Abstract 3196: Combination immunotherapy with PD-1 and CXCR4 blockade activates antitumor immunity against pancreatic neuroendocrine tumors
Authors: Jiang Xiuyun , Sullivan Kevin , Labadie Kevin , Daniel Sara K. , Seo David , Abbasi Arezou , Kim Teresa , Yeung Raymond , Pillarisetty Venu 
Published: August 2019

Abstract
Introduction: Although pancreatic neuroendocrine tumors (PNET) are less common and less deadly than pancreatic adenocarcinoma, they remain a considerable source of morbidity and mortality. The cases increasingly discovered due to advanced cross sectional imaging and the majority of PNETs are malignant when diagnosed. Immunotherapy for PNET has not yet been successfully demonstrated, although we and others have previously shown that they often contain robust T cell infiltrates. We hypothesized that combined immunotherapy could reactivate endogenous antitumor activity in organotypic tumor slice cultures of human PNET.
Methods: Human PNET tumors were collected from the operating room and 250μm slices were cut using a vibratome and place on 0.4μm pore size membrane inserts (6 tumors). Slice culture survival testing was conducted using immunohistochemistry (IHC), the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, or live immunofluorescence imaging. To test the combining immunotherapeutic effects, slices were treated with a PD-1 blocking monoclonal antibody or isotype control, with or without the CXCR4 inhibitor, AMD3100. For time-lapse live imaging, slices were stained with fluorescently conjugated antibodies for CD8 and EpCAM, as well as a reagent that binds to activated caspase 3/7 enzymes to indicate induction of apoptosis. The live slice was first imaged alone to serve as a non-treated control, then anti-PD1 antibody and AMD3100 were applied, and the slice imaged immediately afterward to monitor response after treatment.
Results: We confirmed that cultured slices maintain their baseline morphology and architecture over 9 days in culture. The MTT assay showed stable metabolic activity over the same period. The demonstration of the live and dynamic microenvironment was performed first via live multicolor IF including PNET cells (EpCAM+) and immune cells (both CD45+ and CD4+ cells) within the microenvironment. IHC demonstrated 10% or 12% increased of cleaved-Caspase-3+ cells with combined PD-1 and CXCR4 blockade, as compared to control, after 2 days or 7 days in culture. To confirm the role of cytotoxic T cells, live stained slices were imaged before and after addition of PD-1 blockade and AMD3100. We demonstrated that caspase activation was increased in EpCAM+ cells proximal to CD8+ T cells immediately following combination drug treatment. Furthermore, CD8+ cells proximity to EPCAM+ cells (&lt;20 µm) was significantly increased after addition of PD-1 blockade and AMD3100. There was no difference in the proportion of apoptotic EpCAM+ cells that did not have a CD8+ cell nearby, suggesting that apoptosis induction is specifically due to cytotoxic T cell function.
Conclusion: Our study demonstrates that combination PD-1 and CXCR4 blockade enhances CD8+ T cell migration and antitumor activity in human PNET tumor slice cultures.
Citation Format: Xiuyun Jiang, Kevin Sullivan, Kevin Labadie, Sara K. Daniel, David Seo, Arezou Abbasi, Teresa Kim, Raymond Yeung, Venu Pillarisetty. Combination immunotherapy with PD-1 and CXCR4 blockade activates antitumor immunity against pancreatic neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3196.