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Abstract
Background Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer related death in the U.S, has a 5-year survival rate of only 10%.1 The paucity of T cells in the immune privileged tumor microenvironment (TME) is a major limitation in developing an effective immunotherapy against PDAC.2The cancer genome atlas (TCGA) shows that human PDAC tumors express high levels of vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide (figure 1A), that inhibits effector T cell responses.3 4 We hypothesized that paracrine secretion of VIP in the TME is a targetable mediator of immune paralysis in PDAC, and that pharmacological inhibition of VIP receptor signaling could enhance anti-tumor responses in PDAC.
Methods VIP levels in plasma or cell culture supernatant was determined via VIP-specific enzyme immunoassay. Luciferase transfected KPC (KPC.luc) cells were injected subcutaneously or orthotopically into the pancreas of C57BL/6, CD4KO, or CD8KO mice from Jackson Laboratories. C57BL/6 mice T cell subsets in were depleted post tumor implantation with anti-CD4 and/or anti-CD8 antibodies. Tumor-bearing mice were treated daily with ANT008, a novel VIP receptor antagonist peptide, and/or anti-PD1 monoclonal antibody (MoAb) for 10 days, starting 7-10 days after implantation. T cells isolated from peripheral blood of PDAC patients were expanded 9 days ex vivo in anti-CD3 MoAb coated plates with 30U/ml IL-2 and either control peptide (scrambled VIP sequence) or ANT008. Survival by VIP and VIP receptor expression from the TCGA was clinically correlated.
Results Increased human and mouse PDAC expression correlates with elevated blood levels (figure 1). While the PDAC cancer cell lines express VIP receptors, ANT008 does not have direct cytotoxic effect on cell growth in vitro (figure 2). However, in orthotopic KPC model, treatment with ANT008 & anti-PD-1 significantly decreased tumor growth rate and burden (figure 3) while increasing the intratumoral levels of CD4 and CD8 proliferating T cells (figure 4) via a T cell dependent mechanism (figure 5). Additionally, in ex vivo cultures of T cells isolated from PDAC patients, ANT008 improved the effector properties of T cells via decreasing expression levels of co-inhibitory molecules and decreasing frequency of regulatory T cells (figure 6). Clinically, VIPR1 receptor expression, but not VIP, provides a survival benefit (figure 7).
Overexpression of VIP in PDAC tumors(A) VIP mRNA expression in several solid malignancies, with red arrow pointing at the levels in pancreatic cancer, as obtained from The Cancer Genome Atlas. (B) Blood collected from healthy volunteers (n=26) and consented untreated pancreatic cancer patients before surgery/chemotherapy (n=41) were quantified for levels of VIP. (C) Cell free supernatant collected from B16F10, SM1 and D4M (murine melanoma cell lines) or KPC, MT5 or Panc02 (murine PDAC cell lines) or BXPC3 and Panc1 (human PDAC cell lines), were quantified for VIP (D) C57BL/6 mice were implanted with 1 million B16F10 cells (n=4), D4M cells (n=4) or MT5 cells (n=3) subcutaneously, or with KPC cells in the tail of the pancreas after laparotomy (n=3). When tumor volumes reached 500mm3 for subcutaneous tumors or the tumor flux reached 2 x 1010 photons/sec for orthotopic KPC tumors, mice were sacrificed and the concentration of VIP in the blood was determined. P value for B was calculated using student t test and C and D were calculated by one-way ANOVA followed by Tukey’s post-test. Error bars show mean with standard deviation. *p<0.0001
PDAC cell lines express receptors for VIP(A) Western blot analysis showing constitutive expression of VPAC1, VPAC2 and PACAP receptors in murine and human PDAC cell lines and B16F10, a murine melanoma cell line. GAPDH was used as the loading control. (B) Percentage viability of KPC and Capan02 cells treated with varying concentrations of ANT008 (0.5-5µM) relative to cells treated with 0µM ANT008 for 24, 48 and 72 hours as measured by MTT assay.
Efficacy of ANT008+aPD-1 therapy in in-vivo model5x105 KPC.luc cells on a matrigel were implanted in the tail of pancreas of C57BL/6 mice. Seven days after tumor implantation, mice were randomized and treated with ANT008 and/or aPD-1 until day 25, when they were sacrificed. (A) From each treatment group, one mouse with biggest non-ulcerated tumor was imaged via IVIS imaging (top), sacrificed and imaged with 9.4T MRI scanning (MRI) and their paraffin embedded pancreatic tissues were stained via H&E (bottom). (B) The tumor flux measured via IVIS imaging is plotted with respect to days after tumor implantation. ‘o’ represent mice that were imaged via MRI on day 28 and ‘+’ represent mice that were sacrificed before day 25 due to ulceration. (C) On day 25 mice were sacrificed and the weight of the pancreas was plotted. The brown dashed line represent average weight of pancreas from naïve age matched mice. P value was calculated using one-way ANOVA followed by Tukey’s post-test. Error bars show mean with standard deviation with **p<0.01.
ANT008+aPD-1 therapy increases T cell infiltration5x105 KPC.luc cells were implanted in the tail of the pancreas following laparotomy. Once the tumors were detectable via bioluminescent imaging, mice were randomized into four treatment groups and treated with isotype antibody and scrambled peptide or ANT008 and/or anti-PD-1 until day 25, when the mice were sacrificed, tumor tissues harvested, and formalin fixed. The tissues were stained for nucleus, CD4, CD8 and Ki67 via multiplex immunohistochemistry (A) A representative multiplex IHC image showing T cell infiltration in the different treatment groups .(B) Numbers of CD4, CD8, Ki67+ CD4 and Ki67+ CD8 T cells/mm2 with respect to weight of the pancreas is shown.
ANT008+aPD-1 therapy is T cell dependent5x105 KPC.luc cells were subcutaneously implanted in C57BL/6 or CD4KO (A) and C57BL/6 or CD8KO (B) mice and treated with scrambled+IgG (control) or ANT008+aPD-1 (treated) for 10 days from day 5 or 7 when the tumors were palpable. Mice were monitored for tumor growth and sacrificed when the tumor volume reached 500mm3 or when the tumors ulcerated. P values were calculated using Log rank test. *p<0.0001.
ANT008 improves effector properties of T cellsCryopreserved PBMCs from peripheral blood of consented PDAC patients were thawed and rested overnight at 37°C in a CO2 incubator followed by T cell isolated via negative magnetic sorting. Isolated T cells were seeded on human anti-CD3 coated plates and cultured in media supplemented with 30U/ml human recombinant IL-2 and 3uM of scrambled peptide (SCRAM) or ANT008 for 9 days. On day 9 the cells were counted and stained for CD3, CD4, CD8, Tim-3, Lag-3, PD-1, CD25, FoxP3 and with aqua live/dead viability stain and analyzed via flow cytometry. (A) The gating strategy, (B) yield on da 9 with respect to number of cells seeded on day 0 and C) the percentage of T cells expressing co-inhibitory molecules are shown. P values were calculated by student t test . *p<0.001.
Survival correlates with VPAC1 expression (TCGA)Survival probability analysis and the respective Kaplan Meier curves from TCGA data sets for pancreatic ductal adenocarcinoma (PAAD), stomach adenocarcinoma (STAD), and colorectal adenocarcinoma (CAAD). (A) Combined survival analysis of multiple GI malignancies (PAAD, STAD and COAD) demonstrates increased VPAC1 expression is associated with a 1-year 50% survival benefit (75 vs 50%) (n=560 (high) & 557 (low)), (B) while VIP expression is not (n=540 (high) and 540 (low)). (C) In pancreatic adenocarcinoma, increased VIP receptor (VIPR1) expression trends toward an overall survival benefit (n=91 (high) & 92 (low)), while (D) VIP expression does not.
Conclusions VIP is a targetable mechanism of immune escape in PDAC. Inhibiting VIP receptor signaling improves effector properties of T cells and synergistically improves the anti-tumor response to checkpoint inhibitors in mouse PDAC models.
References
Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin2020;70(1):7-30.
Sahin IH, et al. Immunotherapy in pancreatic ductal adenocarcinoma: an emerging entity?Ann Oncol 2017;28(12):2950-2961.
Gonzalez-Rey E, Anderson P, Delgado M. Emerging roles of vasoactive intestinal peptide: a new approach for autoimmune therapy. Ann Rheum Dis 2007;66(Suppl 3):iii70-6.
Anderson P, Gonzalez-Rey E. Vasoactive intestinal peptide induces cell cycle arrest and regulatory functions in human T cells at multiple levels. Mol Cell Biol 2010;30(10):2537-51.
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