Pancreatic Ductal Adenocarcinoma Microenvironment Research Articles

Multiplexed Imaging Mass Cytometry Analysis Characterizes the Vascular Niche in Pancreatic Cancer.

Oncogenesis and progression of pancreatic ductal adenocarcinoma (PDAC) is driven by complex interactions between the neoplastic component and the tumor microenvironment (TME), which includes immune, stromal, and parenchymal cells. In particular, most PDACs are characterized by a hypovascular and hypoxic environment that alters tumor cell behavior and limits the efficacy of chemotherapy and immunotherapy. Characterization of the spatial features of the vascular niche could advance our understanding of inter- and intra-tumoral heterogeneity in PDAC. Here, we investigated the vascular microenvironment of PDAC by applying imaging mass cytometry using a 26-antibody panel on 35 regions of interest (ROIs) across 9 patients, capturing over 140,000 single cells. The approach distinguished major cell types, including multiple populations of lymphoid and myeloid cells, endocrine cells, ductal cells, stromal cells, and endothelial cells. Evaluation of cellular neighborhoods identified 10 distinct spatial domains, including multiple immune and tumor-enriched environments as well as the vascular niche. Focused analysis revealed differential interactions between immune populations and the vasculature and identified distinct spatial domains wherein tumor cell proliferation occurs. Importantly, the vascular niche was closely associated with a population of CD44-expressing macrophages enriched for a pro-angiogenic gene signature. Together, this study provides insights into the spatial heterogeneity of PDAC and suggests a role for CD44-expressing macrophages in shaping the vascular niche.

Targeted Therapy for Highly Desmoplastic and Immunosuppressive Tumor Microenvironment of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a very poor prognosis. Despite advancements in treatment strategies, PDAC remains recalcitrant to therapies because patients are often diagnosed at an advanced stage. The advanced stage of PDAC is characterized by metastasis, which typically renders it unresectable by surgery or untreatable by chemotherapy. The tumor microenvironment (TME) of PDAC comprises highly proliferative myofibroblast-like cells and hosts the intense deposition of a extracellular matrix component that forms dense fibrous connective tissue, a process called the desmoplastic reaction. In desmoplastic TMEs, the incessant aberration of signaling pathways contributes to immunosuppression by suppressing antitumor immunity. This feature offers a protective barrier that impedes the targeted delivery of drugs. In addition, the efficacy of immunotherapy is compromised because of the immune cold TME of PDAC. Targeted therapy approaches towards stromal and immunosuppressive TMEs are challenging. In this review, we discuss cellular and non-cellular TME components that contain actionable targets for drug development. We also highlight findings from preclinical studies and provide updates about the efficacies of new investigational drugs in clinical trials.

Tumor Stroma as a Therapeutic Target for Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis owing to its desmoplastic stroma. Therefore, therapeutic strategies targeting this tumor stroma should be developed. In this study, we describe the heterogeneity of cancer-associated fibroblasts (CAFs) and their diverse roles in the progression, immune evasion, and resistance to treatment of PDAC. We subclassified the spatial distribution and functional activity of CAFs to highlight their effects on prognosis and drug delivery. Extracellular matrix components such as collagen and hyaluronan are described for their roles in tumor behavior and treatment outcomes, implying their potential as therapeutic targets. We also discussed the roles of extracellular matrix (ECM) including matrix metalloproteinases and tissue inhibitors in PDAC progression. Finally, we explored the role of the adaptive and innate immune systems in shaping the PDAC microenvironment and potential therapeutic strategies, with a focus on immune cell subsets, cytokines, and immunosuppressive mechanisms. These insights provide a comprehensive understanding of PDAC and pave the way for the development of prognostic markers and therapeutic interventions.

Abstract CT016: Immunomodulation of the tumor microenvironment of pancreatic ductal adenocarcinoma with histone deacetylase inhibition: Results of a phase 2 clinical trial of entinostat in combination with nivolumab

Abstract Background The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDA) is characterized by a paucity of cytotoxic tumor-infiltrating lymphocytes, an abundance of immune-suppressive cell populations, and a general resistance to immune checkpoint inhibitor (ICI) therapy. In preclinical models of PDA, the HDAC inhibitor entinostat modulated the transcriptional programming of myeloid cells, reducing their capacity for immunosuppression and sensitizing tumors to ICI therapy. Methods We conducted a single-center, open-label, Simon two-stage, phase II study of entinostat in combination with the PD1 inhibitor nivolumab in patients with advanced PDA. Patients received oral entinostat 5 mg once a week. After a 14-day lead-in with entinostat monotherapy, patients concurrently receive entinostat 5 mg orally once a week plus nivolumab 240 mg intravenously every 2 weeks. After 4 months, therapy was continued with entinostat 5 mg weekly plus nivolumab at a dose of 480 mg fixed dose every 4 weeks until the time of progression or unacceptable toxicities. The primary endpoint was the objective response rate by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Peripheral immune profiles at baseline and on treatment were assessed by high-dimensional mass cytometry by time of flight (CyTOF) and by Luminex of chemokines in the plasma, and changes in the TME were performed on paired baseline and on treatment biopsies by multiplexed immunohistochemistry (mIHC), image cytometry-based quantification, and bulk RNA-seq analysis. Results From November 2017 to November 2020, 27 evaluable patients were enrolled. Three patients had a partial response (PR) by RECIST v1.1 criteria (objective response rate (ORR)) of 11%, 95% CI, 0.024-0.292), with a median duration of response of 10.2 months. Grade ≥3 treatment-related adverse events (TRAEs) were observed in 19 (63%) patients. The most common grade ≥3 TRAEs were decreased lymphocyte count, anemia hypoalbuminemia, and hyponatremia. Dendritic cell (DC) activation, maturation, migration, antigen processing, and presentation were increased in the periphery after entinostat treatment, consistent with HDAC inhibitor mediated myeloid reprogramming. Gene expression analysis of paired baseline and on treatment tumors demonstrated an enrichment in inflammatory response signaling pathways with combination treatment. Conclusions Entinostat and nivolumab demonstrated durable radiological responses in a subset of patients with PDA. Paired tissue and peripheral analysis showed immunomodulation of myeloid cell populations, consistent with the preclinical hypothesis supporting the trial. This study creates a roadmap for this strategy with future combinatorial therapeutic approaches to enhance the clinical benefit in PDA patients further. Citation Format: Marina Baretti, Ludmila Danilova, Jennifer N. Durham, Courtney B. Betts, Leslie Cope, Dimitrios N. Sidiropoulos, Joseph A. Tandurella, Soren Charmsaz, Nicole Gross, Alexei Hernandez, Won J. HO, Chris Thoburn, Rosalind Walker, James Leatherman Leatherman, Sarah Mitchell, Brian Christmas, Daria A. Gaykalova, Srinivasan Yegnasubramanian, Elana J. Fertig, Lisa M. Coussens, Mark Yarchoan, Elizabeth Jaffee, Nilofer S. Azad. Immunomodulation of the tumor microenvironment of pancreatic ductal adenocarcinoma with histone deacetylase inhibition: Results of a phase 2 clinical trial of entinostat in combination with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT016.

Abstract LB316: Endocrine pancreas alteration in pancreatic ductal adenocarcinoma

Abstract Background: The pancreas is central to energy homeostasis, playing essential roles in digestion and regulation of blood glucose. Exocrine pancreas contains large amounts of of hydrolytic enzyme which is responsible for the digestion. Acinar cells, as well as ductal cells, represent the prime components of exocrine pancreas. They are both supposed to be the origin of pancreatic ductal adenocarcinoma(PDAC) and the researches focused on these exocrine cells, as well as other components in pancreatic cancer, are numerous. Islet cells are crucial for glucose homeostasis and their identity are proved to be altered significantly during diabetes by single-cell RNA-sequencing(scRNA-seq). However, the presence or extent of variation of islet cells in PDAC is not established yet. Moreover, due to the low proportion of endocrine cells in pancreas, it is hard to analyze the data of endocrine cells by using published scRNA-seq data of PDAC. As a consequence of this underrepresentation, the function of endocrine cells in PDAC remains unclear. Methods: We used scRNA-seq, multiplex immunohistochemistry (mIHC), high-throughput spatial transcriptomic sequencing (Stereo-seq) to detect the alteration of endocrine pancreas in PDAC of human and mouse model. KPC mice (KrasLSL-G12Dp53LoxPPdx1-CreER) and pancreatic orthotopic transplantation mice were applied to verify the islet cell alteration in multiple stages of PDAC. We also performed islet isolation and purification to enhance the proportion of endocrine cells when preparing single-cell suspension. Cell co-culture experiments were used to determine the interaction between exocrine-endocrine compartments. Results: Through mIHC of islet cells in PDAC tissues and normal pancreas tissues of mice (KPC vs. C57BL/6J) as well as human (PDAC patients vs. healthy donors), we found the decreased proportions of inulin positive (INS+) cells and increased proportions of glucagon positive (GCG+) cells in PDAC tissues. Furthermore, the proportion of pancreatic polypeptide positive (PPY+) cells was significantly elevated in PDAC tissue in both human and murine. Then, we quantified the double-positive cells and found the proportions of INS+GCG+, INS+PPY+ and GCG+PPY+ were markedly increased in PDAC tissue. Additionally, through the experiments in mouse model, we found the islet cells altered gradually within the multiple stage of PDAC(normal-PanIN-PDAC). Finally, it was found that PDAC cells could induce the transdifferentiation of pancreatic α and β cells into GCG+PPY+ and INS+PPY+ double-positive cells, which further promoted PDAC proliferation in a paracrine-dependent manner and formed a reciprocal interaction. Conclusions: Our study systematically maps the alteration of pancreatic endocrine cells in PDAC and elucidates the potential interaction mechanisms between endocrine and exocrine cells during PDAC carcinogenesis. Meanwhile, it is suggested that pancreatic endocrine cells should be considered as an important component of the PDAC microenvironment. Citation Format: Yuan Chen, Xinpeng Yin, Ruiyuan Xu, Rexiati Ruze, Jianlu Song, Chenglin Hu, Chengcheng Wang, Yupei Zhao. Endocrine pancreas alteration in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB316.

Solute carrier family 16 member 1 as a potential prognostic factor for pancreatic ductal adenocarcinoma and its influence on tumor immunity.

Solute carrier family 16 member 1 (SLC16A1) serves as a biomarker in numerous types of cancer. Tumor immune infiltration has drawn increasing attention in cancer progression and treatment. The objective of our study was to explore the association between SLC16A1 and the tumor immune microenvironment in pancreatic ductal adenocarcinoma (PDAC). Data were obtained from The Cancer Genome Atlas. The xCell web tool was used to calculate the proportion of immune cells according to SLC16A1 expression. To further explore the mechanism of SLC16A1, immunity-related genes were screened from differentially expressed genes through weighted gene coexpression network analysis, examined via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, and filtrated using univariate Cox regression and least absolute shrinkage and selection operator regression model combined correlation analysis (P<0.05). Next, CIBERSORT was used to analyze the correlation between immune cells and five important genes. SLC16A1 expression and its clinical role in pancreatic cancer was clarified via immunohistochemical staining experiments. Finally, the effects of SLC16A1 on the results of cancer immunity were evaluated by in vitro experiments. SLC16A1 was overexpressed in PDAC tissues and could be an independent prognostic factor. SLC16A1 was significantly negatively correlated with overall survival and suppressed the tumor immunity of PDAC. In clinic, SLC16A1 expression was significantly positively correlated with tumor progression and poor prognosis. We also found that SLC16A1 could suppress the antitumor ability of CD8+ T cells. SLC16A1 is a biomarker for the prognosis of PDAC and can influence the immune environment of PDAC. These findings provide new insights into the treatment of PDAC.

Abstract 1580: Investigating the tumor microenvironment of pancreatic ductal adenocarcinoma with BRCA2 mutation

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest cancers with a survival rate of less than 12% at 5 years. It is estimated that around 14% of human PDAC contain mutations in genes involved in DNA Damage Repair (DDR), including BRCA1/2, ATM, and others. Additionally, PDAC has an extensive network of extracellular matrix components surrounding the tumor in the stroma, such as cancer-associated fibroblasts, which promote cancer cell growth. The interactions of the tumor with its tumor microenvironment and stroma can bring vulnerabilities that could be targeted if identified. New therapeutic strategies to those targets can be developed for specifically PDAC with DDR mutations. Using publicly available RNA-seq dataset of PDAC patients from The Cancer Genome Atlas (TCGA), I computationally estimated the infiltration of immune and stromal cell types into the tumor. From the analyses, I found that there was a significant difference in the migration of Common Lymphoid Progenitor (CLP) cells into PDAC with mutations to DDR genes. CLP cells usually divide into T-cells, B-cells, and NK cells, suggesting that DDR mutations could cause a more immunogenic environment. There was also a decrease in the infiltration of stromal elements, mainly fibroblasts, into PDAC with mutations to DDR genes. In the experiment I am currently working on, I hypothesize there are also in vivo differences in the infiltration rates of certain immune cell types and fibroblasts into BRCA2-mutated PDAC, since BRCA2 is the most commonly mutated DDR gene in PDAC. For my current experimental design, the mice in the experimental group will be orthotopically implanted with PDAC with Brca2 knockout. The control group will have mice orthotopically implanted with PDAC without Brca2 knockout. After letting the tumors grow for 3 weeks, the mice will be sacrificed and the pancreatic tumors will be collected and stained through trichrome staining. Trichrome staining probes for the stromal cancer-associated fibroblasts. Furthermore, immunohistochemistry (IHC) staining will be done to look for specific populations of cells, such as specific types of fibroblasts, effective and immunosuppressive immune cells including T-cells, B-cells, and NK cells. The aim is to see whether the computational findings in regards to the tumor microenvironment are also reflected in vivo. I will have the data from this experiment by the time of the conference in April 2024. Overall, my project is about investigating the tumor microenvironment and fibroblast abundance in PDAC with BRCA2 knockout mice, using computational data, IHC staining and trichrome staining. Citation Format: Fatimah Al-Musawi, Chang-il Hwang. Investigating the tumor microenvironment of pancreatic ductal adenocarcinoma with BRCA2 mutation [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 1580.

Abstract 5327: Interleukin-1 upregulates dual oxidase 2 expression and ROS production in human pancreatic cancer cells

Abstract Chronic pancreatitis increases the risk of developing pancreatic ductal adenocarcinoma (PDAC) and is associated with enhanced expression of the NADPH oxidase (NOX) isoform dual oxidase 2 (DUOX2). Interleukin-1 (IL-1) is a major upstream pro-inflammatory cytokine secreted by malignant or microenvironmental cells that supports pancreatic inflammation, tumorigenesis, invasiveness, and tumor heterogeneity. Previously, our laboratory demonstrated that the expression of DUOX2, one of seven NADPH oxidase (NOX) family members, is significantly enhanced by a wide variety of pro-inflammatory cytokines, and that DUOX2-related ROS production may contribute to the development of an oxidative, pro-angiogenic microenvironment in PDAC. In the current studies, we have shown that DUOX2 is upregulated and expressed along with its maturation factor, DUOXA2, in several human pancreatic cancer cell lines following exposure to both IL-1α and IL-1ß, two major agonists of IL-1 signaling. Administration of the anti-inflammatory antibody anakinra (an IL-1R1 antagonist) either in conjunction with or after prolonged treatment with IL-1 in vitro ablated induction of DUOX2/DUOXA2 and of IL-8 (CXCL8), a chemokine essential for neutrophil recruitment and a potent promoter of angiogenesis. Increased expression of other NOX family members following IL-1 exposure was not observed. Using CRISPR/Cas9-mediated DUOX2 knockout models, we report that DUOX2 is the major contributor to IL-1-mediated ROS production in PDAC cells. In a panel of PDAC lines, siRNA knockdown of proximal IL-1 signaling nodes, such as MYD88, significantly decreased expression of DUOX2/DUOXA2 and of CXCL8. However, knockdown of IRAK1 or IRAK4, two critical signaling kinases downstream of MYD88, as well as known downstream mediators of IL-1-related transcription (such as NF-κB, STAT1, MEK/ERK), did not affect IL-1-induced DUOX2/DUOXA2 upregulation, suggesting that alternative pathways of IL-1 signaling might be involved in the enhancement of DUOX2 expression by the cytokine. Because IL-1 signaling is known to affect various acetylation/methylation events in epithelial cells, we evaluated the effect of administering the histone acetyltransferase (HAT) inhibitor A-485 with IL-1; we found that A-485 significantly diminished cytokine-induced DUOX2/DUOXA2 upregulation; however, CXCL8 induction was unchanged. These studies suggest that epigenetic modulation of PDAC cells by IL-1 could contribute to DUOX2-induced ROS production and ROS-related inflammatory stress in the PDAC microenvironment. Citation Format: David J. Mallick, Yongzhong Wu, Mariam M. Konaté, Becky Diebold, Smitha Antony, Jennifer L. Meitzler, Guojian Jiang, Jiamo Lu, Krishnendu Roy, James H. Doroshow. Interleukin-1 upregulates dual oxidase 2 expression and ROS production in human pancreatic cancer cells [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 5327.

Abstract 5357: The tumor-intrinsic RNA binding protein HuR is essential for anti-tumor immunity in PDAC

Abstract Immunotherapies have shown limited effectiveness in pancreatic ductal adenocarcinoma (PDAC), despite having success in other cancers. The RNA-binding protein HuR is known to play a critical role in the oncogenesis of PDAC by regulating key mRNA transcripts. Our lab has shown that tumor-intrinsic HuR is overexpressed in PDAC and regulates the tumor microenvironment composition, altering multiple tumor cytokines to release. Thus, we hypothesized that HuR plays a role in immune evasion of PDAC. We disrupted the locus encoding HuR (Elavl1) using CRISPR Cas9 in Kras-p53 mutant-driven (KPC) murine PDAC to assess its function in tumor immune evasion. Here, we report that HuR, a known pro-survival factor for PDAC, has a role in tumor immune evasion by suppressing T cell infiltration and T cell activation. Specifically, HuR-knockout (KO) KPC tumors grew slower compared to KPC wildtype (WT) tumors in an orthotopically implanted model (p-value &amp;lt; 0.0001), even though human PDAC cell lines with HuR-WT and HuR-KO grew at the same rate in NRG mice (in an immune-compromised environment). Accordingly, HuR-KO tumors had more T cell infiltration and activated T cells compared to HuR-WT tumors (p-value = 0.0047). Importantly, T cell depletion (both CD4+ and CD8+) partially rescued the size of HuR-KO tumors. Next, we rescued the expression of HuR in the HuR-KO cell line, and the re-expression partially rescued the tumor size in vivo. This observation validates a HuR-mediated dependent mechanism. Collectively, these data support the hypothesis that HuR suppresses T cell activation and prevents T cells from infiltrating into the PDAC microenvironment, leading to PDAC immune evasion. Ongoing experiments will transduce KPC cell lines with ovalbumin (OVA) antigen, which will selectively activate CD8+ OT-I T cells through their transgenic TCR, to measure the ability of KPC HuR-WT or HuR-KO OVA cells in directly activating CD8+ OT-I T cells. We will also investigate HuR's impact on immune cells' cytokine release and how that will impact the cytotoxicity of T cells. These findings support the ongoing concept that HuR plays an important pro-survival role in PDAC in vivo, and targeting strategies against HuR being developed (e.g., siHuR nano-therapies and small molecule inhibitors) could potentially enhance PDAC sensitivity to immune-based anti-cancer therapies, such as checkpoint blockade and T cell transfer. Citation Format: Yifei Guo, Jennifer M. Finan, Alexandra Q. Bartlett, Jonathan R. Brody, Robert Eil. The tumor-intrinsic RNA binding protein HuR is essential for anti-tumor immunity in PDAC [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 5357.

Abstract 6818: Obesity-mediated perineural invasion promotes PDAC tumorigenesis

Abstract Obesity is a modifiable host factor that has been reported to increase the risk of pancreatic ductal adenocarcinoma (PDAC) by ~60% and the mortality by two-fold, however, the exact mechanisms by which obesity enhances PDAC aggressiveness is incompletely understood. Given that nearly 74% of U.S. adults are overweight or obese (US Center for Disease Control), resolving the complex association between obesity and PDAC may unveil new targets for prevention and/or intervention to decrease incidence and improve outcomes. The prevailing notion is that obesity-induced tumor in situ factors (i.e., oxidative, hypoxic, nutrient, and biochemical) play crucial roles in enhancing cancer cell fitness and survival. However, these concepts have yet to lead to the identification of any actionable target in patients, with obesity-related cancer in general and PDAC in particular. Prior studies and our preliminary data show the presence of a distinctive tumor immune profile in PDAC models under conditions of obesity, which encompasses immune suppressive myeloid and type 2 immune cells, namely M2-like tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs), that inhibit effector CD8+ T cell expansion and function enabling PDAC progression. However, the genesis of this ‘type 2 immune cell’ dominant tumor-immune microenvironment (TIME) of obesity-driven PDAC remains to be fully understood. This led us to take a holistic approach utilizing multiple genetically engineered mouse models (GEMMs), pharmacological inhibitors, transcriptomic analysis (RNA-seq, spatial transcriptomic, and scRNA-seq), fresh PDAC patient samples, and imaging technologies (confocal microscopy) to understand the molecular mechanisms. Our novel mechanistic study using some of the above-mentioned tools shows that obesity-driven PDAC has significantly higher perineural invasion (PNI) that enhances PDAC progression. We found that sympathetic nerve terminals (of the PNI) via neurotransmitter release control the expression of integral cytokines that cause a dramatic remodeling of the TIME. Specifically, the PNI-mediated neuro-adrenergic signaling activates β2-adrenergic receptor (β2-AR) on PDAC cells, leading to increased tumor cell production/proliferation and secretion of the ‘alarmins’ IL-33 and IL-1β. Importantly, we showed that mitigation of adrenergic signaling using two separate approaches: (i) pharmacological antagonists of β2-ARs (β-blockers), and (ii) the use of a thermoneutral model leads to substantial abrogation of obesity-mediated tumor progression. These, and other key data raise the provocative question that reducing adrenergic signaling-dependent IL-33 expression and release favor a pro-inflammatory TIME and significantly increase patient survival in obesity-driven PDAC. Citation Format: Shyamananda Singh Mayengbam, Aftab Alam, Sharon Senchanthisai, Irmak Bektas, Prasenjit Dey. Obesity-mediated perineural invasion promotes PDAC tumorigenesis [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 6818.

Nanodrug-bacteria conjugates-mediated oncogenic collagen depletion enhances immune checkpoint blockade therapy against pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) cancer cells specifically produce abnormal oncogenic collagen to bind with integrin α3β1 receptor and activate the downstream focal adhesion kinase (FAK), protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) signaling pathway. Collectively, this promotes immunosuppression and tumor proliferation and restricts the response rate of clinical cancer immunotherapies. Here, by leveraging the hypoxia tropism and excellent motility of the probiotic Escherichia coli strain Nissle 1917 (ECN), we developed nanodrug-bacteria conjugates to penetrate the extracellular matrix (ECM) and shuttle the surface-conjugated protein cages composed of collagenases and anti-programmed death-ligand 1 (PD-L1) antibodies to PDAC tumor parenchyma. We found the oncogenic collagen expression in human pancreatic cancer patients and demonstrated its interaction with integrin α3β1. We proved that reactive oxygen species (ROS) in the microenvironment of PDAC triggered collagenase release to degrade oncogenic collagen and block integrin α3β1-FAK signaling pathway, thus overcoming the immunosuppression and synergizing with anti-PD-L1 immunotherapy. Collectively, our study highlights the significance of oncogenic collagen in PDAC immunotherapy, and consequently, we developed a therapeutic strategy that can deplete oncogenic collagen to synergize with immune checkpoint blockade for enhanced PDAC treatment efficacy. This work was supported by the University of Wisconsin Carbone Cancer Center Research Collaborative and Pancreas Cancer Research Task Force, UWCCC Transdisciplinary Cancer Immunology-Immunotherapy Pilot Project, and the start-up package from the University of Wisconsin-Madison (to Q.H.).

Abstract 1597: CD144+cancer-associated fibroblasts drive the malignancy of pancreatic cancer via stimulating inflammatory paracrine

Abstract Purpose: Cancer-associated fibroblasts (CAFs) are highly heterogeneous in pancreatic ductal adenocarcinoma (PDAC). We previously reported that CAFs can acquire an endothelial phenotype under stress. Thus, we hypothesized that there are CAFs highly expressing endothelial markers and substantiated a novel subtype of CAFs promoting tumor progression. Experimental design: We performed single-cell RNA sequencing to identify the existence of CD144+CAFs. The prospective and retrospective analysis were combined to figure out the correlation between CD144+CAFs proportion and clinical outcome. The proliferation and invasion assay of cell lines, organoids and pancreatic cancer xenograft model were used to detect the effect of CD144+CAFs on tumor progression. Cytokine array assay, RNA-sequencing, IP-mass spectrum, Ch-IP and luciferase analysis were conducted to elucidate the underlying mechanism. siRNA delivery nanosystem was designed and administrated to precisely target CD144+CAFs in vivo. Results: CD144+CAFs were present in tumor microenvironment of PDAC and breast cancer and patients with a higher CD144+CAFs proportion have worse prognosis in PDAC. CD144+CAFs can promote the metastasis of PDAC through enhancing epithelial-mesenchymal transition. CD144-β-catenin-STAT3 signaling axis was activated and downstream inflammatory secreted cytokines were transcriptionally upregulated to maintain the functions and phenotype of CD144+CAFs. A novel CAF-targeting siRNA delivery nanosystem, loading FAPα and siCDH5, was designed and administrated to precisely target CD144+CAFs, which substantially inhibited the protumoral roles in vivo. Conclusion: CD144+CAFs can promote the growth and metastasis of PDAC by activating CD144-β-catenin-STAT3 signaling axis and stimulating inflammatory secretion. CAF-targeting siRNA delivery nanosystem can inhibit tumor progression by precisely targeting CD144+CAFs. Citation Format: Xugang Sun, Jun Yu, Tiansuo Zhao, Jihui Hao. CD144+cancer-associated fibroblasts drive the malignancy of pancreatic cancer via stimulating inflammatory paracrine [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 1597.

Abstract 6092: Three-dimensional multi-omic analysis of early invasion of human pancreatic ductal adenocarcinoma from IPMN

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer, with only 11% of patients surviving beyond 5 years. Intraductal papillary mucinous neoplasm (IPMN), a precancerous lesion and the most common type of neoplastic pancreatic cyst, presents a critical opportunity for cancer interception, but the drivers of malignant progression in IPMN are still largely unknown. Although the immunosuppressive microenvironment of PDAC has been well characterized, the timing of the development of these immune alterations in IPMNs is not well studied and could provide a rational foundation for cancer immuno-prevention. Moreover, the inception of invasive PDAC and its spread into the microenvironment from IPMN is complex, multifactorial, and, due to its unique anatomy, challenging to analyze with traditional 2-dimensional visualization. We sought to capture the precise point of transition from IPMN to invasive carcinoma utilizing CODA, a supervised deep learning tool for three-dimensional (3D) reconstruction of serially sectioned human tissue, in order to quantitatively assess the molecular and cellular alterations associated with malignant progression. Method: Formalin-fixed paraffin-embedded (FFPE) tissue blocks with PDAC arising from IPMN were serially sectioned, every third slide was stained with H&amp;E, and digitized at 20x magnification. Following pathologist-guided annotations on a subset of H&amp;E slides, CODA generated 3D models of each tissue block, including automated annotation of 9 pancreatic tissue components. Using these annotations, we identified the transition from IPMN to invasive carcinoma in each sample and selected regions for multi-omic profiling based on quantitative features of the cellular microenvironment. Multi-omic profiling of IPMN, transition zone, and PDAC included laser capture microdissection followed by whole exome sequencing to identify somatic DNA alterations, as well as spatial transcriptomics to identify alterations in gene expression in neoplastic cells and spatial proteomics to identify cellular alterations in the surrounding microenvironment. Results: CODA generated accurate annotation on all the H&amp;E slides from each case and robustly identified regions of interest for multi-omic profiling. Spatial proteomic profiling using imaging mass cytometry revealed a decrease in T cell density as IPMNs transition into PDAC. Sub- clustering of the T cell compartment identified decreases in multiple T cell subsets, including activated cytotoxic T cells and helper T cells, in the transition zone compared to IPMN. Conclusion: In this study, we integrated multi-omic profiling with CODA-generated high-resolution 3D tissue maps to identify molecular and cellular drivers of malignant progression in human PDAC. Citation Format: Shalini Datta, Sarah M. Shin, Jessie Kanacharoen, Michael Johannes Pflüger, Katsuya Hirose, André Forjaz, Sarah Graham, Pei-Hsun Wu, Ralph H. Hruban, Denis Wirtz, Won Jin Ho, Ashley L. Kiemen, Laura D. Wood. Three-dimensional multi-omic analysis of early invasion of human pancreatic ductal adenocarcinoma from IPMN [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 6092.

A histone methylation-MAPK signaling axis drives durable epithelial-mesenchymal transition in hypoxic pancreatic cancer.

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) plays a key role in tumor progression and response to therapy. The dense PDAC stroma causes hypovascularity, which leads to hypoxia. Here, we showed that hypoxia drives long-lasting epithelial-mesenchymal transition (EMT) in PDAC primarily through a positive-feedback histone methylation-MAPK signaling axis. Transformed cells preferentially underwent EMT in hypoxic tumor regions in multiple model systems. Hypoxia drove a cell-autonomous EMT in PDAC cells which, unlike EMT in response to growth factors, could last for weeks. Furthermore, hypoxia reduced histone demethylase KDM2A activity, suppressed PP2 family phosphatase expression, and activated MAPKs to post-translationally stabilize histone methyltransferase NSD2, leading to an H3K36me2-dependent EMT in which hypoxia-inducible factors played only a supporting role. Hypoxia-driven EMT could be antagonized in vivo by combinations of MAPK inhibitors. Collectively, these results suggest hypoxia promotes durable EMT in PDAC by inducing a histone methylation-MAPK axis that can be effectively targeted with multi-drug therapies, providing a potential strategy for overcoming chemoresistance.

Cancer-Associated Fibroblasts in Pancreatic Ductal Adenocarcinoma or a Metaphor for Heterogeneity: From Single-Cell Analysis to Whole-Body Imaging.

Pancreatic ductal adenocarcinoma (PDAC) represents a formidable challenge due to its aggressive nature and poor prognosis. The tumor microenvironment (TME) in PDAC, characterized by intense stromal desmoplastic reactions and a dominant presence of cancer-associated fibroblasts (CAFs), significantly contributes to therapeutic resistance. However, within the heterogeneous CAF population, fibroblast activation protein (FAP) emerges as a promising target for Gallium-68 FAP inhibitor positron emission tomography (Ga68FAPI-PET) imaging. Notably, 68Ga-FAPI-PET demonstrates promising diagnostic sensitivity and specificity, especially in conjunction with low tracer uptake in non-tumoral tissues. Moreover, it provides valuable insights into tumor-stroma interactions, a critical aspect of PDAC tumorigenesis not adequately visualized through conventional methods. The clinical implications of this innovative imaging modality extend to its potential to reshape treatment strategies by offering a deeper understanding of the dynamic TME. However, while the potential of 68Ga-FAPI-PET is evident, ongoing correlative studies are essential to elucidate the full spectrum of CAF heterogeneity and to validate its impact on PDAC management. This article provides a comprehensive review of CAF heterogeneity in PDAC and explores the potential impact of 68Ga-FAPI-PET on disease management.

A Comparison of Spatial and Phenotypic Immune Profiles of Pancreatic Ductal Adenocarcinoma and Its Precursor Lesions.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5-year survival rate of 12.5%. PDAC predominantly arises from non-cystic pancreatic intraepithelial neoplasia (PanIN) and cystic intraductal papillary mucinous neoplasm (IPMN). We used multiplex immunofluorescence and computational imaging technology to characterize, map, and compare the immune microenvironments (IMEs) of PDAC and its precursor lesions. We demonstrate that the IME of IPMN was abundantly infiltrated with CD8+ T cells and PD-L1-positive antigen-presenting cells (APCs), whereas the IME of PanIN contained fewer CD8+ T cells and fewer PD-L1-positive APCs but elevated numbers of immunosuppressive regulatory T cells (Tregs). Thus, immunosuppression in IPMN and PanIN seems to be mediated by different mechanisms. While immunosuppression in IPMN is facilitated by PD-L1 expression on APCs, Tregs seem to play a key role in PanIN. Our findings suggest potential immunotherapeutic interventions for high-risk precursor lesions, namely, targeting PD-1/PD-L1 in IPMN and CTLA-4-positive Tregs in PanIN to restore immunosurveillance and prevent progression to cancer. Tregs accumulate with malignant transformation, as observed in PDAC, and to a lesser extent in IPMN-associated PDAC (IAPA). High numbers of Tregs in the microenvironment of PDAC went along with a markedly decreased interaction between CD8+ T cells and cancerous epithelial cells (ECs), highlighting the importance of Tregs as key players in immunosuppression in PDAC. We found evidence that a defect in antigen presentation, further aggravated by PD-L1 expression on APC, may contribute to immunosuppression in IAPA, suggesting a role for PD-L1/PD-1 immune checkpoint inhibitors in the treatment of IAPA.

Ectopic CXCR2 expression cells improve the anti-tumor efficiency of CAR-T cells and remodel the immune microenvironment of pancreatic ductal adenocarcinoma

BackgroundRecent progressions in CAR-T cell therapy against pancreatic ductal adenocarcinoma (PDAC) remain disappointing, which are partially attributed to the immunosuppressive microenvironment including macrophage-mediated T cell repletion.MethodsWe first characterized the expression patterns of macrophage-relevant chemokines and identified CXCR2 as the key factor regulating T cell trafficking and tumor-specific accumulation in PDAC microenvironment. After that, we synthesized and introduced a CXCR2 expression cascade into Claudin18.2 CAR-T cells and compared the behaviors of CAR-T cells in vitro and in vivo. The therapeutic potential of CXCR2 CAR-T was evaluated in two different allogeneic models: subcutaneous allografts and metastatic PDAC models.ResultsThe results showed that CXCR2 CAR-T not only reduced the size of allografted PDAC tumors, but also completely eliminated the formation of metastases. Lastly, we investigated the tumor tissues and found that expression of ectopic CXCR2 significantly improved tumor-targeted infiltration and residence of T cells and reduced the presence of MDSCs and CXCR2 + macrophages in PDAC microenvironment.ConclusionOur studies suggested that ectopic CXCR2 played a significant and promising role in improving the efficiency of CAR-T therapy against primary and metastatic PDAC and partially reversed the immune-suppressive microenvironment.

The Tumor Microenvironment of Pancreatic Ductal Adenocarcinoma

The Tumor Microenvironment of Pancreatic Ductal Adenocarcinoma

Exosomes define a local and systemic communication network in healthy pancreas and pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC), a lethal disease, requires a grasp of its biology for effective therapies. Exosomes, implicated in cancer, are poorly understood in living systems. Here we use the genetically engineered mouse model (ExoBow) to map the spatiotemporal distribution of exosomes from healthy and PDAC pancreas in vivo to determine their biological significance. We show that, within the PDAC microenvironment, cancer cells establish preferential communication routes through exosomes with cancer associated fibroblasts and endothelial cells. The latter being a conserved event in the healthy pancreas. Inhibiting exosomes secretion in both scenarios enhances angiogenesis, underscoring their contribution to vascularization and to cancer. Inter-organ communication is significantly increased in PDAC with specific organs as most frequent targets of exosomes communication occurring in health with the thymus, bone-marrow, brain, and intestines, and in PDAC with the kidneys, lungs and thymus. In sum, we find that exosomes mediate an organized intra- and inter- pancreas communication network with modulatory effects in vivo.

Physico-chemical characterization of the tumour microenvironment of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy that accounts for more than 90% of pancreatic cancer diagnoses. Our research is focused on the physico-chemical properties of the tumour microenvironment (TME), including its tumoural extracellular matrix (tECM), as they may have an important impact on the success of cancer therapies. PDAC xenografts and their decellularized tECM offer a great material source for research in terms of biomimicry with the original human tumour. Our aim was to evaluate and quantify the physico-chemical properties of the PDAC TME. Both cellularized (native TME) and decellularized (tECM) patient-derived PDAC xenografts were analyzed. A factorial design of experiments identified an optimal combination of factors for effective xenograft decellularization. Our results provide a complete advance in our understanding of the PDAC TME and its corresponding stroma, showing that it presents an interconnected porous architecture with very low permeability and small pores due to the contractility of the cellular components. This fact provides a potential therapeutic strategy based on the therapeutic agent size.