Pancreatic Ductal Adenocarcinoma Tumor Microenvironment Research Articles

Effects of losartan and vitamin D on outcomes in metastatic pancreatic adenocarcinoma.

723 Background: Active modification of surrounding stroma is a critical mechanism of tumor cell growth in pancreatic ductal adenocarcinoma (PDAC). There is some early translational evidence that losartan (L) and vitamin D (D) may affect the tumor microenvironment and supportive stroma in PDAC to help restore a physically and biologically tumor-suppressive stroma. These effects of potentially stroma-modifying drugs have not been established in the clinical management of metastatic PDAC (mPDAC), where there have been limited advances in treatment and prognosis remains poor. Methods: This is a single center, retrospective analysis of patients with mPDAC treated at Fox Chase Cancer Center from 2010 to 2019. All data including L and D use were extracted from the medical record. Patient characteristics were compared across subgroups using Chi-square tests, Fisher’s exact tests, and Wilcoxon rank sum tests. The primary outcome was overall survival (OS) from the time of metastatic diagnosis. Kaplan-Meier curves and log-rank tests were used to compare unadjusted OS across subgroups. Cox proportional hazards models were used to characterize OS adjusted for age at metastatic diagnosis, sex, initial stage at diagnosis, performance status, Charlson Comorbidity Index (CCI), hypertension, and renal disease. Results: 518 patients were included; a majority were male (54.1%), White (82.4%), and diagnosed with de novo mPDAC (54.6%). About half of patients had hypertension (53.5%) and few had renal dysfunction (5.6%). 153 (29.5%) patients were taking D and 41 (8%) patients were taking L. Women were more likely to take D than men (p < 0.001) and White patients were more likely to take L than other races/ethnicities (p = 0.006). Median OS since metastatic diagnosis for patients taking L was 11.4 months compared to 9.6 months for patients not taking L (p = 0.07). Median OS for patients taking D was 11.3 months compared to 8.7 months for those not taking D (p = 0.10). A Cox proportional hazards model adjusting for confounders, however, showed that both L (HR 0.66, p = 0.02) and D (HR 0.81, p = 0.04) were significantly associated with improved OS from time of metastatic diagnosis. Conclusions: This large retrospective study demonstrates potential clinical and survival benefit of L and D in conjunction with mPDAC treatment. L and D are safe, well tolerated, and widely used in general medical practice. These results support further investigation of the stroma-modifying potential of L and D, as well as conducting prospective trials with these agents to further validate these findings.

Expression Profiles of Cuproptosis-Related Genes Determine Distinct Subtypes of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent subtype of pancreatic cancer and one of the most malignant tumors worldwide. Due to the heterogeneity of its genomics and proteomics, the prognosis of PDAC remains disappointing despite advances in surgery and medicines. Recently, a novel form of programmed cell death, cuproptosis, was proposed, although its role in PDAC has not been investigated. This study aimed to quantify the expression of cuproptosis-related genes and characterize the novel subtypes of PDAC. To evaluate the pattern of cuproptosis in PDAC, the gene expression data and clinical information of 372 samples were collected from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. A consensus cluster analysis was performed using the transcriptional levels, genetic alterations, and individual prognostic values of seven pre-selected cuproptosis-related genes (DLAT, LIPT1, FDX1, DLD, PDHB, PDHA1, and LIAS) to identify the novel subtypes associated with cuproptosis in PDAC. A univariate Cox regression analysis was used to determine the significant prognostic indicators and cuproptosis scores among the differentially expressed genes (DEGs) between the dividing subclusters, followed by a principal component analysis. The prognostic values, immune profiles, treatment sensitivities, and cuproptosis scores were evaluated between the different subgroups. Seven cuproptosis-related genes showed aberrant expression levels and genetic alterations in the PDAC tumor microenvironment. Among them, LIPT1, LIAS, DLAT, PDHA1, and DLD were significantly correlated with overall survival. Based on the expression profiles of the seven cuproptosis-related genes, three cuproptosis clusters (Clusters A, B, and C) were identified, which were represented by different clinicopathologic features, gene expression levels, and biological processes. A total of 686 DEGs were identified among the three cuproptosis clusters, of which 35 prognosis-related DEGs were selected to further classify the PDAC samples into two subgroups with different survival rates, clinicopathologic features, immune infiltration levels, and drug sensitivities. Higher cuproptosis scores were associated with a significantly poorer prognosis. The cuproptosis subtypes, scores, and relevant genes represent valuable information for assessing the heterogeneity, treatment, and prognosis of PDAC.

Abstract IA013: Age-related changes in pancreatic fibroblasts drive pancreatic cancer growth and progression

Abstract Aging is an independent risk factor for the development of pancreatic ductal adenocarcinoma (PDAC). However, the ways in which aging alters cell populations that will ultimately comprise the PDAC tumor microenvironment (TME) are incompletely understood. PDAC tumors are highly desmoplastic, and fibroblasts alter the behavior of pancreatic cancer cells and impact treatment responses. Here, we show that normal pancreatic fibroblasts acquire tumor-promoting properties during aging using both in vitro and in vivo models. To determine if PDAC tumors may be influenced by an aged microenvironment, we performed orthotopic KPC cell injections into the pancreata of aged (>52-week-old) and young (6-8-week-old) syngeneic C57Bl/6J mice. Aged mice have significantly larger tumors, increased incidence of liver metastases, and decreased survival compared to young mice. We next queried whether aged pancreatic fibroblasts contribute to this increase in tumor growth. We generated a panel of aged (donors >55 years old) and young (donors <35 years old) normal human pancreatic fibroblasts to determine the effects of healthy aging fibroblasts on PDAC cell behavior. Conditioned media from aged human pancreatic fibroblasts significantly increases the proliferation of PDAC cells, suggesting that secreted factors produced by aged fibroblasts enhance cancer cell growth. Next, we evaluated changes in the invasiveness of PDAC cells in a 3D co-culture system with either aged or young pancreatic fibroblasts. PDAC spheroids containing cancer cells and aged fibroblasts exhibit a significant increase in invasiveness compared to those containing young fibroblasts. Aged (>52-week-old) compared to young (6-8-week-old) mouse pancreatic fibroblasts also increase the proliferation and invasiveness of KPC cells in vitro, providing evidence that aged fibroblasts may contribute to the phenotypes observed in vivo experiments using aged mouse models. In conclusion, we show that aged, non-cancer-associated pancreatic fibroblasts have the potential to promote growth, migration, and invasiveness in multiple models of pancreatic ductal adenocarcinoma. We hypothesize that factors secreted by pancreatic fibroblast change during aging and that this aged secretome may serve to establish a tumor-promoting niche for pancreatic cancer cells. Citation Format: Daniel Zabransky, Elizabeth Jaffee, Ashani Weeraratna. Age-related changes in pancreatic fibroblasts drive pancreatic cancer growth and progression [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr IA013.

Fungal mycobiome-mediated immune response: a non-negligible promoter in pancreatic oncogenesis and chemoresistance.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers in humans due to late diagnosis and poor response to treatments. The tumor microenvironment (TME) of PDAC is characterized by a distinctive, suppressive immune profile, which inhibits the protective functions of anti-tumor immunity and thereby contributes to PDAC progression. Recently, the study of Alam et al. discovered for the first time that the intratumoral fungal mycobiome could contribute to the recruitment and activation of type 2 immune cells in the TME of PDAC via enhancing the secretion of a chemoattractant, interleukin (IL-) 33. In this article, we reviewed the important findings of this study. Together with our findings, we synthetically discussed the role of the fungal mycobiome in orchestrating the immune response and thereby modulating tumor progression.

Prognostic value of tumor-associated N1/N2 neutrophil plasticity in patients following radical resection of pancreas ductal adenocarcinoma

BackgroundAs an integral part of the tumor microenvironment (TME), tumor-associated neutrophils play a crucial role in tumor development. The objective of this study was to investigate the plasticity of tumor-associated...

Pancreatic ductal adenocarcinoma holds unique features to form an immunosuppressive microenvironment: a narrative review

Pancreatic ductal adenocarcinoma (PDAC) is the most common type and composes about 90% of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis, which has been considered the least immunogenic cancer for decades. However, this characterization might be over-simplistic, and more sophisticated approaches are needed to develop effective treatment strategies. In this review, we aim to summarize studies involving PDAC immunity in different aspects to provide a multidimensional recognition and comprehensively understanding of the mechanisms underlying the tumor microenvironment (TME) of PDAC. A database search of peer-reviewed articles published in English between 2003 and 2022 in PubMed and the Web of Science was performed. Original articles and review articles relevant to the topic were selected. We emphasized the importance of investigating tumor-infiltrating lymphocytes (TILs) in pancreatic cancer, especially focusing on CD8+ T cells, along with indicating potential therapeutic strategies to turn the immune-cold PDACs into the immune-hot ones.

Abstract PR008: Overcoming PDAC T cell therapy barriers with CD47-targeted costimulatory fusion proteins

Abstract T cell therapy is a promising immunotherapy treatment option that uses genetically modified immune cells (T cells) to eliminate tumors. This approach uses the patients’ own immune cells to generate a “living drug” and can avoid the toxic side effects of other common therapies, such as radiation or chemotherapies. However, the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) can establish several obstacles to protect the tumor from T cells, including delivery of inhibitory and death signals that shut down T cells, usurping metabolic nutrients, and recruiting and/or converting immune cells to inhibitory phenotypes that block the T cell response. CD47 is upregulated in PDAC tumors, and patients with CD47hi tumors exhibit worse survival. CD47 serves as a “don’t eat me” signal by binding to the SIRPα receptor on macrophages to block phagocytosis. CD47 expression also inhibits T cell-mediated tumor rejection by blocking cross-presentation of tumor antigens by SIRPα+ dendritic cells. Reagents that interfere with SIRPα-CD47 signaling, such as monoclonal antibodies, are currently in clinical trials and have been shown to synergize with chemotherapy in PDA models, but native CD47 expression can produce erythrocyte and platelet depletion. Costimulatory signals delivered by surface-bound receptors can initiate gene expression programs that address multiple issues in the PDAC TME by mechanisms such as lowering the threshold of activation, altering metabolic programming, and reducing exhaustion. We develop engineering strategies to deliver costimulatory signals to engineered T cells. Immunomodulatory fusion proteins (IFPs) combine the ectodomain of an inhibitory T cell receptor with an intracellular costimulatory signaling domain and we have shown in proof-of-concept studies that this effectively “replaces a brake with an accelerator” in CD8 T cells. We have found that generating IFPs is not as simple as indiscriminately fusing any two proteins together and we have developed critical design concepts that inform the selection of the fusion site, ectodomain and costimulatory signaling domain pairing, and immunological synapse localization to enhance signaling. To develop a CD47-targeted IFP, we combined the SIRPa ectodomain with the CD28 signaling endodomain and tested this technology with our mesothelin-targeted TCR-T cell platform. In in vitrostudies, SIRPa-CD28 T cells exhibited enhanced proliferation, accumulation, and tumor cytotoxicity. SIRPa IFP-T cells exhibited increased intratumoral accumulation and therapeutic efficacy in the KrasLSL-G12D/+; Trp53LSL-R172H/+;p48Cre/+ (KPC) model, without toxicity or erythrocyte depletion. Here we describe how a thoughtfully engineered fusion protein can “armor” T cells against multiple obstacles and significantly improve therapeutic efficacy against PDAC. Citation Format: Shannon Oda, Leah Schmidt, Ashley Thelen, Cody Jenkins, Edison Chiu, Aitong Ruan, Philip Greenberg. Overcoming PDAC T cell therapy barriers with CD47-targeted costimulatory fusion proteins [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 PR008.

Abstract C058: Remodeling the tumor microenvironment by targeting integrin alpha V beta 3 (αvβ3) expressing cells in pancreatic cancer

Abstract ProAgio is a therapeutic cytotoxin that targets and kills integrin αvβ3-expressing cells, resulting in reduced tumor collagen, decreased formation of new blood vessels in the tumor microenvironment (TME), and prolonged survival in mouse models of pancreatic ductal adenocarcinoma (PDAC). ProAgio is currently being tested in a Phase 1 dose-finding study in patients with solid tumors including pancreas cancers. The cell subsets within the PDAC TME that express integrin αvβ3 and could be subject to ProAgio-mediated killing have not been previously studied. Using the KPC genetically engineered mouse model of PDAC, we found that ProAgio (20 mg/kg daily, x7 days) restrained tumor growth by 20% compared to vehicle treatment without changing the cancer cell proliferation rate. We subsequently re-analyzed publicly available scRNA-seq PDAC datasets to identify cells present in human and mouse PDAC tumors that co-express integrins αv and β3 and could be targets of ProAgio. We found that specific subsets of cancer-associated fibroblasts (CAFs), monocytes and macrophages expressed both integrins. Flow cytometry was performed on cells dissociated from orthotopically implanted KPC-derived tumors treated for 15 days with ProAgio or vehicle to identify TME subsets affected by ProAgio treatment. There was no significant difference in the myofibroblast (myCAF) subset, but an increase in the percentage of inflammatory CAFs (iCAFs) was seen. ProAgio treatment also significantly increased the percentage of conventional dendritic cells which play a pivotal role in antigen recognition and T cell priming. T cell abundance was unchanged, but a decrease in B cells was observed. These data demonstrate that ProAgio treatment modifies specific immune and fibroblast subsets within the PDAC TME. Ongoing studies seek to further delineate the cell subtypes affected by ProAgio and the cytokine and chemokine mediators responsible for these changes. Citation Format: Mayrel Palestino Dominguez, Philip Homan, Xianyu Zhang, Sandra Navas Reyes, Theresa Guerin, Laura Bassel, Zhi-ren Liu, Serguei Kozlov, Christine Alewine. Remodeling the tumor microenvironment by targeting integrin alpha V beta 3 (αvβ3) expressing cells in pancreatic cancer [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 C058.

Abstract PR010: Single-cell sequencing elucidates the effects of chemotherapy on cancer cell heterogeneity and the tumor microenvironment of human pancreatic adenocarcinoma

Abstract The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression. Improving our understanding of the PDAC TME and its role in response to therapy via in-depth single cell characterization will have broad clinical implications for biomarker development and therapeutic design. In this study, we performed single-cell RNA sequencing on freshly collected human PDAC samples of primary (n=16) or metastatic (n=11) origin, either before (n=20) or after (n=7) chemotherapy. We found a heterogeneous mixture of basal and classical Moffitt cancer cell subtypes in all samples, along with distinct cancer-associated fibroblast (CAF) and tumor-associated macrophage (TAM) subpopulations. We identified the major CAF subpopulations as inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs); within these subpopulations were a very few cells expressing immunogenic features which have previously been associated with antigen presenting CAFs (apCAFs). Tumor-associated macrophages (TAMs) could be categorized into two major subpopulations, C1QC+ TAMs or SPP1+ TAMs, each with distinct functional characteristics. For example, phagocytosis-associated gene sets were enriched in C1QC+ TAMs, while angiogenesis-associated gene sets were enriched in SPP1+ TAMs. Comparison of naïve and chemotherapy treated primary PDAC samples revealed that classical and basal-like cancer cells exhibited similar transcriptional responses to chemotherapy; this contrasts with some previous reports which posited a shift towards a basal-like transcriptional program among treated samples. We further noted that treated samples evinced fewer ligand-receptor interactions, particularly between TIGIT on CD8+ T cells and its ligand on cancer cells. We also identified TIGIT, not PD1, as the major inhibitory checkpoint molecule of CD8+ T cells in the PDAC TME. Altogether, our results suggest that chemotherapy impacts the PDAC TME and may further reduce response to immunotherapy. Citation Format: Daniel Weissinger, Emily A. Kawaler, Gregor Werba, Ende Zhao, Despoina Kalfakakou, Surajit Dhara, Grace Oh, Xiaohong Jing, Nina Beri, Lauren Khanna, Tamas Gonda, Paul E. Oberstein, Cristina Hajdu, Cynthia Loomis, Adriana Heguy, Mara H. Sherman, Amanda W. Lund, Theodore H. Welling, Igor Dolgalev, Aristotelis Tsirigos, Diane M. Simeone. Single-cell sequencing elucidates the effects of chemotherapy on cancer cell heterogeneity and the tumor microenvironment of human pancreatic adenocarcinoma [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 PR010.

Abstract C063: Smad4 deletion alters the composition of the tumor microenvironment in pancreatic cancer

Abstract Rationale: Pancreatic ductal adenocarcinoma (PDAC) tumors are notorious for their extensive non-malignant stroma in which the most abundant cells are cancer-associated fibroblasts (CAFs). CAFs are comprised of distinct subtypes with seemingly diverse roles within the PDAC tumor microenvironment (TME) and have been shown to influence disease progression and therapy response. However, most insights into PDAC CAF heterogeneity have been based on KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-Cre) mouse models, which recapitulate the pathophysiology of human PDAC but not its genetic complexity, representing only ~5% of patient tumor mutation profiles. We hypothesized that further understanding of CAF heterogeneity in PDAC tumors with commonly occurring mutations is essential for improving patient stratification and treatment strategies, and consequently, survival of this disease. To start exploring this, we developed and analyzed new PDAC models with deletion of Smad4, which is inactivated in ~30% of PDAC cases, in the context of Kras and Trp53 mutations. Methods: Smad4 deletion was performed using CRISPR-Cas9 technology in pancreatic cancer organoids derived from KPC tumors to generate matched KPC Smad4 knockout organoids. To assess whether this mutation profile modulates the PDAC TME, we cultured pancreatic stellate cells (PSCs), a precursor of CAFs, in media conditioned from KPC and KPC Smad4 knockout organoids and assessed the activation of inflammatory CAF (iCAF) and myofibroblastic CAF (myCAF) markers by RT-qPCR. We also established co-cultures of PSCs and organoids and performed bulk RNA-sequencing on flow-sorted CAFs and cancer cells. Finally, orthotopically-grafted organoid (OGO) transplantation mouse models were generated and pancreatic tumors were evaluated by ultrasound-based imaging, single-cell RNA-sequencing, immunohistochemistry, and flow cytometry. Results: We found that Smad4 deletion in PDAC cells, in the context of Kras and Trp53 mutations, promotes PDAC progression, as expected, and alters the PDAC TME. In particular, Smad4 deletion leads to a decrease in matrix-producing myCAFs, CD105-positive CAFs and antigen-presenting apCAFs, while increasing potentially immunosuppressive iCAFs. Additionally, pancreatic tumors with Smad4 deletion are depleted for macrophages, while enriched for neutrophils. Conclusions and Significance: This study demonstrates how distinct cancer genetics differentially shape the PDAC TME and establishes a workflow for ongoing investigation of additional relevant mutation profiles. This approach could lead to better PDAC patient stratification based on the profile of both cancer and stromal cells for more effective and targeted therapies. Citation Format: Eloise G. Lloyd, Gianluca Mucciolo, Muntadher Jihad, Judhell Sandoval Manansala, Sara Pinto Teles, Giulia Biffi. Smad4 deletion alters the composition of the tumor microenvironment in pancreatic cancer [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 C063.

Abstract C085: Spatial arrangements of immune cells of the pancreatic ductal adenocarcinoma tumor microenvironment correlated with outcomes in the phase 3 APACT trial

Abstract Introduction The newly diagnosed pancreatic ductal adenocarcinoma (PDAC) population has a high unmet need for effective treatments, with median survival of < 1 year. The phase 3 APACT (Adjuvant Pancreatic Adenocarcinoma Clinical Trial) evaluated the use of adjuvant nab-paclitaxel plus gemcitabine vs. gemcitabine in 866 patients with surgically resected PDAC. We explored the tumor microenvironment (TME) of >500 baseline resected APACT tumors to identify TME features that are associated with adverse outcomes. Methods Biopsy analyses included RNA-seq, DNA-seq, and multiplexed immunohistochemistry (mIHC). We quantified, for 533 patient biopsies, via mIHC the spatial arrangement of 7 immune cell types and 2 checkpoint markers relative to tumor and nontumor regions, and pairwise colocalization relative to each other. We identified combinations of biomarkers that correlate significantly with molecular signatures, predefined patient subsets, and overall survival (OS). Hazard ratios (HR) and p-values were computed via a Cox proportional hazards regression model which included resection and lymph node status as covariates. Significantly differentially-expressed transcripts were associated with biomarkers derived from the mIHC via unpaired t-test. Results Higher densities of CD8+ T cells within tumor regions correlated with longer OS (hazard ratio HR=0.76; p=0.03), and higher overall densities of CD163+ macrophages correlated with shorter OS (HR=1.44; p=0.006). We furthermore identified a patient subset (n=72) with a combination of higher CD8+ T cell and lower CD163+ macrophage densities that had a strikingly significant decreased HR of 0.46 (p=0.009). Moreover, patients with a high degree of spatial colocalization between CD8+ T cells and dual-positive CMAF+CD163+ M2-like macrophages observed a significant increased HR of 1.51 (p=0.0006), a biomarker only surpassed by nodal status in significance of correlation with OS (HR=1.9; p=0.00015). While this cellular colocalization was computed to be independent of cell densities, we found that the association of this colocalized pair of cell types with shorter OS was most significant for patients with lower overall CD8+ T cell densities (HR=1.84; p=0.0004). We further identified differentially regulated transcripts associated with this interaction and found specific putative ligand-receptor pairs that were also associated with lower OS. Conclusion A combination of greater infiltration of CD8+ cytotoxic T cells and lower infiltration of macrophages is associated with longer OS in the APACT trial. Moreover, the spatial colocalization between CD8+ T cells and CMAF+ M2 macrophages is associated with shorter OS. These findings were observed across both treatment arms of the study. Future investigation of this apparent interaction, and associated differentially expressed transcripts, may inform management of patients with pancreatic adenocarcinoma and increase effectiveness of PDAC therapies. Citation Format: David J. Reiss, Andrew Browne, Brian Fox, Alexander V. Ratushnyy, Maria Wang, Andrew V. Biankin, Thomas Lila. Spatial arrangements of immune cells of the pancreatic ductal adenocarcinoma tumor microenvironment correlated with outcomes in the phase 3 APACT trial [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 C085.

Abstract C036: Cancer associated fibroblasts are regulators of the tumor microenvironment in human pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a classically heterogeneous tumor for which tumor cells comprise less than 40% of the total tumor mass. To better understand mechanisms of intercellular interactions driving tumor response or resistance to chemotherapy, it is crucial to account for the complex tumor microenvironment (TME). Specifically, cancer associated fibroblasts (CAFs) have been implicated as both a tumor promoting and tumor restraining entity through disease progression and have therefore been a focus of our evolving model system. Methods: Our group created a 3-dimensional (3D), matched coculture system of patient-derived organoids (PDO), CAFs, and immune cells obtained from patients undergoing surgical resection. PDOs grown in 3D preserves the nascent tumor architecture and better replicates the disease in vitro than traditional 2D cell lines. The fully patient-matched coculture model of human PDAC allows for mechanistic interrogation into the PDAC TME by utilizing multiparameter flow cytometry, cellular sorting, and qPCR. Additionally, pharmacotyping experiments using a 5-drug regimen were used to assess chemotherapeutic resistance allowing for targeted questions exploring patient response to standard of care chemotherapy. Results: Using a combination of direct CAF-PDO coculture and PDO with CAF-conditioned media, we examined the direct (coculture) and indirect (conditioned media) impact of CAFs on PDOs. Using flow cytometry, we showed that CAF-conditioned human complete organoid media drives increased proliferation of PDOs when compared to PDOs grown in standard media. This effect was likely due to factors that are secreted from CAFs having a synergistic effect with compounds in the human complete organoid media, as this phenotype was not replicated in traditional serum free conditioned media. In direct coculture experiments of PDOs and CAFs, PDO numbers increased over time from day 2 to day 7 when compared to monoculture of PDOs, likely due to CAF presence. Lastly, to explore how CAFs may alter chemotherapeutic efficacy, PDOs were cocultured with CAFs and assessed for chemotherapeutic resistance. Pharmacotyping results showed that PDOs sorted from the PDO-CAF coculture exhibited a change in IC50 value in comparison to those PDOs that were not cocultured with CAFs. Conclusions: Here we put forth a model of PDO and CAF coculture and demonstrated its application for examining the impact of CAFs on PDAC proliferation and chemotherapeutic response. These data demonstrate the feasibility of establishing a patient-derived matched coculture model and provide the backbone for expanding this model to include representative immune cells. Citation Format: Samantha Guinn, Joseph Tandurella, Jacquelyn Zimmerman, Richard Burkhart, Elizabeth M. Jaffee. Cancer associated fibroblasts are regulators of the tumor microenvironment in human pancreatic ductal adenocarcinoma [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 C036.

Abstract IA005: Generating T cell responses against pancreatic cancer

Abstract The ultimate promise of tumor immunotherapy is dependent on the generation of an effective immune response against cancer, including CD4 and CD8 T cells. However, driving T cell responses in tumors such as pancreatic ductal adenocarcinoma (PDAC) requires overcoming significant hurdles in the tumor microenvironment (TME). Local and systemic suppression of dendritic cell function depresses endogenous T cell immunity against PDAC, which is further exacerbated by tumor cell intrinsic recruitment of suppressive myeloid cells and macrophages to the tumor site. To bypass these barriers, we’ve utilized agonistic anti-CD40 antibody, which mimics the endogenous CD40 ligand, to license and mature dendritic cells and re-educate tumor-associated myeloid cells towards a Th1/M1 phenotype in the PDAC TME. Using the KrasLSL-G12D/+,Trp53LSL-R172H/+,Pdx1-Cre (KPC) genetically engineered mouse model of pancreatic cancer, we show that combining standard-of-care chemotherapy with CD40 agonism promotes clonal T cell activation and expansion locally in the tumor site, resulting in regressions and cures of established tumors. This CD40-induced T cell response was further enhanced by the addition of dual immune checkpoint blockade (anti-PD-1 and anti-CTLA-4), but was independent of classical innate immune sensors such as Type I interferon receptor (IFNAR) and toll-like receptors (TLRs) previously thought to be critical for the generation of T cell immunity against tumors. These studies formed the preclinical rationale for trials investigating the use of agonistic CD40 therapy for patients with resectable or metastatic PDAC, and provided insight for immunological correlates of response in the clinical setting. More recently, we have reported on the negative impact of chemotherapy on the developing anti-tumor T cell response, emphasizing the need for additional investigations into the combination and sequencing of treatment modalities for enhanced outcomes in patients with PDAC. These studies reveal the potency of using an immunologically relevant mouse model to interrogate immune interventions in PDAC for patients with the greatest unmet clinical need. Citation Format: Katelyn T. Byrne. Generating T cell responses against pancreatic cancer [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 IA005.

Abstract C040: CREB-LIF axis drives immune suppression by promoting macrophage polarization in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) remains an aggressive malignancy, and its unique genetic makeup and immunosuppressive tumor microenvironment (TME) produce a lack of response to current therapies. Previously, we have identified Cyclic AMP Response Element Binding protein 1 (CREB) as an oncogenic transcriptional factor that promotes disease aggressiveness, poor survival, and immune suppression. Based on these, we sought to determine the role of tumor intrinsic CREB in promoting immunosuppressive TME in PDAC. Methods: We have generated a genetically engineered mouse model (GEMM) of pancreas-specific CREB deletion (CREBfl/fl) in LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice that phenocopy human PDAC disease. CRISPR/CAS9-based genomic editing was utilized to ablate CREB (CREBKO) in KPC tumor cells. RNA-sequencing analysis was performed in KPC CREB wild type (CREBWT) vs. CREBKO tumor cells to identify CREB-mediated transcriptomic changes. Chromatin immunoprecipitation (ChIP-qPCR) analysis was performed in KPC tumor cells. Syngeneic orthotopic tumor implantation of these cells was performed in the pancreata of mice. Immunophenotyping was accomplished to assess changes in the immune subsets with CREB deletion invivo. Additionally, these tissues were also processed for single-cell RNA (scRNA) transcriptomics analysis to evaluate changes on different cellular constituents. Results: Pancreas-specific CREB deletion in the KPC GEMM led to a significant reduction in the primary tumor burden, liver metastases, and improved overall survival compared to wild-type KPC. In assessing the immune repercussions of CREB deletion in pancreatic tumors, we observed a decreased infiltration of tumor-promoting CD11b+ F4/80+ CD206+ [M2-like tumor-associated macrophages (TAMs)] and a concomitant increase in the antigen-presenting M1-like macrophages (F4/80+MHC-IIhighCD86high). Additionally, scRNA sequencing analysis within the macrophage compartment in CREBKO tumors revealed significant enrichment of M1 hallmark signaling pathways. Also, CREB ablation in these tumors further facilitated increased infiltration of activated effector memory CD8+ T cells and resulted in enhanced adaptive immune response within the PDAC TME. Mechanistically, RNA transcriptomic-based analysis of CREBKO tumor cells revealed, Leukemia inhibitory factor (LIF) as one of the downstream targets of CREB. ChIP qPCR analysis after CREB1 pulldown confirmed its occupancy on LIF promoter regulatory region. Incubation of macrophages with CREBWT conditioned media in the presence of LIF neutralizing antibody or blocking its receptor expression using EC359 pushed these macrophages towards an M1-like phenotype, confirming its role as a mediator of tumor cell macrophage crosstalk. Conclusion: These findings broaden our understanding of the tumor cell-intrinsic role of CREB and provide new insights into its molecular underpinnings in fostering immunosuppressive profile by promoting skewness of TAMs towards M2 phenotype in PDAC. Citation Format: Siddharth Mehra, Vanessa T. Garrido, Samara Singh, Iago De Castro Silva, Anna Bianchi, Luis A. Nivelo, Nilesh U. Deshpande, Austin R. Dosch, Zhou Zhiqun, Supriya Srinivasan, Christine I. Rafie, Ifeanyichukwu C. Ogobuiro, Xi Chen, Alejandro Villarino, Jashodeep Datta, Nipun B. Merchant, Nagaraj Nagathihalli. CREB-LIF axis drives immune suppression by promoting macrophage polarization in pancreatic cancer [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 C040.

Abstract C052: Identifying mediators of perineural invasion in pancreatic cancer using spatial transcriptomics

Abstract Intratumoral nerves play important and versatile roles in cancer initiation, progression, recurrence, treatment-resistance, metastasis, morbidity, and mortality for many malignancies but the diverse molecular mechanisms underlying tumor-nerve crosstalk remain largely unknown. One of the differentiating hallmarks of pancreatic ductal adenocarcinoma (PDAC) is an exceptionally high frequency of perineural invasion (PNI), a histopathologic manifestation of tumor-nerve crosstalk whereby cancer cells recruit, migrate towards, and envelop or invade peripheral nerves. Evidence for some neurochemicals/neurotrophins involved in PNI have been uncovered, but most of the underlying work was limited by a lack of cell-type specificity, spatial context, and fragmented focus on individual pathways. To address these shortcomings, we set out to comprehensively identify cell-type specific genes spatially linked to PNI in patient tumors and then dissect the functional roles of these genes through live imaging of dorsal root ganglia (DRG) sensory neurons incubated in conditioned media from cancer cell organoids overexpressing candidate genes via CRISPR activation (CRISPRa). First, we performed whole transcriptome digital spatial profiling (NanoString GeoMx) on twelve custom tissue microarrays (n=288 cores) derived from intratumorally-matched malignant regions with and without PNI in primary resected PDAC specimens (n=31 patients). Differential gene expression (DE) analysis (FDR < 0.001) for PNI demonstrated that for malignant cells there were 271 enriched and 65 depleted genes, and for fibroblasts there were 16 enriched and 27 depleted genes. We further evaluated associations between PNI and expression of malignant subtypes previously identified from single-nucleus RNA-seq applied to 43 primary resected PDAC specimens. We found that malignant cells engaged in PNI were enriched in the mesenchymal, basaloid and neural-like progenitor (NRP) subtypes and depleted in the classical subtype. To test these associations functionally, we generated isogenic murine organoid lines (KrasG12D/+;Trp53FL/FL;R26-dCas9-VPR) overexpressing subtype-driving transcription factors and collected conditioned media. DRG sensory neurons demonstrate enhanced and suppressed growth kinetics when grown in NRP and classical conditioned media, respectively; mesenchymal and basal-like conditioned media do not appear to influence growth kinetics. These results suggest that while mesenchymal, basaloid, and NRP cells likely all play a role in cancer cell invasion of nerves, NRP cells may have an additional role in tumor-nerve tropism. Additional experiments exploring the functional effects of the top enriched and depleted genes from the DE analysis are ongoing. We anticipate that this study will provide a high-resolution understanding of critical intercellular interactions in the PDAC tumor microenvironment that facilitate PNI and tumor-nerve crosstalk more broadly to guide novel therapeutic strategies. Citation Format: William L. Hwang, Jennifer Su, Jimmy A. Guo, Carina Shiau, Jaimie L. Barth, Hannah I. Hoffman, Prajan Divakar, Jason W. Reeves, Eric Miller, Grissel Cervantes-Jaramillo, William Freed-Pastor, Vanessa Funes, Jennifer Y. Wo, Theodore S. Hong, Carlos Fernandez-del Castillo, Lei Zheng, Andrew J. Aguirre, David T. Ting, Mari Mino-Kenudson, Tyler Jacks. Identifying mediators of perineural invasion in pancreatic cancer using spatial transcriptomics [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 C052.

Abstract A017: Phase 1b Study of 2nd line vactosertib plus oxaliplatin with 5FU/LV (FOLFOX) in patients with metastatic pancreatic ductal adenocarcinoma (PDAC) who have failed first-line gemcitabine with nab-paclitaxel

Abstract Background: Despite of recent advances in chemotherapies, PDAC remains aggressive malignancies and the leading cause of cancer-related death in the world. TGF-β is strongly involved in the tumor microenvironment in PDAC, and dysregulation of TGF-β signaling is a frequent molecular disturbance in progression and metastasis. Vactosertib is an orally bioavailable TGF-β signaling inhibitor that targets the TGF-β type I receptor kinase. In in vivo studies, vactosertib in combination with FOLFOX improves pancreatic cancer survival by suppressing cell migration, invasion, and EMT, highlighting a potential clinical application of this approach for PDAC patients. Based on this preclinical study, we develop a phase 1b study to determine the RP2D and to evaluate the safety of vactosertib in combination with FOLFOX in patients with metastatic PDAC who have failed first-line gemcitabine with nab-paclitaxel. Methods: Eligible patients have histologically confirmed PDAC who have failed first-line gemcitabine/nab-paclitaxel with adequate organ function and performance status. This study is composed of two parts; dose escalation and dose expansion. In the dose escalation part, different dose levels of vactosertib (50 mg bid, 100 mg bid, and 200 mg bid) for escalation were tested, starting with dose level 0 (DL 0, 100 mg bid) with 3 to 6 subjects recruited in each cohort. DL -1 was only planned to test when DL 0 was unacceptable. In the dose expansion part, an additional backfill cohort was planned to open for determination of the final RP2D. Patients in each cohort were planned to receive vactosertib 50-200 mg orally twice per day 1-5 & day 8-12 with oxaliplatin 85 mg/m2 on day 1, LV 200mg/m2 IV on day 1, 5FU 200mg/m2 bolus on day 1 and continuous 5-FU 2400mg/m2 infusion over 48 hours every 2 weeks. The primary endpoints were to determine RP2D and to evaluate safety of this combination. The key secondary endpoints were progression free survival (PFS), overall response rate (ORR), disease control rate (DCR) based on RECIST 1.1 and overall survival. Results: A total of 16 patients were enrolled, 3 in DL 0, 4 in DL 1 and 9 in DL 1 backfill cohort. No dose limiting toxicities (DLTs) were observed and the RP2D was established as vactosertib 200 mg orally twice per day 1-5 & day 8-12 with FOLFOX. The vactosertib related adverse events (AEs) of any grade included fatigue, nausea, vomiting and anorexia. Three of 13 patients (23.1%) had partial response and 5 (38.5%) stable disease as best response, with clinical benefit rate of 61.5 % in DL 1, while there were no PR or SD in DL 0. Median PFS was 4.2 months [95% confidence interval (CI), 1.3–7.1] and median OS was 9.3 months [95% confidence interval (CI), 4.86–13.7]. Conclusion: In this phase Ib study, we demonstrated the feasibility and safety of adding vactosertib to FOLFOX in 2nd line setting, which needs to be further investigated. Updated results, including PK analyses, safety profiles, and clinical outcomes will be presented. This study was prospectively registered on ClinicalTrials.gov, NCT03666832. Citation Format: Joon Oh Park, Seung Tae Kim, Eunji Hong, Jung Yong Hong, Young Suk Park, Seong-Jin Kim. Phase 1b Study of 2nd line vactosertib plus oxaliplatin with 5FU/LV (FOLFOX) in patients with metastatic pancreatic ductal adenocarcinoma (PDAC) who have failed first-line gemcitabine with nab-paclitaxel [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 A017.

Abstract C055: The IL1RAP-blocking antibody nadunolimab disrupts pancreatic cancer cell and fibroblast crosstalk, reduces recruitment of myeloid cells and inhibits tumor growth

Abstract IL1RAP is expressed by tumor and stromal cells in pancreatic ductal adenocarcinoma (PDAC). Signaling by IL1 through the IL1R1/IL1RAP complex promotes cancer progression and contributes to the immune suppressive microenvironment in PDAC. The IL1RAP-blocking antibody nadunolimab blocks the signaling of both IL-1a and IL-1b and is currently evaluated in a phase I/IIa clinical study for PDAC (NCT03267316). Cancer-associated fibroblasts (CAFs) are a primary constituent of the PDAC stroma and has previously been shown to be regulated by IL-1. The aim of this study was to explore the functional consequences of nadunolimab treatment on the crosstalk between tumor cells and CAFs. Co-cultures of the PDAC cell line BxPC3 and pancreatic CAFs induced major changes in gene expression of both cell types as determined by RNA sequencing, indicating an extensive communication between the two cell types. Inclusion of nadunolimab to the co-cultures resulted in only 6 differentially expressed genes (padj<0.05) in the BxPC3 cells but 294 differentially expressed genes (padj<0.05) in CAFs compared to an isotype control antibody. Among the nadunolimab-downregulated genes were several cytokines, including CXCL1, CXCL2, CXCL3, CXCL6, IL8 and CCL2 (padj<0.05). Hence, we next measured cytokine concentrations in the co-culture medium and confirmed that nadunolimab treatment resulted in significant reductions of CXCL1, LIF, IL8 and CSF3 (p<0.05). We also found reduced levels of CCL2 (p=0.059). To identify which biological processes were affected by nadunolimab, we performed gene set enrichment analysis (GSEA). Nadunolimab induced a gene expression signature in the CAFs with negative enrichments of mononuclear cell migration (padj 0.003) and monocyte chemotaxis (padj 0.003). In line with these findings, conditioned media from co-cultures treated with nadunolimab exhibited reduced capacity to stimulate migration of peripheral blood monocytes in transwell assays (p=0.033). Interestingly, blockade of IL1b only using a neutralizing anti-IL1b antibody did not affect cell migration, suggesting that the broader blockage of cytokine signaling by nadunolimab was required to reduce monocyte migration. To assess whether the effects of IL1RAP-blockade by nadunolimab on PDAC-CAF crosstalk is relevant for tumor growth in vivo, PDAC cells and fibroblasts or PDAC cells alone were subcutaneously inoculated in Balb/c nude mice. Notably, treatment with nadunolimab reduced tumor growth in mice transplanted with a mixture of BxPC3 and CAFs (N=10 and N=8, p=0.035) but not in mice transplanted with BxPC3 cells only. This study demonstrates that antibody-based blockade of IL1RAP by nadunolimab disrupts interactions between PDAC cells and CAFs resulting in substantial global transcription changes in the CAFs, reduced recruitment of monocytes and decreased PDAC tumor growth in vivo. These findings suggest that targeting IL1RAP has a major impact on the PDAC tumor microenvironment and reveals new anti-tumor mechanisms of nadunolimab treatment. Citation Format: Nils Hansen, Pablo Peña, Finja Hansen, Petter Skoog, Susanne Larsson Faria, Karin von Wachenfeldt, Carl Högberg, Camilla Rydberg Millrud, David Liberg, Marcus Järås. The IL1RAP-blocking antibody nadunolimab disrupts pancreatic cancer cell and fibroblast crosstalk, reduces recruitment of myeloid cells and inhibits tumor growth [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 C055.

Abstract C061: Alternative polyadenylation regulates gene expression within the pancreatic cancer tumor microenvironment

Abstract A characteristic of pancreatic ductal adenocarcinoma (PDAC) is that about 90% of its tumor volume can be made up of dense, fibrotic stroma. Multiple studies have shown that this dense stroma strongly influences disease progression and drug delivery. Amongst the cells that make up the stroma, Cancer Associated Fibroblasts (CAFs) are important regulators directly affecting cancer progression and therapeutic resistance. CAFs are a heterogeneous population, including inflammatory, myofibroblastic, and antigen-presenting CAFs, each with specific functional contributions to the cancer phenotype. While we have been able to define CAFs phenotypically, we lack a deeper understanding of the mechanisms underlying their transcriptional heterogeneity and ultimately their contribution in driving PDAC progression. Our lab has previously shown that 3’UTR-Alternative Polyadenylation (APA) is a key mechanism underlying gene dysregulation in PDAC. APA machinery makes use of multiple Poly A Sites (PAS) in a transcript and leads to the formation of distinct isoforms of mRNA with varying lengths, commonly referred to as short or long isoforms. This lengthening and shortening of mRNA modulates mRNA stability, protein expression, and localization, thus directly affecting cellular processes including proliferation, metastasis, and migration. We have now extended these findings to an analysis of APA events within the tumor microenvironment (TME). We find that 3’ UTR shortening is significantly associated with inflammatory CAFs (iCAFs), and that these APA events are correlated with gene expression changes in iCAF marker genes. We hypothesize that APA is a crucial driver of heterogeneity within the tumor microenvironment of PDAC. We have begun to elucidate the mechanisms underlying these gene regulatory changes in vitro using human-derived CAF cell lines via genetic and pharmacological inhibition of APA. Understanding these mechanisms may help us find druggable targets that may eventually affect the overall outcomes of PDAC patients. Citation Format: Aditi H Chaubey, Michael E Feigin. Alternative polyadenylation regulates gene expression within the pancreatic cancer tumor microenvironment [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 C061.

Abstract C048: Benzodiazepines modify the pancreatic ductal adenocarcinoma tumor microenvironment

Abstract Benzodiazepines (BZDs) are a class of drugs commonly prescribed to pancreatic ductal adenocarcinoma (PDAC) patients to treat anxiety, anticipatory nausea prior to chemotherapy, and insomnia. We discovered that approximately 1 in 4 pancreatic cancer patients are prescribed BZDs and that significant differences in survival outcomes are associated with the type of BZD prescribed. Lorazepam is associated with poorer recurrence-free survival (HR 3.83 (1.53,9.57)) while alprazolam is associated with improved recurrence-free survival (HR 0.38 (0.16,0.92)). We hypothesize that these differences in survival result from off-target activation of the proton-sensing GPCR, GPR68, by n-unsubstituted BZDs, such as lorazepam. GPR68 is preferentially expressed in PDAC cancer-associated fibroblasts (CAFs). We found that n-unsubstituted BZDs commonly prescribed to PDAC patients potentiate activation of GPR68 in the presence of acidity, subsequently promoting secretion of IL-6 by CAFs in a GPR68-dependent manner. Conversely, we found that n-substituted BZDs, such as alprazolam, decrease IL-6 secretion by CAFs in a GPR68-independent manner. Using a syngeneic KPC transplant model, we found that tumors from mice treated with lorazepam had increased expression of inflammatory signaling pathways and ECM-related genes by RNA sequencing. Histologically, lorazepam-treated tumors had increased levels of collagen, alpha-smooth muscle actin staining, and high levels of ischemic necrosis in the centers of the tumors. Overall, these findings indicate that benzodiazepines can modify the PDAC tumor microenvironment, which may subsequently impact chemotherapeutic efficacy and patient survival. Citation Format: Abigail C. Cornwell, Abdulrahman A. Alahmari, Arwen A. Tisdale, Kathryn Maraszek, Swati Venkat, Michael E. Feigin. Benzodiazepines modify the pancreatic ductal adenocarcinoma tumor microenvironment [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 C048.

Abstract C007: Targeting vasoactive intestinal peptide receptor signaling enhances T-cell mediated anti-tumor response to immune checkpoint therapy in pancreatic ductal adenocarcinoma

Abstract Current immune checkpoint therapies including anti-CTLA-4 and anti-PD1 have limited efficacy in the treatment of pancreatic ductal adenocarcinoma (PDAC), largely due to a paucity of effector T cells within the tumor microenvironment (TME). In our study, we show that vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, is over-expressed in human PDAC tumors. Thus, we hypothesized that paracrine production of VIP by tumor cells within the PDAC TME constitutes an immune checkpoint pathway that limits the antitumor activity of VIP-receptor (VIP-R) expressing T cells. We first evaluated whether inhibiting VIP-R signaling by peptide-based VIP-R antagonist affects the growth of PDAC cells in vitro. Treatment of PDAC cells with increasing concentrations of VIP-R antagonist did not affect the viability of PDAC cell lines in vitro, except for a transient effect in MT5 albeit not significant. Furthermore, depletion of VIP receptor 2 (VPAC2) in a PDAC cell line, Panc02, by CRISPR-Cas9 KO had similar growth rates in vitro with a slight growth delay in vivo compared to the parental cell line. These data suggest that the expression of VPAC2 on PDAC cells may not confer a significant autocrine effect on tumor growth impacted by VIP signaling. On the contrary, human T cells cultured with VIP-R antagonists had enhanced activation, decreased proportions of Tregs, and exhausted T cells co-expressing PD-1, Tim-3, and Lag-3. Next, we performed pharmacological inhibition with VIP-R antagonists in tumor-bearing mice to model T cell homing, activation, and antitumor responses in preclinical murine models of PDAC. Daily subcutaneous injections of VIP-R antagonists had synergistic antitumor activity when combined with anti-PD-1 therapy with decreased tumor burden and improved survival. In contrast to the limited single-agent activity of anti-PD1 or VIP-R antagonists, combination therapy eliminated tumors in up to 40% of animals. Anti-tumor responses were T cell-dependent, as the combination therapy failed to improve survival in CD4 or CD8 deficient mice using knock-out strains and antibody depletion. In addition, tumor-free mice were resistant to tumor re-challenge, indicating the generation of anti-cancer immunological memory. In correspondent to these data, combination therapy led to enhanced T cell activation in vivo with increased antigen-specific T cells, decreased frequencies of Tregs as well as enhanced T cell infiltration to the tumor site as observed by infiltration of adoptively transferred GFP+ T cells into PDAC tumors. Taken together, our findings show that VIP-VIPR signaling is a targetable mechanism of immune escape in PDAC. Inhibiting VIP-R signaling improves T cell effector properties and enhances the responsiveness of PDAC to immune checkpoint therapy. Further pharmacokinetic and toxicology studies are underway. Citation Format: Tenzin Passang, Sruthi Ravindranathan, Jian-Ming Li, Shuhua Wang, Michael Brandon Ware, Hanwen Zhang, Gregory B. Lesinski, Edmund K. Waller. Targeting vasoactive intestinal peptide receptor signaling enhances T-cell mediated anti-tumor response to immune checkpoint therapy in pancreatic ductal adenocarcinoma [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 C007.