Abstract 3692: Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment
Authors: Jiang Xiuyun , Seo David , Chang Jae , Nigjeh Eslam , Pan Sheng , Jalikis Florencia , Pillarisetty Venu 
Published: July 2017

Introduction

Pancreatic ductal adenocarcinoma (PDA) remains a deadly disease that is rarely cured. Given the recent successes with immunotherapy for other malignancies and the fact that PDA is heavily infiltrated by effector T cells, we postulate that accurate modeling of the PDA immune microenvironment would allow us to unveil mechanisms of immunosuppression that could be overcome for therapeutic benefit.

Methods

PDA tumors were collected from the operating room and transported in cold media to the laboratory, where 250μm slices were cut using a vibratome and placed on 0.4μm pore size membrane inserts pre-coated with collagen gel and culture. Proliferation was measured using MTT assay and weight-adjusted optical density. Slices were fixed in 4% PFA for imaging after live immunofluorescence (IF) staining or for subsequent immunohistochemistry (IHC) prior to imaging. For proteomic analysis, slices were digested with trypsin and analyzed by mass spectrometry. To assess for immune cell migration into the tumor slices, isolated autologous splenocytes were stained with CFSE and added to the slice culture and imaged using IF after 6 days.

Results

Using light microscopy, we confirmed that cultured slices maintain their baseline morphology, architecture and surface area over 9 days in culture. The MTT assay showed stable growth over the same period; IHC for Ki-67 and cleaved Caspase-3 corroborated a similar pattern of proliferation and apoptosis after 6 days culture when compared to day 1. T cells (CD3+, CD8+ and FOXP3+), macrophages (CD68+, CD163+, and HLA-DR+), and stromal myofibroblasts (αSMA+) were confirmed to be present through day 6 using IHC. Quantitative proteomic analysis of over 3000 proteins showed that only 2-3% of proteins showed significant changes of abundance greater than 2 fold after 6 days in culture. However, when slice were challenged with cytotoxic drugs (staurosporine and cyclohexamide), there was a time- and dose-dependent response in proliferation and apoptosis compared with controls.

The demonstration of the live and dynamic microenvironment was performed first via live multicolor IF including PDA cells (EpCAM+), fibroblasts (fibronectin+) and immune cells (both CD11b+ and CD8+ cells) within the microenvironment. Furthermore, co-culture of CFSE-labeled splenocytes on top of slices for 6 days. Z-stacked live confocal imaging confirmed their migration from the surface into the slice microenvironment.

Conclusion

Our study demonstrates that the PDA slice culture maintains original tumor microenvironment, and overall proteomic production and dynamic function response in vitro. The slice culture model thus represents a novel platform to evaluate both pharmaceutical and cell-based immunotherapies for mechanisms of action and potential synergism, effects on specific cell types of the microenvironment, and tumor-specific immunogenic potential.

Citation Format: Xiuyun Jiang, David Seo, Jae Chang, Eslam Nigjeh, Sheng Pan, Florencia Jalikis, Venu Pillarisetty. Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3692. doi:10.1158/1538-7445.AM2017-3692