Downregulating Heparanase-Induced Vascular Normalization: A New

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Downregulating Heparanase Induced Vascular Normalization: a New Approach to Increase the Bioavailability of Chemotherapeutics in Solid Tumors Xudong Zhang, Yuxin Wang, Manman Xie, Christopher Corbett, Sunil Singhal, Bo Dai, Jianquan Wang, Qingqing Ding, Qian Lu, and Yiqing Wang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00628 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018

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Molecular Pharmaceutics

The normalized blood vessels after downregulating HPSE 70x35mm (300 x 300 DPI)

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Downregulating Heparanase Induced Vascular Normalization: a New Approach to Increase the Bioavailability of Chemotherapeutics in Solid Tumors ※

‡※

§

§

∥

Xudong Zhang†, , Yuxin Wang , , Manman Xie†, Christopher Corbett , Sunil Singhal , Bo Dai , Jianquan Wang†, Qingqing Ding⊥, Qian Lu† *, and Yiqing Wang †,* †

Department of Biomedical Engineering, College of Engineering and Applied Sciences, Nanjing University, 22 Hankou Road, Nanjing, Jiangsu Province 210093, China. ‡

Department of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, China.

§

Department of Surgery, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA. ∥

Drum Tower Clinical Medical College of Nanjing Medical University, Nanjing, Jiangsu 210000, China. ⊥

Department of Geriatric Gastroenterology, the First Affiliated Hospital of Nanjing Medical University

*

corresponding: [email protected]; [email protected]

※

these authors contributed equally to this work.

Keywords：Heparanase, Vascular Normalization, Bioavailability, Chemotherapeutics


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ABSTRACT Downregulating heparanase has been shown to reduce tumor angiogenesis and prevent chemoresistance, and it is becoming an appealing approach to treat solid tumors. However, little attention has been given to its underlying antitumor mechanisms, especially the relationship between heparanase and vascular development in solid tumors which is not yet fully understood. In this study, we found that downregulating heparanase through orthotopic injection of heparanase small interfering RNA could not only reduce vascular density, but more importantly lead to vascular normalization in solid tumors. Consequently, this may lead to more efficient delivery of chemotherapeutic agents. These findings provide the basis for developing new approaches to treat solid tumors with a combination of heparanase inhibitors and chemotherapeutics.

INTRODUCTION Heparanase (HPSE) is a mammalian endoglycosidase which degrades heparan sulfate (HS) in the extracellular matrix (ECM)1, leading to the breakdown of extracellular barriers for cell invasion and the release of HS-binding growth factors (e.g. vascular endothelial growth factor [VEGF], basic fibroblast growth factor [bFGF] and hepatocyte growth factor [HGF]2. Recent studies have shown that HPSE induces tumor angiogenesis3 by promoting the release of pro-angiogenic factors that are preserved and protected by heparan sulfate proteoglycans (HSPGs) in the ECM. HPSE degrades the HS chains of HSPGs and releases HS-bound bFGF and VEGF4. The HSbFGF complex subsequently binds FGF receptor (FGFR) and forms the HS-FGF-FGFR complex which activates FGFR related signaling pathways in the endothelium. The vital role of HPSE in tumor development makes it an important target in cancer therapy. Currently, there are four HPSE inhibitors (PI-88, Roneparstat, M402, and PG545) undergoing clinical investigation5,6.

According to the literature, HPSE is upregulated in response to chemotherapy in cancer patients, and tumor cells acquire chemoresistance partly due to autophagy7. Malignant cells overexpressing HPSE are more resistant to cisplatin treatment, and lowering expression of HPSE can improve this situation8. Researchers believe that the addition of HPSE inhibitors can


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potentially overcome initial chemoresistance, and provide a better prognosis for cancer patients than those treated with conventional chemotherapy alone7. In fact, we have found that HPSE is closely linked with vascular morphology. Tumor vasculature always displays an abnormal structure, manifested as leaky, disorganized vessels with limited permeability inside tumors9. This allows for the dissemination of cancer cells and dramatically impairs drug delivery and efficacy. Normalized vasculature in solid tumors will not only prevent the tumor cells from leaking out of blood vessels, but also has improved permeability which enhances the delivery of small molecule chemotherapeutics. To our knowledge, there is no report showing that downregulating HPSE is able to normalize vascular structures in solid tumors.

In this contribution, we downregulated HPSE using an orthotopic injection of HPSE small interfering RNA (siRNA), which is a commonly used method to alter enzyme expression levels locally10–12. Our results demonstrated that downregulating HPSE led to vascular normalization in solid tumors, and consequently improved chemotherapeutic bioavailability.

EXPERIMENTAL SECTION Reagents and Chemicals Cholesterol-conjugated HPSE siRNA for in vivo RNA interference was purchased from RiboBio Co. (Guangzhou, China). Fetal calf serum (FBS) was purchased from Biological Laboratories (Kibutz Beit Haemek, Israel). Dulbecco's modified eagle medium (DMEM), medium 199 (M199) and 0.25% trypsin were purchased from Gibco BRL (Grand Island, NY, USA). Matrigel was obtained from BD Biosciences (Bedford, MA, USA). Antibodies against VEGF, p-AKT, pPI3K, p-src, p-VEGFR2, CD31, GAPDH and peroxidase-conjugated secondary antibodies were purchased from Bioworld Technology (St Louis, MN, USA). Antibodies against HPSE and endostatin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). FITClabeled α-SMA antibody was obtained from eBioscience (San Diego, CA, USA). DAPI, FITC and TRITC-conjugated secondary antibodies were purchased from KeyGEN BioTECH (Nanjing,


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China). Doxorubicin (DOX) and Evans Blue were obtained from Sigma-Aldrich (St Louis, MO, USA). The other chemicals and reagents were of analytical grade.

Cell Culture All experiments protocols, methods and procedures were performed in accordance with relevant guidelines and regulations of the Medical Ethics Review Committee of Nanjing Maternal and Child Health Hospital of Nanjing Medical University, China.

Mouse melanoma cell line B16F10 was obtained from ATCC (Manassas, VA, USA), and grown in DMEM (10% FBS). Human umbilical vein endothelial cells (HUVECs) were kindly provided by Nanjing Medical University (Nanjing, China). The cells were harvested and seeded into 25 cm2 flasks and cultured with M199 consisting of 20% (v/v) FBS, 2 mM L-glutamine and 7.5 mg/ml endothelial cell growth supplement (Sigma) at 37°C in a humidified atmosphere containing 5% CO2. Medium was changed every two days. HUVECs were treated with fresh HPSE siRNA medium for 48 h prior to subsequent experiments.

Mouse Subcutaneous Xenotransplanted Tumor Model Study All experiments protocols, methods and procedures were performed in accordance with relevant guidelines and regulations of the Committee on the Care and Use of Laboratory Animals and the related ethical regulations of Nanjing University. ICR mice (male; age 5-6 week; weight 18±2g) were purchased from Jiesijie Lab-animal Company in Shanghai (Animal License No. SCXK (HU) 2013-0006). The mice were kept in a standard laboratory environment and given water and food freely. 12 ICR mice were randomly divided into 2 groups and inoculated with B16F10 cells (5×106 cells in 100 µL PBS per mouse) into the left foreleg axilla. When tumor volume was approximately 40 mm3, the mice were intratumorally injected with 100 µL PBS (control group) or 2.5 nM cholesterol-conjugated HPSE


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siRNA twice a week. Tumor volume was measured at the same time and was estimated by the following equation: V = 1/2 × larger diameter × (smaller diameter) 2.

Immunofluorescence Assay Tumor cell slices were prepared after all cells were treated with HPSE siRNA medium for 48 h. For solid tumors, frozen tissue slices were prepared after a 28-day HPSE siRNA treatment. Next, immunofluorescence assays were performed as described previously. Slices were fixed with 4% paraformaldehyde for 1 h, washed with PBS 3 times and blocked with 5% BSA-PBS solution for over 1 h. Then slices were incubated with 200 µl of primary antibodies against HPSE, VEGF, endostatin, CD31 or α-SMA overnight at 4°C. After washing, they were exposed to FITC and/or TRITC-conjugated secondary antibodies. DAPI staining was used to recognize the cell nucleus after slices were permeabilized with 1% Triton X-100. Images were observed and captured using a laser scanning confocal microscope (Zeiss LSM710, Germany). At least five fields were chosen randomly to count for fluorescence intensity and vessel area of each section by image J (Image J 1.51j8, Wayne Rasband, NIH, USA). For HPSE, the value was defined as the mean fluorescence intensity (MFI) ratio of HPSE and DAPI: (HPSE/DAPI), HPSE siRNA/ (HPSE/DAPI). The Control, DOX, VEGF, and endostatin were processed as above. For CD31 and α-SMA, mean vessel area was selected by image J, and the pericyte coverage of blood vessels was estimated by the ratio of the area of α-SMA to CD31.

Vessel Permeability Assay and DOX Quantification Evans blue (0.5%) was intravenously injected into mice in the control and HPSE siRNA groups (3 mice each group, 250 µL). All mice were sacrificed 30 minutes after injection. Tumors in different groups were collected and weighed. The dye was extracted from tumors by formamide (2%, 55°C, overnight) and quantified by the UV absorption intensity（610 nm）.


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For the quantification of DOX, 3 mice were used in each group. Tumor-bearing ICR mice were injected intravenously with DOX (10 mg/kg) 4 hours before sacrifice and tumor mass slices were prepared. At least five fields were chosen randomly to count for fluorescence area of each section by image J.

Western Blot Assay The expression of p-AKT, p-PI3K, p-src, p-VEGFR2 and GAPDH were detected by western blot analysis as described previously. GAPDH was used as an internal standard. After different treatments, total cellular protein was extracted with RIPA lysis buffer (KeyGEN BioTECH, Nanjing, China) for 20 min at 4°C. The lysates were clarified by centrifugation at 12,000 rpm for 10 min at 4°C. The concentrations of protein in the supernatants were determined using bicinchoninic acid protein assay kit (KeyGEN BioTECH). Equal amounts of protein lysate were separated by 8% SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 10% non-fat dry milk for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C. Subsequently, they were washed five times with PBS Tween-20 (PBST) buffer and incubated with secondary antibodies for 1h at room temperature. Immunoreactive bands were visualized using film exposure with enhanced chemiluminescence detection reagents (KeyGEN BioTECH).

Statistical Analyses All analyses were conducted in OriginPro 2016 Version 9.3.226 (OriginLab Corporation, Northampton, USA) and IBM SPSS Statistics Version 22 (IBM Corporation, New York, USA). All Images were processed by image J (Image J 1.51j8, Wayne Rasband, NIH, USA). All data were expressed as mean ± SD which were obtained from at least three independent experiments. Comparisons between multiple groups were performed using independent sample t-test. Values of p < 0.05 were considered to be statistically significant.


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RESULTS Downregulating HPSE Inhibited Tumor Growth. HPSE is becoming an important target in cancer therapy. To determine whether HPSE is involved in tumor development, we downregulated HPSE by directly injecting cholesterolconjugated HPSE siRNA10–12. siRNA targets and mediates cleavage of complementary mRNA transcripts, subsequently repressing target gene expression and compromising the function of target genes13. As shown in Figure S1, siRNA (2.5 nM intratumoral injection, three times/week) dramatically reduced HPSE expression (92%) in solid tumors, leading to a 37.84% decrease in tumor volume (Figure 1A). There was no significant toxicity in major organs induced by the intratumoral injection of HPSE siRNA (Figure S2), and no decrease in body weight in the siRNA-treated group (Figure 1B), indicating low toxicity of HPSE siRNA.

Figure 1. HPSE siRNA inhibited tumor growth. A) Effect of HPSE siRNA on tumor volume. B) Effect of HPSE siRNA on body weight of mice. (*P

Downregulating Heparanase-Induced Vascular Normalization: A New

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