolytic virus such as an oncolytic adenovirus expressing relaxin (YDC002) is an innovative treatment option for combating chemoresistant pancreatic cancer. Here, we examined the ability of combined treatment with gemcitabine and YDC002, which degrades extracellular matrix (ECM), to efficiently treat chemoresistant and desmoplastic pancreatic cancer. Gemcitabine alone exhibited similarly low cytotoxicity toward pancreatic cancer cells throughout the concentration range (1e50 mM) used, whereas the combination of YDC002 and a subtherapeutic dose of gemcitabine (0.01e0.05 mM) resulted in potent anticancer effects through effective induction of apoptosis. Importantly, YDC002 combined with gemcitabine significantly attenuated the expression of major ECM components including collagens, fibronectin, and elastin in tumor spheroids and xenograft tumors compared with gemcitabine alone, resulting in potent induction of apoptosis, gemcitabine-mediated cytotoxicity, and an oncolytic effect through degradation of tumor ECM. Our results demonstrate that YDC002 can selectively degrade aberrant ECM and attenuate the ECM-induced chemoresistance observed in desmoplastic pancreatic tumor, resulting in a potent antitumor effect through effective induction of apoptosis. © 2017 Elsevier B.V. All rights reserved.

Keywords: Adenovirus Relaxin Gemcitabine Extracellular matrix Pancreatic cancer

Introduction Pancreatic cancer, one of the most aggressive types of cancer, has the highest mortality rate. Because the pancreas is located in a deep retroperitoneal site and no specific symptoms are observable at early stages of pancreatic cancer, diagnosis at a surgically resectable stage is uncommon [1,2]. In advanced stages of pancreatic cancer, the only treatment option available is chemotherapy, which often combines gemcitabine with other chemotherapeutics [3,4]. However, these chemotherapeutics are highly toxic and lack therapeutic efficacy. Specifically, clinical beneficial response to gemcitabine are observed in approximately 25% of cases, but even this limited therapeutic efficacy rapidly declines owing to

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* Corresponding author. Fax: þ82 32 890 2462. ** Corresponding author. Fax: þ82 2 2220 4850. E-mail addresses: [email protected] (C.-O. Yun), (S.-S. Hong). 1 These authors have contributed equally to this work.

[email protected]

long-term tolerance, resulting in a median overall survival of 6 months [5,6]. Therefore, a novel strategy is needed to improve the therapeutic efficacy of gemcitabine for the treatment of pancreatic cancer. Oncolytic adenovirus (Ads), which selectively replicate in cancer cells, are a promising alternative to conventional therapy for cancer treatment and have been extensively examined in clinical trials [7]. A major advantage of oncolytic Ads is their excellent safety profile, demonstrated in multiple studies on patients with ovarian cancer, melanoma, soft tissue or primary bone sarcoma, and other neoplasms [8e10]. However, the antitumor efficacy of oncolytic Ads is often insufficient due to variable expression levels of the coxsackievirus-adenovirus receptor (CAR) on tumor cells [11]. Although a number of studies have sought to improve and enhance entry of oncolytic Ads into tumor cells by switching the Ad5 fiber knob [12,13], the inability of the virus to effectively spread throughout a large tumor remains a major problem [14]. Connective tissue and extracellular matrix (ECM) components play a prominent role in inhibiting viral spread following

http://dx.doi.org/10.1016/j.canlet.2017.03.009 0304-3835/© 2017 Elsevier B.V. All rights reserved.

Please cite this article in press as: K.H. Jung, et al., Oncolytic adenovirus expressing relaxin (YDC002) enhances therapeutic efficacy of gemcitabine against pancreatic cancer, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.03.009

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administration of Ads [14]. Indeed, degradation of ECM by proteins such as relaxin and decorin enhances viral distribution within a tumor [15e17]. Furthermore, ECM components are abundantly expressed in pancreatic cancer, and desmoplastic reaction has been proposed as a substantial barrier that prevents efficient diffusion of chemotherapeutic agents, leading to poor disease management [18]. Notably, resistance to gemcitabine is associated with ECMmediated signaling in pancreatic cancer cells [19]. For these reasons, degradation of ECM components may be an important strategy for effective treatment of pancreatic cancer, which possesses a dense collagen-rich and fibrotic tumor microenvironment. Relaxin, a peptide hormone that softens the uterine cervix, vagina, and interpubic ligaments in preparation for parturition, has been used to degrade and suppress the expression of tumor ECM proteins [20]. Specifically, relaxin attenuates the synthesis of interstitial collagens and upregulates collagenase expression. In a mouse tumor model, chronic administration of relaxin was shown to induce collagen degradation and increase intratumoral diffusion of macromolecules [21]. In addition, Brown et al. have reported that chronic relaxin treatment significantly increases diffusive transport of immunoglobulin G and dextran molecules into solid tumors [21]. We have previously demonstrated that relaxin-expressing oncolytic Ads induce greater viral spread and promote viral persistence within solid tumor through degradation of tumor ECM [22]. Given the important ECM-degrading role of relaxin, we hypothesized that combining a relaxin-expressing oncolytic Ad (YDC002) with the conventional chemotherapeutic agent gemcitabine would enhance the efficacy of gemcitabine against pancreatic cancer. In this study, we observed extensive desmoplasia in pancreatic tumor spheroids and xenograft tumors. We further explored the combination of gemcitabine with YDC002 as a strategy for improving the anticancer effect of both therapeutic agents through remodeling the ECM-rich tumor microenvironment of pancreatic cancer. Our findings demonstrated that YDC002 induces ECM degradation in the tumor, thereby facilitating the intratumoral distribution of gemcitabine and the virus, leading to a potent antitumor effect against highly aggressive and desmoplastic pancreatic cancer. Materials and methods Generation of relaxin-expressing Ad The oncolytic Ad, YDC002, was propagated and purified as described in our previous studies [22e25]. All viruses were propagated in HEK293 cells and purified by CsCl density purification, dissolved in storage buffer (10 mM/L Tris, 4% sucrose, 2 mM/L MgCl2), and stored at 80  C. Viral particle numbers were calculated from measurements of absorbance at 260 nm (A260), where one absorbance unit is equivalent to 1012 viral particles (VP)/mL, and infectious titers [plaque-forming unit (PFU)/mL] were determined by limiting dilution assays performed in HEK293 cells; the multiplicity of infection (MOI) was calculated from infectious titers [26]. Cells, materials, and human samples The human pancreatic cancer cell lines (PANC-1, MIAPaCa-2, and BxPC-3) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). PANC-1 and MIAPaCa-2 cells were grown in Dulbecco's modified Eagle's medium (DMEM), and BxPC-3 cells were grown in Roswell Park Memorial Institute-1640 (RPMI-1640) medium. All media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cell cultures were maintained at 37  C in a CO2 incubator with a controlled humidified atmosphere composed of 95% air and 5% CO2. Gemcitabine [4-amino-1-(2-deoxy-2,2-difluoro-b-D-erythro-pentofuranosyl) pyrimidin2(1H)-on] was purchased from LTK Corporation (Wilmington, DE). PDAC tumor samples were obtained from formalin-fixed and paraffin-embedded tissue blocks collected for clinical purposes at Yonsei University Hospital. The PDAC diagnosis, histologic subtype, and TNM staging were determined by a board-certified staff pathologist at Yonsei University Hospital (Supplementary Table 1).

and YDC002 alone or together for 72 h; phosphate-buffered saline (PBS) was used as a control. Sensitivity to gemcitabine was assessed by determining cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays as described previously [27]. Briefly, cells were treated with gemcitabine (0.01, 0.05, and 0.1 mM) and/or YDC002 (0.1, 0.5, 1, and 2 MOI) for 72 h, after which 200 mL of MTT (Sigma, St. Louis, MO) in PBS (2 mg/mL) was added to each well. After incubating for 4 h at 37  C, the supernatant in each well was discarded, and the precipitate was dissolved in dimethyl sulfoxide (DMSO). The absorbance of each well was then analyzed at 540 nm using a microplate reader. All assays were performed in triplicate. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay MIAPaCa-2 and PANC-1 cells were first plated onto chamber slides at a density of 5  104 cells per chamber. At 24 h post-incubation, cells were infected with YDC002 at 0.5 or 1 MOI, and incubated at 37  C for 48 h. Apoptotic cells were identified by detecting cleaved DNA using an In Situ ApopTag kit (Chemicon International, Temecula, CA) according to the manufacturer's instructions. Apoptotic cells were visually identified in 10 randomly selected fields and photographed at a magnification of 400. More than 2000 cells were counted to calculate the percentage of TUNEL-positive cells (apoptotic cell ratio). Apoptosis in vivo was assessed by detecting apoptotic cells in formalin-fixed tissue sections prepared and processed as described previously [28]. Tumor sections (8 mm thick) were treated with proteinase K (20 mg/mL) for 15 min, and endogenous peroxidase was blocked by incubating with 3% hydrogen peroxide in PBS for 10 min. Samples were washed three times in distilled water and incubated for 10 min at room temperature with terminal deoxynucleotidyl transferase buffer. Excess buffer was then drained, and samples were incubated for 1 h at 37  C with terminal transferase and biotin-16-dUTP. Samples were then rinsed four times with PBS and incubated for 1 h at 37  C with a 1:400 dilution of peroxidase-conjugated streptavidin. Slides were rinsed with PBS and incubated for 5 min with 3, 3’-diaminobenzidine (DAB). Sections were then washed three times with PBS, and counterstained with methyl green. Quantitative reverse transcription-polymerase chain reaction (PCR) Total RNA was extracted from pancreatic cancer cells using Trizol reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer's instructions. RNA in samples was quantified by spectrophotometry, and RNA integrity was assessed using agarose gel electrophoresis and ethidium bromide staining. RNA samples were then diluted in RNase-free water and stored at 70  C until use. cDNA was synthesized from total RNA (5 mg) by reverse transcription using an RNA PCR kit (Takara Bio Inc., Otsu, Japan) in accordance with the manufacturer's instructions. Collagen I and collagen III mRNAs were detected using oligonucleotide primers and TaqMan probes obtained from Applied Biosystems (PerkineElmer/PE Applied Biosystems, Foster City, CA) and purchased in a ready-for-use form as Assays-on-Demand Gene Expression Products; 18S was used as an internal control. The TaqMan probes were labeled at the 50 end with the reporter dye FAM and at the 30 end with a minor groove binder and nonfluorescent quencher. Quantitative reverse transcriptionpolymerase chain reaction (RT-qPCR) was performed in triplicate for each sample using a Step One Plus Real-Time System (Applied Biosystems, Foster City, CA). Each 20 mL reaction contained 10 mL of TaqMan Fast Universal Master Mix (Applied Biosystems, Darmstadt, Germany), 1 mL of Gene Expression Mix and 2 mL of cDNA diluted in 7 mL of RNase-free water. Thermal cycling conditions were 20 s at 95  C, followed by 40 cycles of 5 s at 95  C and 20 s at 60  C. Fold changes in target gene mRNA relative to the endogenous 18S control were calculated as described previously [29]. Assessment of therapeutic effects by combination of YDCOO2 and gemcitabine in pancreatic tumor spheroids A MIAPaCa-2 xenograft tumor model was established by subcutaneously injecting 5  105 cells into the abdomen of 6-week-old male nude mice (Orient-Bio, Korea). After tumors reached a volume of ~150e200 mm3 (3e4 weeks), tumor tissue was surgically excised, blood and necrotic tissue were removed from the tissue, and tumor fragments (1e2 mm in diameter) were dissected with sterile 21-gauge needles. These explants were plated individually on 1.45% agarose-coated 24-well plates and cultured in DMEM supplemented with 10% FBS. Cultures received fresh medium every 2 or 3 days for 2 weeks. Only explants that became spherical (i.e., spheroids) with a diameter of approximately 2-mm were used for subsequent experiments. For infection of tumor spheroids with oncolytic Ads, 100 mL of culture medium was removed from each tumor spheroid culture dish by aspiration, and 50 mL of YDC002 (1  109 VP) was added. The next day, spheroids were treated with gemcitabine (20 mM), and 5 days later, each tumor spheroid was assessed by immunohistochemistry and Masson's trichrome (MT) staining. Each experiment was carried out three to four times with three replicates per group, and the results were similar across all experiments.

Cell viability assay The cancer cell-killing effects of gemcitabine and YDC002 were assessed by seeding pancreatic cancer cells (MIAPaCa-2, BxPC-3, and PANC-1) onto 96-well plates at a density of 5  103 cells per well, and treating them with gemcitabine

Picrosirius red staining and image analysis Collagen in tumor spheroids was detected using picrosirius red staining and was semi-quantitatively evaluated by calculating the fibrotic area using an image

Please cite this article in press as: K.H. Jung, et al., Oncolytic adenovirus expressing relaxin (YDC002) enhances therapeutic efficacy of gemcitabine against pancreatic cancer, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.03.009

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Fig. 1. Gemcitabine resistance of pancreatic cancer cells and aberrant ECM accumulation in pancreatic cancer patient-derived tumor spheroids. A, The cancer cell-killing effects of gemcitabine in the pancreatic cancer cells (MIAPaCa-2, PANC-1, BxPC-3) were examined by MTT assay. Cells were seeded in 96-well culture plates, and treated with different concentrations of gemcitabine for 72 h. Data are presented as means ± SD of triplicate independent experiments. B, Aberrant ECM accumulation in pancreatic cancer patient. Patient-derived pancreatic tumor was stained with H&E and MT. Magnifications: 50, 100 and 400.

analysis system as described previously [30], with some modifications. Images of picrosirius red-stained sections, which showed fibrosis in red and the parenchyma in grey, were captured under 0 magnification. After applying an interactive thresholding method, the image was converted into a binary image. The

two-dimensional patterns were measured by directly counting pixels in the binary images. The total area was the sum of the area of microscopic fields, including parenchyma and fibrosis. For each slide, the area of fibrosis was evaluated in 20 consecutive high-power fields, and averaged.

Please cite this article in press as: K.H. Jung, et al., Oncolytic adenovirus expressing relaxin (YDC002) enhances therapeutic efficacy of gemcitabine against pancreatic cancer, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.03.009

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K.H. Jung et al. / Cancer Letters xxx (2017) 1e12 Tumor xenograft study All animal experiments were performed in accordance with the guidelines of the INHA Institutional Animal Care and Use Committee (INHA IACUC) at Inha University Medical School. Pancreatic tumor xenograft models were established by subcutaneously injecting 6-week-old male BALB/c nude mice (Orient Bio) with 3  105 MIAPaCa-2 or 5  105 BxPC-3 and PANC-1 cells. After tumors reached approximately 50e100 mm3 in size, mice were randomly divided into four groups (n ¼ 7/group): (1) YDC002 (100 mL, 2  106 PFU/mice, intratumoral injection); (2) gemcitabine (1.5 mg/kg, intraperitoneal injection); (3) combined YDC002 and gemcitabine, administered at the same dose and administration route used for single treatments; and (4) PBS control. For individual treatments, mice were treated every other day for a total of three treatments. For the combination of YDC002 and gemcitabine, YDC002 was treated on days 1, 3 and 5, and gemcitabine was delivered on days 2, 4, and 6. Tumor size was measured every 2 days, and tumor volume was calculated using the formula, 0.5  length  width2. Tumor tissues were harvested 60 days after the initial viral injection, fixed in 10% formalin, and embedded in paraffin (CellPath, Newton, UK). Immunohistochemistry Tissue sections were blocked with normal goat or horse serum (Vector Laboratories, Burlingame, CA) for 1 h, and then incubated at 4  C overnight with antibodies against collagen I, collagen III, collagen IV, fibronectin, elastin, or cleaved caspase-3 (Cell Signaling Technology, Beverly, MA), diluted 1:50. After washing, sections were incubated with biotinylated secondary antibodies (1:100) for 1 h. Immunoreactive proteins were detected by incubating sections with an avidinebiotin peroxidase complex solution ABC kit (Vector Laboratories). After washing with PBS, proteins were visualized by incubating sections with DAB for 15 min, followed by counterstaining with hematoxylin. At least three random fields of each section were examined at 400 magnification and analyzed using a computer image analysis system (Media Cybernetics. Rockville, MD). Statistical analysis Data are expressed as means ± standard deviation (SD). Statistical analyses were performed using analysis of variance (ANOVA) and unpaired Student's t-tests, as appropriate p-values < 0.05 were considered statistically significant.

Results Gemcitabine resistance of pancreatic cancer cell lines Sensitivity and resistance to gemcitabine are attributable to cellular events involved in the drug response that affect the intracellular availability and ability of gemcitabine and its ability to trigger apoptosis [31]. To analyze the sensitivity of three human pancreatic cancer cells (PANC-1, MIAPaCa-2, and BxPC-3) toward gemcitabine, we assessed cell viability following exposure to different concentrations (0.05e50 mM) of gemcitabine. As shown in Fig. 1A, treatment with 1 mM gemcitabine decreased cell viability by 43.4% in MIAPaCa-2 cells, 30.4% in BxPC-3 cells, and 28.6% in PANC1 cells. Exposure of cells to higher doses of gemcitabine (5e50 mM) had no additional effect beyond that observed at 1 mM, indicating that pancreatic cancer cells are refractory to gemcitabine treatment. Aberrant ECM accumulation in primary pancreatic tumors Pancreatic cancer is characterized histologically by an abundance of ECM, commonly referred to as desmoplasia [32]. Indeed, the accumulation of ECM components disrupts the normal architecture of pancreatic tissue, resulting in an abnormal configuration of blood and lymphatic vessel [32]. One factor that contributes to therapeutic resistance of pancreatic cancer is the rigidity of the ECM, which impedes the delivery of drugs to cancer cells.

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Therefore, we assessed the accumulation of ECM components in primary pancreatic tumors derived from three patients using H & E and MT staining. As shown in (Fig. 1B and Supplementary Fig. 1), collagen, a major ECM component, occupied large areas in tumor tissues of pancreatic cancer patients, confirming the desmoplasia described in previous reports. These results reinforce the idea that abundant ECM accumulation in pancreatic tumor may function as a physical barrier against perfusion of chemotherapeutics such as gemcitabine, and contribute to the chemoresistance of pancreatic cancer. Induction of apoptotic cell death and inhibition of collagen expression in pancreatic cancer cells by combination treatment with YDC002 and gemcitabine Our previous study reported that YDC002 induces apoptotic cell death in various types of cancer cells [22,33,34]. To determine whether YDC002 enhances the cancer cell-killing effect of gemcitabine, we analyzed the viability of pancreatic cancer cells by MTT assay following combined treatment with YDC002 and gemcitabine. Cells were treated with gemcitabine (0.01 or 0.05 mM) with or without YDC002 (1 or 2 MOI for MIAPaCa-2; 0.1 or 0.5 MOI for PANC-1). The combination of YDC002 with gemcitabine significantly inhibited cell growth compared with gemcitabine or YDC002 alone (Fig. 2A). Because the combined treatment significantly reduced cell viability, we next investigated the apoptotic effect of the combination treatment. Consistent with MTT assay results, the combination treatment with YDC002 and gemcitabine resulted in more apoptotic cell death compared with either treatment alone, suggesting that the potent cancer cell-killing effect of combination treatment was mediated by increased induction of apoptosis (Fig. 2B). Desmoplasia in pancreatic cancer is characterized by collagen-rich tumor and fibrillary collagen (collagen I and collagen III), which accelerates tumor progression and functions as a physical barrier that prevents drug diffusion, leading to abysmal therapeutic outcomes [35]. Therefore, we assessed whether combined treatment with YDC002 and gemcitabine is capable of overcoming ECM-induced chemoresistance by modulating major ECM components such as collagen I and III in pancreatic cancer. As shown in Fig. 2C, YDC002 significantly attenuated mRNA expression of collagen I and III in pancreatic cancer cells compared with control or treatment with gemcitabine alone. Importantly, the combination of YDC002 and gemcitabine potently reduced collagen expression levels, demonstrating that YDC002 can degrade aberrant tumor ECM via inhibiting collagen synthesis. ECM degradation and induction of apoptosis in tumor spheroids by combination treatment with YDC002 and gemcitabine To further assess the therapeutic effect of combination treatment, we utilized organotropic tumor spheroids cultured from xenograft tumor tissues, which closely emulate in vivo physiologic parameters, such as multicellular architecture, physical barriers to drug transport, and ECM composition. Abundant deposition of ECM components, closely resembling those shown in pancreatic cancer patient-derived tumors, was observed in pancreatic tumor spheroids, demonstrating that tumor spheroids closely emulate the desmoplasia of clinical pancreatic tumors (Fig. 3A). MT and

Fig. 2. Cancer cell killing effects, induction of apoptosis, and inhibition of collagen mRNA expression by the combination of gemcitabine and YDC002 in MIAPaCa-2 and PANC-1 pancreatic cancer cells. A, Cells were treated with gemcitabine and/or YDC002, and cell viability was determined by MTT assay. B, Induction of apoptosis by combination treatment was assessed by TUNEL assay following treatment of cells with gemcitabine and/or YDC002. C, Inhibition of collagen mRNA expression in pancreatic cancer cells by combined treatment with YDC002 and gemcitabine. MIAPaCa-2 and PANC-1 cells were treated with gemcitabine and/or YDC002 for 72 h; quantitative data are expressed as the fold change relative to untreated controls. Magnifications, 400. Data are represented as means ± SD of triplicates (*p < 0.05, **p < 0.01, and ***p < 0.001).

Please cite this article in press as: K.H. Jung, et al., Oncolytic adenovirus expressing relaxin (YDC002) enhances therapeutic efficacy of gemcitabine against pancreatic cancer, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.03.009

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picrosirius red staining revealed that YDC002 or combination treatment induced a significant decrease of ECM components compared with controls or gemcitabine treatment alone. Combination treatment induced significantly greater tumor cell apoptosis than either gemcitabine or YDC002 single treatment, suggesting that the decrease of ECM induced by YDC002 can enhance the chemotherapeutic efficacy of gemcitabine in a highly chemoresistant pancreatic cancer. Combination treatment resulted in a 7.4-fold higher induction of apoptosis than YDC002 alone in MIAPaCa-2 tumor spheroids, markedly higher than the 1.3-fold increase observed in cell experiments, suggesting that tumor spheroids with an intact tumor ECM are more refractory to gemcitabine. These results demonstrate the significance of desmoplasia in attenuating the efficacy of therapeutics against pancreatic cancer. To assess the mechanism underlying ECM depletion, we analyzed the effect of each treatment on intratumoral expression level of the major ECM components, collagen I/III, fibronectin, and elastin, by immunohistochemistry (Fig. 3B). Combination treatment decreased the levels of collagen I by 95%, collagen III by 98%, fibronectin by 72%, and elastin by 80% compared with controls, indicating that this treatment paradigm results in ECM loss by attenuating the expression of ECM components. These results demonstrate that ECM-induced chemoresistance can be ameliorated through YDC002-mediated disruption of the ECM, allowing combination treatment to produce potent anticancer effects through efficient induction of tumor cell apoptosis.

Antitumor effects of combination treatment with YDC002 and gemcitabine in a pancreatic cancer xenograft model To evaluate the antitumor effects of combination treatment in vivo, we assessed tumor growth inhibition in three different pancreatic cancer xenograft models following treatment with YDC002 and gemcitabine, alone or in combination. As shown in Fig. 4, tumors in the control group grew rapidly; treatment with gemcitabine alone produced a trend toward a delay in tumor growth, but this difference did not reach statistical significance. These results are in good agreement with previous reports and our in vitro results illustrating the highly chemoresistant nature of pancreatic tumors. In contrast, YDC002 induced significant inhibition of tumor growth in all pancreatic cancer models compared with treatment with vehicle or gemcitabine alone. Combination treatment significantly attenuated tumor burden (tumor weight and volume) compared with single treatment with gemcitabine or YDC002, suggesting that oncolytic Ads can restore the chemosensitivity of pancreatic tumors toward gemcitabine and enhance antitumor effects. Tumor volumes in the combination treatment group measured 60 days after the beginning of treatment were 4.4-, 3.5-, or 5.8-fold lower in MIAPaCa-2, BxPC-3, and PANC-1 xenograft models, respectively, compared with those in the group treated with gemcitabine alone. No significant changes in body weight and liver toxicity-related biomarkers were observed (Supplementary Fig. 2), indicating that combination treatment possesses a good safety profile even with multiple administrations of oncolytic Ads and gemcitabine. Taken together, these results demonstrate that the combination of a relaxin-expressing oncolytic Ad and gemcitabine is capable of inducing potent antitumor effects in highly desmoplastic and chemoresistant pancreatic tumors.

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Induction of apoptosis and ECM disruption by the combination of YDC002 and gemcitabine in pancreatic cancer xenograft models To further confirm that the combination of YDC002 and gemcitabine effectively inhibits tumor growth by inducing apoptosis, we investigated the expression level of cleaved caspase-3 and the number of TUNEL-positive cells in MIAPaCa-2 tumor tissues. Combined treatment with YDC002 and gemcitabine increased cleaved caspase-3 expression and TUNEL-positive cell numbers compared with controls and single-treatment groups (Fig. 5). Moreover, combination treatment significantly attenuated intratumoral expression of the major ECM components, collagen I, collagen IV, fibronectin, and elastin (Fig. 6), which are associated with inhibition of drug diffusion in tumor beds. These results demonstrate that YDC002-mediated loss of ECM can restore chemosensitivity toward gemcitabine in desmoplastic pancreatic tumors. Discussion With a 5-year relative survival rate of 7%, pancreatic cancer is the only major cancer with a survival rate in the single digits, because most cases are unresectable at the time of diagnosis [36]. At any stage of disease progression, pancreatic cancer responds very poorly to systemic treatments owing to its intrinsic and extrinsic drug resistance, which culminate in complex therapyresistant phenotypes over time [37]. The intrinsic drug resistance of pancreatic tumor cells involves mechanisms such as activation of anti-apoptotic signaling pathways. Extrinsic drug resistance arises from tumor cell interactions with the complex tumor microenvironment, including the ECM and stromal compartments, as well as activation of alternative escape pathways. Several pancreatic cancer cell lines, including PANC-1 and MIAPaCa-2, are intrinsically resistant toward gemcitabine, a standard drug of choice for the treatment of pancreatic cancer [38]. In accord with these reports, high dose (50 mM) gemcitabine treatment exhibited a cancer cellkilling efficacy similar to that of low-dose (0.1e1 mM) gemicitabine in a panel of the aforementioned gemcitabine-resistant pancreatic cancer cells (Fig. 1A), confirming the intrinsic drug resistant attributes of pancreatic cancers. Oncolytic Ads chemosensitize cancer cells through various mechanisms [39,40]. For example, the Ad E1A protein causes infected cells to transit the cell cycle into S-phase, which rapidly chemosensitizes these dividing tumor cells toward DNA damaging chemotherapeutics. In the present study, the combination of the relaxin-expressing and Ad E1B19kDA-deleted oncolytic Ad, YDC002, together with a subtherapeutic dose of gemcitabine (0.01 or 0.05 mM) resulted in a potent cancer cell-killing effect that surpassed that of either treatment alone, demonstrating that YDC002 is capable of sensitizing highly chemoresistant pancreatic cancer cells to gemcitabine (Fig. 2A). Furthermore, the combination treatment induced highly efficient apoptosis of pancreatic cancer cells (Fig. 2B). These findings are in good agreement with previous reports in which an Ad E1B19kDa gene-deleted oncolytic Ad sensitized chemoresistant pancreatic cancer cells and led to increased chemotherapeutic-induced tumor cell apoptosis [41,42]. Additionally, the Ad E1A gene is a potent inducer of p53 protein expression in infected cells, which can increase cellular sensitivity toward drug-induced and p53-dependent apoptosis [43].

Fig. 3. ECM degradation and induction of apoptosis by the combination of gemcitabine and YDC002 in pancreatic tumor spheroids. A, Representative images of MT, picrosirius red, and TUNEL staining. Organotropic tumor spheroids cultured from xenograft tumor tissues (~2 mm in diameter) were treated with YDC002 (1  109 VP); gemcitabine (20 mМ) was added the next day. Magnification, 400. B, Immunohistochemistry for collagen I, collagen III, fibronectin, and elastin. Microscopic images were analyzed semi-quantitatively, and the expression levels of ECM components were expressed as fold change relative to untreated controls. Data are presented as means ± SD (n ¼ 5e7; ***p < 0.001).

Please cite this article in press as: K.H. Jung, et al., Oncolytic adenovirus expressing relaxin (YDC002) enhances therapeutic efficacy of gemcitabine against pancreatic cancer, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.03.009

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Fig. 5. Induction of apoptosis by the combination of gemcitabine and YDC002 in pancreatic cancer xenograft models. Tumors were excised and processed for immunohistochemical and histological analysis. Induction of apoptosis in tumor tissues was assessed by TUNEL assay and immunohistochemical analysis of cleaved caspase-3. Magnification, 400. Data are presented as means ± SD (*p < 0.05, **p < 0.01, and ***p < 0.001).

One main component of the extrinsic drug resistance mechanisms of pancreatic cancer is the dense desmoplastic reaction in tumor tissue, which inhibits diffusion and dispersion of anticancer therapeutics [22,37,44]. Additionally, changes in the level of ECM proteins are associated with increased invasion and migration of

tumor cells [45]. As shown in Fig. 1B, MT staining of pancreatic cancer patient-derived tumor samples confirmed the extensive accumulation of collagens, a major component of ECM, throughout the tumor tissue. Importantly, three-dimensional (3D) pancreatic tumor spheroids also exhibited extensive accumulation of dense

Fig. 4. Inhibition of tumor growth by the combination of gemcitabine and YDC002 in pancreatic cancer xenograft models. AeC, Tumor volume and weight measurements in MIAPaCa-2, BxPC-3, and PANC-1 pancreatic cancer xenograft models. All tumor xenografts were established on the flank of mice by subcutaneous injection of MIAPaCa-2, BxPC-3, or PANC-1 cells. Tumors were injected with gemcitabine (intraperitoneal) and/or YDC002 (intratumoral); PBS-injected mice were used as a control. Tumor size was measured every two days. Data are presented as means ± SD (n ¼ 5e7; *p < 0.05, **p < 0.01, and ***p < 0.001).

Please cite this article in press as: K.H. Jung, et al., Oncolytic adenovirus expressing relaxin (YDC002) enhances therapeutic efficacy of gemcitabine against pancreatic cancer, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.03.009

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Fig. 6. Inhibition of ECM component accumulation by the combination of gemcitabine and YDC002 in pancreatic cancer xenograft models. Levels of the major ECM components, collagen I, collagen IV, fibronectin, and elastin in tumor tissues were analyzed by immunohistochemistry. Magnification, 400. Data are presented as means ± SD (**p < 0.01 and ***p < 0.001).

Please cite this article in press as: K.H. Jung, et al., Oncolytic adenovirus expressing relaxin (YDC002) enhances therapeutic efficacy of gemcitabine against pancreatic cancer, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.03.009

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ECM layers, which closely emulated the desmoplastic reaction of patient-derived pancreatic tumors (Fig. 3A). Notably, gemcitabineinduced apoptosis was markedly lower in 3D tumor spheroids than 2D cultures, implying that the ECM- and tumor microenvironmentmediated extrinsic drug resistance of pancreatic cancer is a more critical determinant of the low efficacy of systemically administered gemcitabine than intrinsic resistance (Figs. 2B and 3A). These findings are in good agreement with previous reports, demonstrating that 3D cultured tumor cells, which emulate the complex tumor microenvironment, are more resistant to treatment with chemotherapeutics and thus are better suited for drug screening purposes than their 2D counterparts [46]. Relaxin selectively inhibits production of excessive ECM proteins through dysregulation of collagen expression and promotes degradation of ECM during aberrantly upregulated fibrotic processes, such as cancer-induced ECM formation [44,47]. As shown in Fig. 3C, YDC002 significantly suppressed mRNA expression of collagen type I and III, two predominant components of the tumor stroma that constitute 80% of the tumor mass [48], supporting the idea that oncolytic Ad-mediated expression of relaxin can inhibit collagen expression in pancreatic cancer cells. Similarly, efficient inhibition of collagen type I/III and other major ECM components were observed in tumor spheroids treated with YDC002 (Fig. 3). ECM degradation is mostly mediated by YDC002 and its expression of relaxin in tumor tissue as gemcitabine does not degrade ECM. Thus, the expression of major ECM components in combination treatment group is expected to be similar to those of YDC002 monotherapy (Fig. 3). Effective breakdown of the ECM by YDC002 enhanced gemcitabine-induced apoptosis, likely through increased penetration and diffusion of the drug, leading to synergistic induction of apoptosis, confirming that extrinsic chemoresistance to gemcitabine can be overcome through combination treatment with YDC002. Trends similar to those observed in tumor spheroids, where gemcitabine treatment alone induced negligible tumor growth inhibition, were observed in all ECM-rich pancreatic tumor xenograft models. By contrast, combination treatment with YDC002 and gemcitabine elicited potent antitumor effects that surpassed those of either single treatment, confirming the sensitizing effect of YDC002 on chemoresistant pancreatic xenografts (Fig. 4). Moreover, combination treatment showed a good safety profile, producing effects on body weight similar to those caused by treatment with either gemcitabine or YDC002 alone. Notably, the potent anticancer and proapoptotic effects induced by combination treatment were positively associated with the degree of ECM loss in tumor tissue (Figs. 5 and 6), suggesting that removal of this physical barrier improves diffusion of gemcitabine and oncolytic Ads. This is in good agreement with previous reports demonstrating that expression of ECM-degrading genes enhances drug diffusion as well as viral spread of oncolytic virus in tumor tissue [37]. Collectively, our results demonstrate that the combination of YDC002 and gemcitabine effectively overcomes both intrinsic and extrinsic drug resistance of pancreatic cancer to elicit potent anticancer effects. YDC002 effectively sensitized chemoresistant and desmoplastic pancreatic tumors toward gemcitabine treatment through effective loss of the tumor ECM, resulting in augmented gemcitabine-induced tumor cell apoptosis with no observable side effects. We foresee that the combination of YDC002 and gemcitabine could be a promising strategy for overcoming tumor-induced drug resistance and inducing potent antitumor effects in highly desmoplastic pancreatic cancers in a clinical setting. Acknowledgments This research was supported by grants from the National Research Foundation (NRF) of Korea (2015R1A2A1A10054108,

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