Abstract 2919: Ultrasound-mediated neuropilin-1 shRNA minicircle delivery inhibits tumour growth in an orthotopic human pancreatic adenocarcinoma model

Abstract

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumour desmoplasia/fibrosis, which is partially responsible for its aggressive nature. The pro-fibrotic cytokine TGFβ1 promotes fibroblast accumulation in several pathological disorders, including cancer via endothelial-to-mesenchymal transition (EndMT), thereby implicating EndMT as a potential therapeutic target. Previous studies have demonstrated angiogenic co-receptor, neuropilin-1 (NRP-1) as a co-receptor for TGFβ1 in mediating several oncogenic processes. NRP-1 expression and TGFβ1 signaling have been shown to be up-regulated in PDAC. However, the therapeutic benefits of targeting NRP-1 in PDAC remain unexplored. We therefore hypothesized that targeting NRP-1 in PDAC may inhibit TGFβ1-mediated EndMT, tumour perfusion and tumour growth. Methods: Orthotopic tumours were generated in RNU athymic rats from BxPC-3 human pancreatic cancer cells using the implantation technique. Animals were delivered at day 28 post surgery with either empty minicircle-DNA (150 μg) or shNRP-1 minicircle-DNA (200 μg) using ultrasound targeted microbubble destruction (UTMD). For UTMD, ultrasound was transmitted using a phased array transducer (S3, Sonos 5500) at 1.3 MHz and a transmit power of 0.9 W (120 V, 9 mA), and at a pulsing interval of 5 s. Contrast enhanced ultrasound perfusion imaging was performed on day 28 (gene delivery) and day 56 (end-point) with standardized analyses. Tumour tissues and remote organs were collected for evaluation of tumour volume and other downstream analyses like real-time polymerase chain reaction (RT-PCR), immunoblotting, immunohistochemistry and Masson's trichrome staining (for fibrosis). Results: RT-PCR and immunoblotting demonstrated successful silencing of NRP-1 in shNRP-1 minicircle group. Immunohistochemistry demonstrated a typical ductal morphology and substantial collagen content within the tumours. Histological assessments further revealed reduced NRP-1 expression and reduced fibrosis in shNRP-1 minicircle group. Significant changes were observed in EndMT markers at transcript level and protein level in shNRP-1 minicircle delivered animals compared to empty minicircle. At day 56, profound reductions were observed in tumour volumes in shNRP-1 minicircle delivered animals. Tumour area and perfusion were found to be greater and more complete throughout the tumour in empty minicircle group, while reduced area and perfusion was observed in shNRP-1 minicircle group. At day 56, tumour microvascular blood flow and blood volume were markedly reduced in the shNRP-1 minicircle group. Conclusions: Overall, these in vivo results suggest that loss of NRP-1 leads to reduced fibrosis and tumour growth in PDAC possibly through limiting EndMT and NRP-1 mediated angiogenesis, making NRP-1 as a potential therapeutic target against PDAC. Citation Format: Pratiek N. Matkar, Krishna K. Singh, Gerald J. Prud’homme, David W. Hedley, Howard Leong-Poi. Ultrasound-mediated neuropilin-1 shRNA minicircle delivery inhibits tumour growth in an orthotopic human pancreatic adenocarcinoma model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2919.