Pancreatic Ductal Adenocarcinoma Tumor Cells Research Articles

Dual-crosslinking gelatin-hyaluronic acid methacrylate based biomimetic PDAC desmoplastic niche enhances tumor-associated macrophages recruitment and M2-like polarization

The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) is characterized by deposition of desmoplastic matrix (including collagen and hyaluronic acid). And the interactions between tumor-associated macrophages (TAMs) and tumor cells play a crucial role in progression of PDAC. Hence, the appropriate model of tumor cell-macrophage interaction within the unique PDAC TME is of significantly important. To this end, a 3D tumor niche based on dual-crosslinking gelatin methacrylate and hyaluronic acid methacrylate hydrogels was constructed to simulate the desmoplastic tumor matrix with matching compressive modulus and composition. The bionic 3D tumor niche creates an immunosuppressive microenvironment characterized by the downregulation of M1 markers and upregulation of M2 markers in TAMs. Mechanistically, RNA-seq analysis revealed that the PI3K-AKT signaling pathway might modulate the phenotypic balance and recruitment of macrophages through regulating SELE and VCAM-1. Furthermore, GO and GSEA revealed the biological process of leukocyte migration and the activation of cytokine-associated signaling were involved. Finally, the 3D tumor-macrophage niches with three different ratios were fabricated which displayed increased M2-like polarization and stemness. The utilization of the 3D tumor niche has the potential to provide a more accurate investigation of the interplay between PDAC tumor cells and macrophages within an in vivo setting.

Abstract 1577: Cancer associated fibroblast - tumor cell crosstalk enhances epithelial to mesenchymal transition and promotes a classical to basal switch in human pancreatic ductal adenocarcinoma

Abstract Introduction: Cancer associated fibroblasts (CAF) drive a complex tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) through incompletely understood interactions. Two major CAF subtypes, myofibroblastic (myCAF) and inflammatory (iCAF), dominate the tumor landscape and have been defined by gene expression profiles. There is significant plasticity between subtypes and alterations in CAF behavior likely drive changes in tumorigenicity and immune evasion. Additionally, PDAC tumor cells usually present with a predominant classical or basal phenotype that is associated with clinical outcomes. We hypothesized that CAF phenotype impacts tumor cell gene expression and alters the clinically-relevant tumor subtyping. Methods: We interrogated cellular crosstalk in the PDAC TME using a novel three-dimensional, patient-matched coculture system of patient-derived organoids (PDO) and CAFs, established from 12 patients undergoing surgical resection. Molecular characterization included bulk RNA sequencing, proteome profiling (107 factor), flow cytometry, multiplex RNA Scope, imaging mass cytometry (IMC), and RT-qPCR. Results: PDO gene expression analysis from bulk RNAseq demonstrated CAF coculture induced phenotypic shifts in the Moffitt subtype from a classical to a basal phenotype. Further, CAF coculture enhanced epithelial to mesenchymal transition (EMT) across all patients, even those with a baseline basal phenotype. We hypothesized this could be due to both cell-cell crosstalk and secreted factors. We therefore treated PDO with CAF-conditioned media (CM) and demonstrated that CM is sufficient to drive the observed classical-to-basal shift by RT-qPCR. Secretome analysis of CM derived from 3 CAF lines identified HGF, FGF19, and IL-8 as factors contributing to this transcriptional change. Next, we used spatial - omics (transcriptomics and proteomics) to examine the validity of these findings in source patient tissue. Using RNA Scope, we discovered distinct tumor neighborhoods expressing basal mRNAs KRT17 and KRT6a surrounded by CAFs compared to those expressing classical genes TFF1 and GATA6. We then used IMC to further explore CAF subtypes in patient tumors and found FAP+ myCAF regions corresponded to basal tumor regions, while CXCL12+ iCAFs were found in classical regions. Conclusions: PDAC tumor biology is likely driven by interactions between cancer cells and CAFs. Potential mediators of these interactions include both cell-cell contact and secreted factors. We introduce a novel approach combining in vitro coculture with tissue-based assays, demonstrating that modern culture techniques preserve cell type heterogeneity and plasticity. Together, these data demonstrate that a CAF-rich TME drives tumor cell plasticity not only towards EMT but also a more basal disease phenotype. Citation Format: Samantha Guinn, Brayan Perez, Jignasha Patel, Jae Lee, Daniel Zabransky, Won Ho, Elana J. Fertig, Emma Kartalia, Joseph Tandurella, Richard Burkhart, Jacquelyn W. Zimmerman, Elizabeth Jaffee. Cancer associated fibroblast - tumor cell crosstalk enhances epithelial to mesenchymal transition and promotes a classical to basal switch in human pancreatic ductal adenocarcinoma [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 1577.

Abstract 4043: Nanoparticle-mediated combination therapy to synergistically harness Type I interferons and senescence in the pancreatic tumor microenvironment

Abstract We seek to combine a unique innate immunomodulatory nanoparticle (NP) system with RAS inhibitors for a multi-faceted therapy that mitigates the formidable drug delivery challenges in pancreatic ductal adenocarcinoma (PDAC). PDAC has quickly risen to the third most deadly cancer largely due to its fibrotic, desmoplastic, and immunosuppressive tumor microenvironment (TME) that limits delivery and efficacy of chemo- and immunotherapeutics. We have engineered unique lipid-based NPs that co-encapsulate agonists of the Stimulator of Interferon Genes (STING) and Toll-like Receptor 4 (TLR4) pathways. NPs can be safely delivered in the systemic blood circulation to achieve TME deposition, uptake by innate antigen-presenting cells (APCs), and robust, synergistic production of Type I interferons by dual STING/TLR4 activation. Here, we hypothesize that combination therapy of proinflammatory NPs with tumor senescence-inducing RAS inhibitors, tremetinib and palbociclib, will not only augment CD8+ T cell recruitment to the TME but enhance their sustained activation by mitigating local immunosuppression. In mice bearing orthotopic transplant KPC tumors or KPC GEMMs, combination NP/inhibitor treatment promoted significant NP delivery to tumors, APC and natural killer (NK) cell activation, and CD8+ T cell-mediated clearance, compared to monotherapies and untreated controls. Unexpectedly and strikingly, these studies also showed that, besides innate immune cells, combination therapy also promoted Type I interferon and other proinflammatory cytokine production by PDAC tumor cells, which are otherwise notoriously unresponsive to current state-of-the-art treatments. Further, in KPC GEMMs, 25% of mice exhibited apparent complete responses following combination treatment, which, to our knowledge, is challenging to achieve in this model. Current studies include the mechanistic elucidation of immune- and tumor-intrinsic factors that govern efficacy of our combination therapy. In conclusion, these findings strongly corroborate the use of this NP-based system as a platform therapy for similar drugs and across other aggressive cancers. They also strongly make the case for the rational design of combination therapies to achieve synergistic therapeutic outcomes with minimal systemic toxicities. Citation Format: Prabhani Atukorale, Marcus Ruscetti, Loretah Chibaya, Christina Lusi, Kelly DeMarco, Griffin Kane, Meghan Brassil, Chaitanya Parikh, Katherine Murphy. Nanoparticle-mediated combination therapy to synergistically harness Type I interferons and senescence in the pancreatic tumor microenvironment [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 4043.

Abstract 1576: The heterogeneity between pancreatic ductal adenocarcinoma with & without lymphovascular invasion

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignant solid tumors, with a five-year survival rate less than 10%. Lymphovascular invasion (LVI) is a poor prognostic indicator for PDAC, yet the difference between tumors with or without LVI is unclear. Methods We used scRNA-seq analysis on the PDAC tissue of 24 patients obtained from the National Genomics Data Center (Project number: PRJCA001063) to compare T-cell, macrophage, & cancer-associated fibroblast (CAF) heterogeneity between LVI-positive & LVI-negative PDAC. To validate the scRNA-seq results, complementary techniques, including multiple immunofluorescence assays & flow cytometry, were used. Additionally, we analyzed the characteristics of LVI-positive PDAC tumor cells. Results LVI-positive PDAC thrives in an immunosuppressive microenvironment. We discovered that CD8+ T cells were more deficient in LVI-positive PDAC than in LVI-negative PDAC. Furthermore, the effective & cytotoxic markers (such as GZMB & IFN-γ) of CD8+ T cells also decreased in LVI-positive PDAC. M2 macrophage polarization was found to be higher in LVI-positive PDAC compared to LVI-negative PDAC. In addition, we discovered a more fibrotic TME signature in LVI-positive PDAC. LVI-positive tumor cells, in particular, had a mesenchymal transition phenotype & a hypoxic microenvironment. Conclusions This study adds to our understanding of the intricate heterogeneity seen in LVI-positive PDAC. It sheds light on the distinct characteristics of LVI-positive PDAC & advances our understanding of its underlying biology. Citation Format: Yiping Zou, Yongjie Xie, Song Gao, Tianxing Zhou, Jun Yu, Jihui Hao. The heterogeneity between pancreatic ductal adenocarcinoma with & without lymphovascular invasion [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 1576.

Abstract 1134: Dissecting pancreatic cancer tumor-immune microenvironment crosstalk using spatial transcriptomics

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) often fails to respond to immune therapies due to various factors, including the role of Epithelial to Mesenchymal Transition (EMT) plasticity in conferring broad resistance to diverse therapies. However, the relationship between cancer cell heterogeneity and the tumor immune microenvironment remains unclear. To address this, we utilized single cell RNA-seq and spatial transcriptomics to uncover the landscape of these cell-cell interactions in human PDAC. Here, we applied to specimens from a clinical trial testing Losartan, an indirect TGF-beta inhibitor with FOLFIRINOX chemotherapy and nivolumab (anti-PD1) in a randomized multi-institutional clinical trial for PDAC (NCT03563248). Methods: Using the NanoString GeoMx Digital Spatial Profiler, we selected multiple regions of interest in formalin-fixed paraffin-embedded (FFPE) human PDAC specimens. Immunofluorescent antibody-guided isolation of RNA and protein from cancer cells (pan-cytokeratin), cancer-associated fibroblasts (alpha-SMA), and immune cells (CD45) were performed. Utilizing the whole transcriptome assay (WTA; 18,000+ protein-coding genes) and a new IO Proteome Atlas (IPA; 500+ plex proteins), we ventured to understand the relationship between tumor cells and the surrounding microenvironment. Results: PDAC cells, CAFs, and immune cells were successfully characterized using NanoString GeoMx in clinical trial specimens. Analysis revealed associations between cancer cell plasticity, TGF-beta signaling mesenchymal subtypes in PDAC cells. These TGF-beta enriched mesenchymal PDAC cells were enriched in Arm 1 (FOLFIRINOX) compared to Arm 2 (FOLFIRINOX+losartan). Immune deconvolution analysis identified variations in immune infiltrates, showing an anti-correlation between macrophages and T-cells. The IPA analysis revealed lower MHC I protein levels in short compared to long term survivors in Arm 3. Conclusions: Spatial transcriptomics and proteomics reveal insights into the spatial relationship between PDAC tumor cell EMT plasticity, CAFs, and immune infiltrates. This enables the discovery of novel immune response biomarkers and potential therapeutic avenues to target tumor and microenvironment interactions. Trial Registration: DF/HCC protocol 18-179: Losartan and Nivolumab in Combination With FOLFIRINOX and SBRT in Localized Pancreatic Cancer. NCT03563248 Ethics Approval: All studies presented were approved by the Dana-Farber/Harvard Cancer Center IRB protocols 18-179. Citation Format: Amaya Pankaj, Michael J. Raabe, Bidish K. Patel, Evan R. Lang, Joshua R. Kocher, Katherine H. Xu, Linda T. Nieman, William L. Hwang, Alec C. Kimmelman, David P. Ryan, Theodore S. Hong, Martin J. Aryee, David T. Ting. Dissecting pancreatic cancer tumor-immune microenvironment crosstalk using spatial transcriptomics [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 1134.

Abstract 1602: Leukemia inhibitory factor (LIF)-factor XIIIA mediated macrophage-stromal crosstalk in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy marked by a highly immunosuppressive tumor microenvironment (TME). Macrophages are known to promote immunosuppression and exhibit a profibrotic transcriptional profile with potential involvement in extracellular matrix (ECM) remodeling. Previously, we identified hyperactivation of cancer cell intrinsic cyclic AMP response element binding protein 1 (CREB) regulates leukemia inhibitory factor (LIF) to mediate macrophage infiltration and polarization within the TME of PDAC. Subsequently, we observed that disrupting LIF-LIF receptor (LIFR) signaling therapeutically not only diminishes the infiltration of tumor-associated macrophages (TAMs) but also results in a decrease in ECM deposition. This study delves into the potential regulatory pathway through which LIF induces remodeling of the ECM within the PDAC TME. Methods: We targeted LIF-induced macrophage-stromal crosstalk using the LIFR antagonist (EC359) in an orthotopically tumor implanted LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice model of PDAC. Sirius red staining in tumor sections was performed to assess ECM (collagen) deposition. Moreover, we performed RNA transcriptomics-based analysis of recombinant (rLIF) induced bone marrow derived macrophages harvested from syngeneic C57BL/6 mice. The Cancer Genome Atlas (TCGA) pancreatic cancer (PAAD) data set along with single-cell RNA sequencing (scRNA seq) were employed to assess the expression of major downstream targets of LIF. To validate the RNA-seq results, we conducted immunohistochemical analysis (IHC) on mouse PDAC tissue sections and quantitative polymerase (qPCR) analysis on RAW 264 macrophage cell line treated with rLIF or KPC CREBWT PDAC tumor cells conditioned media. Results: We observed a drastic decrease in both TAM infiltration and, of note, ECM deposition in orthotopically implanted KPC pancreas tumor mice tissue treated with EC359 as compared to vehicle. Mechanistically, our RNA-seq analysis unveiled a significant upregulation of Factor XIIIA (FXIIIa), a major downstream target of LIF. Notably, TCGA data revealed that FXIIIa mRNA expression is upregulated across PDAC tumors marked by poor survival outcomes. Moreover, scRNA seq and IHC established that TAMs highly express FXIIIa in the PDAC TME. In exploring the effect of LIF-LIFR signaling in the ECM, mouse RAW 264.7 macrophage lines incubated in high LIF conditions confirmed transcriptional upregulation of ECM-associated proteins such as FXIIIa as well as fibronectin. Conclusion: Our investigation provides valuable insights into the possible mechanism by which LIF triggers ECM remodeling, thereby contributing to the desmoplastic stroma. Significantly, our findings propose that LIF-regulated FXIIIa signaling in macrophages serves as a plausible mediator of the LIF-induced ECM remodeling in PDAC. Citation Format: Nishah Jaferi, Siddharth Mehra, Varun Krishnamoorthy, Sudhakar Jinka, Anna Bianchi, Vanessa T. Garrido, Luis A. Nivelo, Ban Yuguang, Nagaraj S. Nagathihalli. Leukemia inhibitory factor (LIF)-factor XIIIA mediated macrophage-stromal crosstalk in pancreatic cancer [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 1602.

Abstract 7217: Orthogonal targeting of NAD metabolism and ErbB2/ErbB3 signaling in pancreatic adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is now the third leading cause for cancer related deaths in the US with a five-year survival of ~10% that has improved little over the past 20 years. Thus, there is a critical need to fully understand mechanisms of PDAC tumorigenesis in order to develop novel and effective therapies. Nicotinamide Adenine Dinucleotide (NAD), a critical co-factor and energy source required for cellular processes such as DNA repair and epigenetic regulation is frequently upregulated in PDAC. We recently reported a novel oncogenic axis connecting the NAD-dependent epigenetic regulator and oncogene C-terminal Binding Protein (CtBP) with the EGFR family growth factor receptors ErbB2/ErbB3 (ErbB2/3) in PDAC cells and tumors. Indeed, previous reports have shown ~30% of human PDAC tumors overexpress ErbB3, and our work has shown that PDAC tumor cell growth relies on maintenance of ErbB3 expression. Moreover, we show that the majority of PDAC cells with high levels of ErbB3 expression are uniquely sensitive to the ErbB2-targeted tyrosine kinase inhibitor lapatinib. In addition, we have recently shown that co-targeting NAD and CtBP with small molecule inhibitors is an effective and synergistic therapeutic strategy in PDAC model systems. Methods and Results: In the current study, we demonstrate NAD-dependent regulation of ErbB2/3 signaling in PDAC cells in vitro. Upon depletion of NAD using the NAD salvage pathway inhibitor GMX1778 in human PDAC cells, we observed a striking coordinate decrease in the levels of both ErbB2 and ErbB3. Furthermore, stable knockdown of the limiting enzyme of the complementing Preiss-Handler NAD synthetic pathway, Nicotinamide Adenine Phosphoribosyl Transferase, also resulted in robust downregulation of ErbB2/3 protein levels, suggesting a direct linkage in PDAC cells between the status of NAD metabolism and synthesis and activity of the key growth factor receptors ErbB2/3. Conclusions: Our findings highlight the novel connection between NAD and ErbB2/3 in PDAC. Furthermore, our data suggests combining chemical or biologic inhibitors of ErbB2/3 with an inhibitor of NAD synthesis may result in highly synergistic cytotoxic effects in PDAC cells and PDAC preclinical models. Such data could support the future development of a human PDAC clinical trial repurposing agents targeting NAD synthesis and ErbB2/3 that are already FDA-approved or currently in clinical testing. Citation Format: Kranthi Kumar Chougoni, Nari Kim, Martin M. Dcona, Diana T. Dcona, Gautam K. Malhotra, Steven R. Grossman. Orthogonal targeting of NAD metabolism and ErbB2/ErbB3 signaling in pancreatic adenocarcinoma [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 7217.

Innate and adaptive immune-directed tumour microenvironment in pancreatic ductal adenocarcinoma.

One of the most deadly and aggressive cancers in the world, pancreatic ductal adenocarcinoma (PDAC), typically manifests at an advanced stage. PDAC is becoming more common, and by the year 2030, it is expected to overtake lung cancer as the second greatest cause of cancer-related death. The poor prognosis can be attributed to a number of factors, including difficulties in early identification, a poor probability of curative radical resection, limited response to chemotherapy and radiotherapy, and its immunotherapy resistance. Furthermore, an extensive desmoplastic stroma that surrounds PDAC forms a mechanical barrier that prevents vascularization and promotes poor immune cell penetration. Phenotypic heterogeneity, drug resistance, and immunosuppressive tumor microenvironment are the main causes of PDAC aggressiveness. There is a complex and dynamic interaction between tumor cells in PDAC with stromal cells within the tumour immune microenvironment. The immune suppressive microenvironment that promotes PDAC aggressiveness is contributed by a range of cellular and humoral factors, which itself are modulated by the cancer. In this review, we describe the role of innate and adaptive immune cells, complex tumor microenvironment in PDAC, humoral factors, innate immune-mediated therapeutic advances, and recent clinical trials in PDAC.

Abstract C101: Calcium signaling rewiring activates epithelial-to-mesenchymal transition

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States and is often not diagnosed until it has already metastasized. Calcium and downstream signaling cascades contribute to epithelial-to-mesenchymal transition (EMT) and facilitate metastasis formation in PDAC. Ca2+/calmodulin-dependent kinase-II (CaMK2), a downstream effector kinase activated by Ca2+, has previously been shown in vitro to be an important mediator of a partial/hybrid EMT program that gives tumor cells high metastatic proclivity. We hypothesize that CaMK2 is a critical node in the partial EMT state and thus deleted Camk2b in vivo to study the effect of its loss on tumor progression and metastasis. Methods: We generated a genetically engineered mouse model where Camk2b was knocked out in the KPCY autochthonous PDAC mouse model (herein KPCY-Camk2bcKO). Isolated cell lines were treated with the EMT-inducing ligand TGF-b to study the effect of Camk2b on tumor cell plasticity. Results: Prior in vitro work indicated that CAMK2 is required for hybrid EMT and thus we expected KPCY-Camk2bcKO mice to have fewer metastases and live longer. Surprisingly, the genetic deletion of Camk2b accelerated tumor formation and reduced overall survival relative to KPCY controls. Despite dying sooner, there was no difference in overall tumor burden in KPCY-Camk2bcKO mice. Pathological and immunofluorescence analysis of KPCY-Camk2bcKO tumors showed an increased number of poorly differentiated regions and EMT as indicated by loss of the epithelial cell marker ECADHERIN (E-CAD) in YFP lineage-labeled tumor cells. In stark contrast to prior work, the loss of Camk2b increased tumor cell plasticity, potentially highlighting a rewiring of calcium signaling circuits in the Camk2b-null state. To this end, KPCY-Camk2bcKO tumor cell lines displayed a baseline mesenchymal morphology in vitro and had lower expression of Cdh1 (the gene encoding E-CAD) mRNA. Using a flow cytometry-based approach, we also found baseline E-CAD protein levels to be diminished relative to KPCY lines. Additionally, KPCY-Camk2bcKO tumor cells were more responsive to the EMT-inducing ligand TGF-b and downregulated their E-CAD levels more than KPCY cells. Notably, treatment with low dose TGF-b also dramatically reduced E-CAD expression in KPCY-Camk2bcKO but not KPCY cells, indicating that the knockout cells are easily pushed towards a mesenchymal state. Conclusions: While CAMK2 has been studied previously as a facilitator of the EMT program, this work paradoxically demonstrates that Camk2b loss enhances cellular plasticity within PDAC. In this context, we speculate that loss of Camk2b rewires calcium signaling to drive EMT. In future studies, we will investigate the effect of Camk2b loss on the metastatic cascade and uncover mechanisms through which Camk2b loss makes PDAC tumor cells more plastic. We will also begin to characterize the non-cell autonomous roles of CAMK2B in crosstalk with the tumor microenvironment. Citation Format: Jessica Peura, Yamini Ogoti, Jason Pitarresi. Calcium signaling rewiring activates epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C101.

Circular RNA circTMEM59 inhibits progression of pancreatic ductal adenocarcinoma by targeting miR-147b/SOCS1: An in vitro study

PurposeThis study aimed to detect the role and mechanism of circTMEM59 in pancreatic ductal adenocarcinoma (PDAC). Methods66 paired PDAC tissues and normal samples were harvested from patients diagnosed and undergoing pancreatic cancer surgery in our hospital. The expression of circTMEM59 in PDAC tissues and cell lines was detected. Based on bioinformatics information, the circTMEM59 mimics, miR-147b mimics, miR-147b inhibitor and si-suppressor of cytokine signaling 1 (SOCS1) were transfected into PDAC cells. The expression levels of circTMEM59, miR-147b and SOCS1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR). RNA interaction was confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Cell invasion and proliferation were evaluated by Transwell and Cell Counting Kit-8 (CCK-8) assays. The protein expression was detected by Western blot. ResultsCircTMEM59 was confirmed to be downregulated in PDAC tumor tissues and cells. Low expression of circTMEM59 was closely correlated with the short survival time and poor clinicopathological characteristics. By up-regulating the expression of circTMEM59 in PDAC cells, cell proliferation, invasion and epithelial-mesenchymal transition (EMT) were inhibited. More importantly, miR-147b could be sponged by circTMEM59, and knockdown of miR-147b inhibited progression of PDAC cells. Further study revealed that SOCS1 was targeted by miR-147b. SOCS1 expression was negatively related to miR-147b expression and positively related to circTMEM59 expression in PDAC tissues. Upregulated miR-147b and downregulated SOCS1 could rescue the effects of circTMEM59 on cell proliferation, EMT and invasion. ConclusionOur data indicated that circTMEM59 inhibited cell proliferation, invasion and EMT of PDAC by regulating miR-147b/SOCS1 axis.

Characterization of tumor-associated endothelial cells and the development of a prognostic model in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by a complex tumor microenvironment. Angiogenesis is of paramount importance in the proliferation and metastasis of PDAC. However, currently, there are no well-defined biomarkers available to guide the prognosis and treatment of PDAC. In this study, we investigated the interactions between tumor-associated endothelial cells (TAECs) and tumor cells in PDAC, and identified a specific subset of TAECs characterized by high expression of COL4A1. COL4A1+ endothelial cells interact with tumor cells through the COLLAGEN signaling pathway to promote tumor cell proliferation, migration, and invasion. We also observed activation of HOXD9 in COL4A1+ endothelial cells. Based on these findings, we developed a prognostic model called TaEMS, which accurately predicts patient prognosis. TaEMS identified high-risk patients enriched in cell cycle-related pathways and low-risk patients enriched in focal adhesions, smooth muscle regulation, and immune pathways. Moreover, high-risk patients displayed a reduced level of immune cell infiltration, indicating the presence of a “cold tumor” phenotype. Overall, our study uncovered an intricate crosstalk between TAECs and tumor cells in PDAC, emphasizing the role of HOXD9 and highlighting the potential of TaEMS as a prognostic biomarker for precise therapies.

Plasminogen deficiency suppresses pancreatic ductal adenocarcinoma disease progression.

Pancreatic ductal adenocarcinoma(PDAC) is a highly fatal metastatic disease associated with robust activation of the coagulation and fibrinolytic systems. However, the potential contribution of the primary fibrinolytic protease plasminogen to PDAC disease progression has remained largely undefined. Mice bearing C57Bl/6-derived KPC (KRasG12D , TRP53R172H ) tumors displayed evidence of plasmin activity in the form of high plasmin-antiplasmin complexes and high plasmin generation potential relative to mice without tumors. Notably, plasminogen-deficient mice (Plg- ) had significantly diminished KPC tumor growth in subcutaneous and orthotopic implantation models. Moreover, the metastatic potential of KPC cells was significantly diminished in Plg- mice, which was linked to reduced early adhesion and/or survival of KPC tumor cells. The reduction in primary orthotopic KPC tumor growth in Plg- mice was associated with increased apoptosis, reduced accumulation of pro-tumor immune cells, and increased local proinflammatory cytokine production. Elimination of fibrin(ogen), the primary proteolytic target of plasmin, did not alter KPC primary tumor growth and resulted in only a modest reduction in metastatic potential. In contrast, deficiencies in the plasminogen receptors Plg-RKT or S100A10 in tumor cells significantly reduced tumor growth. Plg-RKT reduction in tumor cells, but not reduced S100A10, suppressed metastatic potential in a manner that mimicked plasminogen deficiency. Finally, tumor growth was also reduced in NSG mice subcutaneously or orthotopically implanted with patient-derived PDAC tumor cells in which circulating plasminogen was pharmacologically reduced. Collectively, these studies suggest that plasminogen promotes PDAC tumor growth and metastatic potential, in part through engaging plasminogen receptors on tumor cells.

Evaluation of Tumor Fibrosis in Pancreatic Ductal Adenocarcinoma by 2-D Shear Wave Elastography: A Pilot Study

The aim of the work described here was to investigate the association of the stromal proportion with the elasticity obtained by 2-D shear wave elastography (SWE) and the diagnostic value of elasticity in evaluating tumor stromal fibrosis in pancreatic ductal adenocarcinoma (PDAC). Patients who met inclusion criteria underwent pre-operative 2-D SWE examination and intra-operative determination of hardness by palpation from July 2021 to November 2022, and the post-operative specimens were used to evaluate pathological features including the tumor stromal proportion. A receiver operating characteristic curve was created to evaluate its diagnostic value in differentiating the degree of tumor stromal fibrosis. The 2-D SWE measurements in pancreatic lesions were successful in 62 of 69 patients (89.9%). A total of 52 eligible participants were enrolled for subsequent correlation analysis. Elasticity correlated well with tumor stromal proportion (rs=0.646) and number of tumor cells (rs=-0.585) in PDAC. Moreover, pancreatic elasticity determined by 2-D SWE, palpation-determined hardness and tumor stromal proportion were well correlated with each other. Two-dimensional SWE could clearly distinguish mild and severe stromal fibrosis, and its diagnostic performance was better than that determined by palpation even though the difference was not statistically significant (p=0.103). The elasticity of PDAC obtained using 2-D SWE was closely related to stromal proportion and tumor cellularity and could clearly be used to diagnose the degree of stromal fibrosis, which indicates that 2-D SWE can be a non-invasive predictive imaging biomarker in personalization of therapy and monitoring of treatment.

Integrated analysis revealed hypoxia signatures and LDHA related to tumor cell dedifferentiation and unfavorable prognosis in pancreatic adenocarcinoma: Hypoxia in PDAC

Pancreatic ductal adenocarcinoma (PDAC) is a highly heterogeneous cancer with limited understanding of its classification and tumor microenvironment. Here, by analyzing single-nucleus RNA sequencing of 43, 817 tumor cells from 15 PDAC tumors and non-tumor, we find that hypoxia signatures were heterogeneous across samples and were potential regulators for tumor progression and more aggressive phenotype. Hypoxia-high PDAC tends to present a basal/squamous-like phenotype and has significantly increased outgoing signaling, which enhances tumor cell stemness and promotes metastasis, angiogenesis, and fibroblast differentiation in PDAC. Hypoxia is related to an extracellular matrix enriched microenvironment, and increased possibility of TP53 mutation in PDAC. TP63 is a specific marker of squamous-like phenotype, and presents elevated transcriptome levels in most hypoxia PDAC tumors. In summary, our research highlights the potential linkage of hypoxia, tumor progression and genome alteration in PDAC, leading to further understand of the formation of inter-tumoral and intra-tumoral heterogenous in PDAC. Our study extends the understanding of the diversity and transition of tumor cells in PDAC, which provides insight into future PDAC management.

YAP/BRD4-controlled ROR1 promotes tumor-initiating cells and hyperproliferation in pancreatic cancer.

Tumor-initiating cells are major drivers of chemoresistance and attractive targets for cancer therapy, however, their identity in human pancreatic ductal adenocarcinoma (PDAC) and the key molecules underlying their traits remain poorly understood. Here, we show that a cellular subpopulation with partial epithelial-mesenchymal transition (EMT)-like signature marked by high expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) is the origin of heterogeneous tumor cells in PDAC. We demonstrate that ROR1 depletion suppresses tumor growth, recurrence after chemotherapy, and metastasis. Mechanistically, ROR1 induces the expression of Aurora kinase B (AURKB) by activating E2F through c-Myc to enhance PDAC proliferation. Furthermore, epigenomic analyses reveal that ROR1 is transcriptionally dependent on YAP/BRD4 binding at the enhancer region, and targeting this pathway reduces ROR1 expression and prevents PDAC growth. Collectively, our findings reveal a critical role for ROR1high cells as tumor-initiating cells and the functional importance of ROR1 in PDAC progression, thereby highlighting its therapeutic targetability.

Abstract 5173: BET inhibition reprograms pancreatic cancer associated fibroblasts to promote anti-tumor immunity

Abstract BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most malignant neoplasm of the pancreas, and is refractory to most therapeutic strategies, including immunotherapy. We have previously demonstrated that loss of CDKN2A in PDAC tumor cells promotes an immunologically-cold tumor microenvironment, which can be counteracted through the use of bromodomain and extraterminal domain inhibitors (BETi). The immunosuppressive PDAC tumor microenvironment is also characterized by a dense desmoplastic stroma, rich in cancer associated fibroblasts (CAFs). These CAFs are a highly plastic population of cells, comprised of myofibroblastic, inflammatory, and antigen-presenting CAFs (myCAFs, iCAFs, and apCAFs, respectively). Previously research has suggested BETi can alter the function of CAFs, suggesting this therapeutic strategy may be effective at targeting both the tumor and stromal compartments of PDAC. Here we hypothesize that BETi enhances anti-tumor immunity in CDKN2A-deficient PDAC by modulating the phenotype and function of PDAC CAFs. METHODS: Isogenic wild-type and CDKN2A-deficient KPCluc pancreatic cancer cell lines were used in conjunction with KPCluc-derived murine CAF cells. CAF spheroids were used to evaluate the effects of BETi on CAF phenotype (as measured by flow cytometry) and function (as measured by NanoString analysis, checkpoint ligand expression by flow cytometry, and T cell migration assays). Additionally, the impact of BETi-induced modulation of CAF phenotype and function on wild-type and CDKN2A-deficient tumor cell spheroids was measured by flow cytometry. RESULTS: Treatment of pancreatic CAFs with BETi induced expression of genes associated with DNA damage responses and type I IFN expression. Additionally, while CAF supernatants suppressed T cell migration in vitro, treatment with BETi restored T cell migration across a transwell. This was due in part to DNA damage/STING/NF-κB signaling induced in PDAC CAF following BETi. Supernatants from BETi-treated PDAC CAFs were found to modulate PD-L1 expression on PDAC tumor cells. Direct treatment of CAFs with BETi induced a dramatic dose- and time-dependent polarization of CAF spheroids towards a myCAF phenotype. However, while BETi induced DNA damage/STING/NF-κB signaling in CAF cells, this signaling did not contribute towards this myCAF polarization, as treatment of CAF spheroids with BETi and STING or NF-κB inhibitors did not impact myCAF polarization. Additional mechanisms of polarization are currently being evaluated and will be updated at presentation. CONCLUSIONS: BET inhibition modulates CAF cell phenotype and function to promote anti-tumor immune responses. Taken together with the impact of BET inhibition on CDKN2A-deficient PDAC tumor cells, these data suggest BETi is a rational treatment approach to promote anti-tumor immunity in this genetically defined immunotherapy resistant patient population. Citation Format: Trisha Minocha, Alison Thomas, Brian M. Olson. BET inhibition reprograms pancreatic cancer associated fibroblasts to promote anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5173.

Abstract 3627: Inhibition of tumor cell-autonomous p38 MAPK suppresses IL1α-mediated inflammatory tumor-stromal crosstalk in pancreatic adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) continues to face major therapeutic challenges due to its innate and acquired chemoresistance. Oncogenic, activating KRAS mutations facilitate extensive cytokine-mediated crosstalk between tumor cells and the fibrotic stroma, producing a pro-inflammatory and immunosuppressive tumor microenvironment (TME). We have identified interleukin-1α (IL1α), downstream of KRAS, as a critical mediator of the inflammatory response due to its pleiotropic effects on cancer-associated fibroblast (CAF) activation and immune evasion. However, the regulatory mechanisms of IL1α expression remain incompletely understood. We identified p38 stress-associated MAPK α (p38α) as a central, KRAS-driven regulatory pathway involved in IL1α production within PDAC tumor cells. Methods: Differential gene expression analyses were performed on patient data from The Cancer Genome Atlas (TCGA) was queried for overactive pathways downstream KRAS in “high-IL1A” PDAC cases. Inhibition of p38α was achieved pharmacologically with pexmetinib and genetically with an shRNA lentiviral system in human and murine PDAC cell lines. ChIP-qPCR and co-immunoprecipitation were performed on a human PDAC cell line with and without p38α inhibition. Single-cell RNA sequencing (scRNA-seq) and flow cytometry were performed on tumors from a PDAC murine model, Ptf1aCre/+; LSL-KrasG12D/+; Tgfbr2flox/flox (PKT), treated with or without pexmetinib. PKT mice were treated daily with pexmetinib, gemcitabine and paclitaxel chemotherapy, or combination therapy for downstream analysis and survival studies. Results: Both pharmacologic and genetic inhibition of p38α significantly reduced IL1A transcription and protein levels in human and murine PDAC tumor cell lines. Furthermore, p38α inhibition reduced binding of Sp1 and NF-κB to the IL1A promoter and prevented IL1α-mediated polarization of inflammatory CAFs in vitro. In PKT mice, p38α inhibition reduced tumor cell-specific Il1a and inflammatory CAF gene signatures by scRNA-seq, and favorably altered the infiltrating immune populations by flow cytometry. Lastly, pexmetinib with chemotherapy significantly reduced tumor burden and improved overall survival in a PDAC murine model. Conclusions: These findings provide a new therapeutic opportunity to target the p38α MAPK pathway for suppression of IL1α-mediated stromal activation and combination with chemotherapy to overcome therapeutic resistance by modulating the stromal and immune microenvironment in PDAC. Citation Format: Samara Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Anna Bianchi, Vanessa Tonin Garrido, Zhiqun Zhou, Haleh Amirian, Edmond W. Box, Jashodeep Datta, Nagaraj Nagathihalli, Nipun B. Merchant. Inhibition of tumor cell-autonomous p38 MAPK suppresses IL1α-mediated inflammatory tumor-stromal crosstalk in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3627.

Trop-2 is a ubiquitous and promising target in pancreatic adenocarcinoma

BackgroundTrop-2 is overexpressed in tumor cells of various cancers, including pancreatic ductal adenocarcinoma (PDAC), and has emerged as a potent therapeutic target. We evaluated Trop-2 expression both at the transcriptomic and protein levels, and its correlation with tumor features and patients’ outcomes in a large cohort of PDAC. MethodsWe included patients undergoing pancreatic resection for PDAC in 5 academic hospitals in France and Belgium. Transcriptomic profiles were obtained from FFPE tissue samples, with paired primary -25and metastatic lesions when available. Protein expression was evaluated by immunohistochemistry (IHC) using tissue micro-arrays. Results495 patients (male 54%, median age 63 years) were included between 1996 and 2012. Trop-2 mRNA expression was significantly associated to tumor cellularity, but no association with survival nor with any clinical or pathological features was observed, with tumor cells showing an overall high expression among every subgroup. Trop-2 mRNA expression was maintained between primary and metastatic lesion in all 26 paired samples evaluated. In 50 tumors assessed by IHC, 30%, 68% and 2% harbored a high, medium, or low Trop-2 expression score, respectively. Trop-2 staining was significantly associated to mRNA expression, but not to survival or any pathological features. ConclusionsOur results suggest Trop-2 overexpression as a ubiquitous marker of PDAC tumor cells and thus a promising therapeutic target to evaluate in these patients.

Novel banana lectin CAR-T cells to target pancreatic tumors and tumor-associated stroma

BackgroundCell therapies for solid tumors are thwarted by the hostile tumor microenvironment (TME) and by heterogeneous expression of tumor target antigens. We address both limitations with a novel class of...

Stroma-targeting strategies in pancreatic cancer: a double-edged sword.

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer with limited treatment options and terrible long-term survival, and it is expected to become the second leading cause of cancer-related death by 2030. One reason why this cancer is so aggressive and resistant is the formation of dense stroma that surrounds the neoplastic epithelium, which promotes tumor progression, invasion, metastasis, and resistance. The three major components of PDAC stroma are extracellular matrix (ECM), cancer-associated fibroblasts (CAFs), and vasculature. The dense ECM acts as a natural physical barrier, impeding drug penetration to PDAC tumor cells. Consequently, the method that combines stroma-targeting with anticancer therapy may be a viable alternative for increasing drug penetration. Additionally, blood vessels are key entities of the tumor stroma, serving as a pathway for nutrition as well as the only way for chemical medicines and immune cells to act. Finally, PDAC CAFs and tumor cells have crosstalk effects in the tumor microenvironment, where they are responsible for enhanced matrix deposition. In this review, we aim to provide an overview of our current comprehension of the three key components of PDAC stroma and the new promising therapeutic targets for PDAC.