The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among β cells, yet testing this in vivo in the intact pancreas is challenging. Robo ...βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets have reduced synchronized intra-islet Ca2+ oscillations among β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by an immunosuppressive tumor microenvironment enriched with cancer-associated fibroblasts (CAF). This study used a ...convergence approach to identify tumor cell and CAF interactions through the integration of single-cell data from human tumors with human organoid coculture experiments. Analysis of a comprehensive atlas of PDAC single-cell RNA sequencing data indicated that CAF density is associated with increased inflammation and epithelial-mesenchymal transition (EMT) in epithelial cells. Transfer learning using transcriptional data from patient-derived organoid and CAF cocultures provided in silico validation of CAF induction of inflammatory and EMT epithelial cell states. Further experimental validation in cocultures demonstrated integrin beta 1 (ITGB1) and vascular endothelial factor A (VEGFA) interactions with neuropilin-1 mediating CAF-epithelial cell cross-talk. Together, this study introduces transfer learning from human single-cell data to organoid coculture analyses for experimental validation of discoveries of cell-cell cross-talk and identifies fibroblast-mediated regulation of EMT and inflammation.
Adaptation of transfer learning to relate human single-cell RNA sequencing data to organoid-CAF cocultures facilitates discovery of human pancreatic cancer intercellular interactions and uncovers cross-talk between CAFs and tumor cells through VEGFA and ITGB1.
Tumor involvement of major vascular structures limits surgical options in pancreatic adenocarcinoma (PDAC), which in turn limits opportunities for cure. Despite advances in locoregional approaches, ...there is currently no role for incomplete resection. This study evaluated a gelatinized neoantigen-targeted vaccine applied to a grossly positive resection margin in preventing local recurrence. Incomplete surgical resection was performed in mice bearing syngeneic flank Panc02 tumors, leaving a 1 mm rim adherent to the muscle bed. A previously validated vaccine consisting of neoantigen peptides, a stimulator of interferon genes (STING) agonist and AddaVax
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(termed PancVax) was embedded in a hyaluronic acid hydrogel and applied to the tumor bed. Tumor remnants, regional lymph nodes, and spleens were analyzed using histology, flow cytometry, gene expression profiling, and ELISPOT assays. The immune microenvironment at the tumor margin after surgery alone was characterized by a transient influx of myeloid-derived suppressor cells (MDSCs), prolonged neutrophil influx, and near complete loss of cytotoxic T cells. Application of PancVax gel was associated with enhanced T cell activation in the draining lymph node and expansion of neoantigen-specific T cells in the spleen. Mice implanted with PancVax gel demonstrated no evidence of residual tumor at two weeks postoperatively and healed incisions at two months postoperatively without local recurrence. In summary, application of PancVax gel at a grossly positive tumor margin led to systemic expansion of neoantigen-specific T cells and effectively prevented local recurrence. These findings support further work into locoregional adjuncts to immune modulation in PDAC.
Personalized cancer vaccines aim to activate and expand cytotoxic antitumor CD8+ T cells to recognize and kill tumor cells. However, the role of CD4+ T cell activation in the clinical benefit of ...these vaccines is not well defined. We previously established a personalized neoantigen vaccine (PancVAX) for the pancreatic cancer cell line Panc02, which activates tumor-specific CD8+ T cells but required combinatorial checkpoint modulators to achieve therapeutic efficacy. To determine the effects of neoantigen-specific CD4+ T cell activation, we generated a vaccine (PancVAX2) targeting both major histocompatibility complex class I- (MHCI-) and MHCII-specific neoantigens. Tumor-bearing mice vaccinated with PancVAX2 had significantly improved control of tumor growth and long-term survival benefit without concurrent administration of checkpoint inhibitors. PancVAX2 significantly enhanced priming and recruitment of neoantigen-specific CD8+ T cells into the tumor with lower PD-1 expression after reactivation compared with the CD8+ vaccine alone. Vaccine-induced neoantigen-specific Th1 CD4+ T cells in the tumor were associated with decreased Tregs. Consistent with this, PancVAX2 was associated with more proimmune myeloid-derived suppressor cells and M1-like macrophages in the tumor, demonstrating a less immunosuppressive tumor microenvironment. This study demonstrates the biological importance of prioritizing and including CD4+ T cell-specific neoantigens for personalized cancer vaccine modalities.
Successful pancreatic ductal adenocarcinoma (PDAC) immunotherapy necessitates optimization and maintenance of activated effector T cells (Teff). We prospectively collected and applied multi-omic ...analyses to paired pre- and post-treatment PDAC specimens collected in a platform neoadjuvant study of granulocyte-macrophage colony-stimulating factor-secreting allogeneic PDAC vaccine (GVAX) vaccine ± nivolumab (anti-programmed cell death protein 1 PD-1) to uncover sensitivity and resistance mechanisms. We show that GVAX-induced tertiary lymphoid aggregates become immune-regulatory sites in response to GVAX + nivolumab. Higher densities of tumor-associated neutrophils (TANs) following GVAX + nivolumab portend poorer overall survival (OS). Increased T cells expressing CD137 associated with cytotoxic Teff signatures and correlated with increased OS. Bulk and single-cell RNA sequencing found that nivolumab alters CD4+ T cell chemotaxis signaling in association with CD11b+ neutrophil degranulation, and CD8+ T cell expression of CD137 was required for optimal T cell activation. These findings provide insights into PD-1-regulated immune pathways in PDAC that should inform more effective therapeutic combinations that include TAN regulators and T cell activators.
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•Prospectively collected PDAC specimens from a neoadjuvant platform clinical trial•Identified sensitivity and resistance mechanisms to anti-PD-1 therapy in PDAC•Informed studies of additional immune-modulating agents in the ongoing platform trial•Generated hypotheses of reprogramed TME signals for combination immunotherapy strategies
Li et al. perform multi-omic analyses on pre- and post-treatment specimens from a pancreatic cancer neoadjuvant platform trial, and identify sensitivity and resistance mechanisms associated with anti-PD-1 combination therapy. Results associate tumor-associated neutrophils with poor outcomes but CD137+CD8+ T cells with better outcomes, suggesting treatment strategies for future interventions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
BiTE (bispecific T-cell engager) immune therapy has demonstrated clinical activity in multiple tumor indications, but its influence in the tumor microenvironment remains unclear. CLDN18.2 is ...overexpressed in solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC), both of which are characterized by the presence of immunosuppressive cells, including regulatory T cells (Tregs) and few effector T cells (Teffs).
We evaluated the activity of AMG 910, a CLDN18.2-targeted half-life extended (HLE) BiTE molecule, in GC and PDAC preclinical models and cocultured Tregs and Teffs in the presence of CLDN18.2-HLE-BiTE.
AMG 910 induced potent, specific cytotoxicity in GC and PDAC cell lines. In GSU and SNU-620 GC xenograft models, AMG 910 engaged human CD3+ T cells with tumor cells, resulting in significant antitumor activity. AMG 910 monotherapy, in combination with a programmed death-1 (PD-1) inhibitor, suppressed tumor growth and enhanced survival in an orthotopic Panc4.14 PDAC model. Moreover, Treg infusion enhanced the antitumor efficacy of AMG 910 in the Panc4.14 model. In syngeneic KPC models of PDAC, treatment with a mouse surrogate CLDN18.2-HLE-BiTE (muCLDN18.2-HLE-BiTE) or the combination with an anti-PD-1 antibody significantly inhibited tumor growth. Tregs isolated from mice bearing KPC tumors that were treated with muCLDN18.2-HLE-BiTE showed decreased T cell suppressive activity and enhanced Teff cytotoxic activity, associated with increased production of type I cytokines and expression of Teff gene signatures.
Our data suggest that BiTE molecule treatment converts Treg function from immunosuppressive to immune enhancing, leading to antitumor activity in immunologically “cold” tumors.
This study, by using clinically relevant mouse models, shows that the Claudin 18.2 bispecific T-cell engager treatment can engage antitumor effector T cells and also convert regulatory T cells from an immunosuppressive T-cell subtype to an antitumor T-cell subtype for treating pancreatic cancer and gastric cancer.
Abstract
Purpose: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dismal prognosis, low ductal cancer cellularity and a dominant tumor microenvironment (TME) in response to malignant ...degeneration. Modern single-cell RNA sequencing (scRNA-seq) platforms fundamentally improved the opportunities to analyze PDAC biology through isolation of diverse cell types including ductal, mesenchymal, myeloid and lymphoid populations. Published scRNA-seq data of PDAC patients provide innovative and astounding insights, but are limited by cohort size and intrinsically vulnerable to internal biases. Herein, we present a human single-cell PDAC atlas which links the previous sequencing efforts aiming for increasing depth and robustness with the combined analysis of transcriptomes. Methods: We selected scRNA-seq data of all patients with PDAC from six publicly available datasets (published between 2019-2020). Altogether, 61 different human samples with 142,807 cells were integrated into one dataset leveraging 15,219 genes, which were conclusively identical between all datasets based on the utilized nomenclature in the provided raw data. In addition, we extracted 16 samples with 31,587 cells from control pancreas specimens which were included in three out of the six datasets. The analyses were performed using the R statistics and Python environments utilizing established software including Monocle3 and Seurat. Results: After computational preprocessing of the integrated dataset, cell types were identified based on differentially expressed and canonical markers. The generated PDAC atlas consists of 26% ductal cancer, 2% ductal normal, 12% mesenchymal (stellate cells and cancer-associated fibroblasts), 18% myeloid, 19% lymphoid and 23% other cells (including acinar and endocrine cells). Copy number variation analyses confirmed the discrimination between cancer and normal ductal cells. Certain subpopulations within cell types were mapped based on the expression of supervised gene sets. Within the ductal cancer cell population, the Classical and Basal-like Moffitt signatures coexisted in the majority of patients with distinct ratios and predominance, which were associated with differences in the TME composition. Furthermore, the presence of myofibroblasts and inflammatory fibroblasts could be quantified at the patient-level. The reconstruction of intercellular signaling between ductal cancer cells and several TME components revealed potential ligands, receptors and transcription factors that may functionally define routes and polarity of cross-talk in PDAC. Conclusion: This human scRNA-seq atlas is the largest available dataset of patients with PDAC while harmonizing previously published data. It is engineered to analyze current knowledge gaps with increased rigor and, most importantly, overcomes obstacles related to bulk transcriptome sequencing data. Molecular characteristics of the ductal cancer cells and TME components inferred from the presented framework are promising to identify disease- and patient-specific signaling, key regulators, and therapeutic targets.
Citation Format: Benedict Kinny-Köster, Melissa R. Lyman, Dimitrios N. Sidiropoulos, Melanie Loth, Alexandra B. Puscek, Laura D. Wood, Jin He, Jun Yu, Richard A. Burkhart, Elizabeth M. Jaffee, Jacquelyn W. Zimmerman, Elana J. Fertig. A human single-cell RNA sequencing atlas of pancreatic ductal adenocarcinoma enables harmonized cell type calling and comprehensive analyses of potential intercellular signaling abstract. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-111.
The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among beta cells, yet testing this in vivo in the intact pancreas is challenging. ...Robo betaKO mice, in which the genes Robo1 and Robo2 are deleted selectively in beta cells, provide a unique model of altered islet spatial architecture without loss of beta cell differentiation or islet damage from diabetes. Combining Robo betaKO mice with intravital microscopy, we show here that Robo betaKO islets have reduced synchronized intra-islet Ca.sup.2+ oscillations among beta cells in vivo. We provide evidence that this loss is not due to a beta cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca.sup.2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.
Abstract Objective: Due to the challenge for intratumoral administration, innate agonists have not made it beyond preclinical studies for efficacy testing in most of tumor types. Pancreatic ductal ...adenocarcinoma (PDAC) has a T-cell excluded or deserted tumor microenvironment. Innate agonist treatments may serve as a T cell priming mechanism to sensitize PDACs to anti-PD-1 antibody (a-PD-1) treatment. Design: Using a transplant murine model with spontaneously formed liver metastasis and also the genetically engineered KPC mouse model that spontaneously develops PDAC, we compared the antitumor efficacy between intrahepatic/intratumoral and intramuscular systemic administration of BMS-986301, a next-generation STING agonist. Flow cytometry, Nanostring, and cytokine assays were used to evaluate local and systemic immune responses. Results: The study demonstrated that administration of STING agonist systemically via intramuscular injection is equivalent or potentially superior to its intratumoral injection in inducing both effector T cell response and antitumor efficacy. Compared to intratumoral administration, T cell exhaustion and immunosuppressive signals induced by systemic administration were attenuated. Nonetheless, either local or systemic treatment of STING agonist was associated with increased expression of CTLA-4 in the tumors. However, the combination of a-PD-1 and anti-CTLA-4 antibody with systemic STING agonist demonstrated the antitumor efficacy in the KPC mouse spontaneous PDAC model. Our study also demonstrated the feasibility and antitumor efficacy of systemic administration of BMS-986299, a new NLRP3 agonist. Conclusion: For the first time, our study supports the clinical development of innate agonists via systemic administration, instead of local administration, for treating PDAC. Citation Format: Keyu Li, Junke Wang, Birginia Espinoza, Yirui Xiong, Nan Niu, Jianxin Wang, Noelle Jurcak, Noah Rozich, Arsen Osipov, MacKenzie Henderson, Vanessa Funes, Melissa Lyman, Alex B. Blair, Brian Herbst, Mengni He, Jialong Yuan, Diego Trafton, Chunhui Yuan, Michael Wichroski, Xubao Liu, Yuquan Wei, Lei Zheng. Overcome the challenge for intratumoral injection of STING agonist for pancreatic cancer by systemic administration abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6736.
Abstract
The evolution of pancreatic ductal adenocarcinoma (PDAC) from tumor initiation to metastases requires years to decades, providing a window of opportunity for the prevention of premalignant ...progression. One of the first genetic mutations to occur early in development is in KRAS, an oncogene that is expressed by over 90% of PDACs. KRAS mutations (mKRAS) are attractive targets as they are recurrent hot–spot drivers that are expressed by, and drive the growth of all PDAC cells. Preclinical data provide compelling evidence that targeting mKRAS proteins using a Listeria-based vaccination approach in KPC mouse model results in prevention of progression from precursor lesions to PDAC. Based on these data, we hypothesize that targeting mKRAS in combination with bypassing early immune–suppressive signals will slow or even halt the progression of precursor lesions to PDA in subjects who are at high–risk. We have thus developed a clinical–grade long peptide vaccine targeting the six most common mKRAS proteins (G12V, G12R, G12A, G12C, G12D, and G13D) in PDAC patients. To establish initial safety and immunogenicity, our KRAS vaccine has been tested in 11 patients thus far in a Phase 1 clinical trial (NCT04117087) in combination with checkpoint blockade in patients who have undergone surgery and peri–operative chemotherapy and remained disease–free on imaging. Longitudinal immune data demonstrates an induction of activated and poly-functional mKRAS-specific CD4+ and CD8+ T cells, as well as effector and central memory T cells post–vaccination. In parallel, we performed paired single-cell RNA and TCR sequencing in PBMCs from a patient to obtain greater resolution of the vaccine–induced cytotoxic anti–tumor T cells. We captured 66,869 immune cells across three timepoints for analysis. Notably, we similarly observed an expansion of central memory CD4+ and CD8+ T cells post–vaccination. Using a novel computational algorithm (developed in collaboration with Dr. Elana Fertig’s lab) that takes into account physiochemical similarity of known mKRAS TCRs, we used our single–cell dataset to identify mKRAS-reactive TCRs` in the periphery that were functionally validated in vitro using CRISPR-Cas12a-based genome editing of human T cells. Overall, these studies suggest the induction of de novo, high quality mutant KRAS–specific T cells in the peripheral blood post–vaccination. Based on these immune data, we are enrolling onto a second clinical trial testing our mKRAS peptide vaccine in patients who are at high risk for developing PDAC (NCT05013216). These high-risk groups include those with strong family histories of PDAC or known pathogenic germline variants with a lifetime risk of up to 10% of developing PDA. Our preliminary data in our first three vaccinated patients shows an induction of mKRAS–specific T cell responses in the peripheral blood. Further studies characterizing the T cell response will lay the foundation for peripheral T cell–based biomarkers that could be used to predict response in a prevention setting.
Citation Format: Neeha Zaidi, Amanda Huff, Emily Marcisak-Davis, Daniel Haldar, Thatcher Heumann, Max Konig, Brian Mog, Janelle Montagne, Gabriella Longway, Lalitya Andaloori, Melissa Lyman, Ludmila Danilova, Julie Nauroth, Luciane Kagohara, Elana Fertig, Nilo Azad, Elizabeth Jaffee. Intercepting pancreatic cancer development with mutant KRAS-targeted immunotherapy abstract. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr IA013.
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