PDAC Tissue Research Articles

Fatty acid oxidation is critical for the tumorigenic potential and chemoresistance of pancreatic cancer stem cells

BackgroundWe have previously demonstrated the significant reliance of pancreatic Cancer Stem Cells (PaCSCs) on mitochondrial oxidative phosphorylation (OXPHOS), which enables versatile substrate utilization, including fatty acids (FAs). Notably, dysregulated lipid scavenging and aberrant FA metabolism are implicated in PDAC progression.Methods & resultsOur bioinformatics analyses revealed elevated expression of lipid metabolism-related genes in PDAC tissue samples compared to normal tissue samples, which correlated with a stemness signature. Additionally, PaCSCs exhibited heightened expression of diverse lipid metabolism genes and increased lipid droplet accumulation compared to differentiated progenies. Treatment with palmitic, oleic, and linolenic FAs notably augmented the self-renewal and chemotherapy resistance of CD133+ PaCSCs. Conversely, inhibitors of FA uptake, storage and metabolism reduced CSC populations both in vitro and in vivo. Mechanistically, inhibition of FA metabolism suppressed OXPHOS activity, inducing energy depletion and subsequent cell death in PaCSCs. Importantly, combining a FAO inhibitor and Gemcitabine treatment enhanced drug efficacy in vitro and in vivo, effectively diminishing the CSC content and functionality.ConclusionTargeting FAO inhibition represents a promising therapeutic strategy against this highly tumorigenic population.

Abstract 4435: Taurine metabolism as a marker for pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC), known for its aggressive nature and poor prognosis, presents a significantly altered metabolic landscape compared to normal pancreatic tissue, a feature that has been the focus of recent research but remains underexplored in actual pancreatic tissues. In this study, we undertook an untargeted metabolomics analysis to compare the metabolic profiles of PDAC with matched normal tissues, revealing a notable elevation in taurine levels in PDAC. This increase was further validated through immunohistochemistry in separate PDAC and normal tissue samples. To delve deeper, we conducted a comprehensive bioinformatics analysis, integrating transcriptomic and proteomic data, which identified the taurine metabolism pathway as significantly altered, with ADO emerging as a pivotal gene in this process. Further investigations, including gene modulation and xenograft models, demonstrated that ADO knockdown curtails cancer cell proliferation and tumor growth, potentially impacting the Raf-MEK-ERK signaling pathway. Clinically, the ADO-Taurine axis was found to have substantial prognostic significance, correlating with recurrence rates and survival in PDAC patients. These insights collectively suggest that the ADO-Taurine axis, through its modulation of taurine metabolism, presents as a promising biomarker for PDAC prognosis and a potential avenue for therapeutic development. Citation Format: Hoonsik Nam, Woohyung Lee, Ha T. Nguyen, Linh X. Mai, Song Cheol Kim, Sunghyouk Park. Taurine metabolism as a marker for 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 4435.

Abstract A007: Expression and activity of Rac, Cdc42, and their downstream effector PAK in Puerto Rican pancreatic cancer patients

Abstract Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed after the localized tumor has metastasized, which decreases its survival rate to a grim 3%. Mortality from PDAC is also increasing in Puerto Rico with lower survival rates than in the US. Therefore, it is pivotal to identify viable targets for the late stages of PDAC. The small GTPase Ras is the most mutated oncogene in PDAC. Oncogenic Ras, as well as multiple oncogenic cell surface receptors, activate the related GTPases Rac and Cdc42, which in turn activate p21-activated kinase (PAK) by autophosphorylation (p-PAK), to drive cancer cell invasion, and thus metastasis. Rac and Cdc42 are overexpressed in 21% of PDAC patients and contribute to poor prognosis. To determine the feasibility of treating PDAC with Rac/Cdc42 inhibitors that we are developing, this study aims to identify the p-PAK levels and Rac/Cdc42 expression in PDAC tissue from Puerto Rican patients. The hypothesis is that p-PAK levels, as well as Rac/Cdc42 expression, are selectively elevated in high-grade invasive PDAC. Immunohistochemistry for p-PAK and immunofluorescence for Rac/Cdc42 were conducted in fixed, paraffin-embedded PDAC biopsy samples of Puerto Rican patients. Preliminary results demonstrate that higher p-PAK levels are correlated with stage 3 and stage 4 tissue, and Rac and Cdc42 are expressed in ductal regions of PDAC tissue. Taken together, p-PAK has potential as a diagnostic biomarker for high-grade PDAC and will aid patient stratification in the planned clinical trials for our lead Rac/Cdc42 inhibitor, MBQ-167. Citation Format: Anamaris Torres-Sanchez, Jahdiel Berrios-Rodriguez, Jerry Cruz-Rodriguez, Jasmin Contreras-Santini, Victor Carlo, Suranganie Dharmawardhane. Expression and activity of Rac, Cdc42, and their downstream effector PAK in Puerto Rican pancreatic cancer patients [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 A007.

Abstract A110: Clec4f is expressed in a subset of primary PDAC cells and confers invasive behavior

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a particularly deadly disease with a 5-year survival rate of just 12%. Poor survival is due in part to late detection of aggressive disease coincident with distant metastases, most often in the liver. While multiple proteins have been implicated in driving PDAC metastasis, effective targeted therapeutics have yet to be developed. Therefore, understanding factors contributing to PDAC liver metastasis has significant implications for diagnosis and treatment of the disease. Evidence has emerged to suggest that a subpopulation of cells with a pre-disposition for successful seeding and outgrowth in the liver are cultivated at the primary site prior to dissemination. Investigating public datasets generated from mouse models of PDAC liver metastasis, we noted upregulation of C-type Lectin Domain Family 4 Member F (Clec4f), a galactose-binding receptor. Importantly, we also detected Clec4f expression in a subset of primary PDAC tumor epithelial cells, which established our hypothesis that Clec4f is a novel regulator of PDAC dissemination. Preliminary in vitro invasion assays demonstrate that PDAC cells engineered to overexpress Clec4f exhibit more invasive behavior compared to control cells, displaying a more mesenchymal phenotype upon confluence in 2D culture and increased invasive protrusion when grown as 3D spheroids compared to control cells. To better examine tumor-intrinsic Clec4f-expressing PDAC cells in isolation, we sorted Clec4f-expressing cells from a mouse PDAC tumor via FACS to culture in vitro. However, upon establishment in culture, the sorted cells lost Clec4f expression. Importantly, Clec4f expression in other cell types has been shown to be dependent on niche-derived TGFβ signaling, which has also been shown to regulate tumor cell invasiveness and dissemination in PDAC. This led us to probe for Clec4f-expressing tumor cells in a mouse model of PDAC lacking TGFβ receptor 2 (PKT model). In the PKT model, we observed no Clec4f-expressing tumor cells, which suggested that niche-derived TGFβ signaling is required for the upregulation of Clec4f in PDAC. Using a variety of established cellular markers, we also determined that Clec4f was co-expressed in a subset of Synaptophysin (Syp) positive enteroendocrine cells in PDAC tissue. We are currently working to determine which niche components in the tumor microenvironment provide the necessary signals to upregulate Clec4f expression in PDAC tumor cells. We expect the outcome of this study to shed light upon pro-dissemination niche dynamics in PDAC and how they contribute to the deadly process of liver metastasis. Citation Format: Bailey A. Bye, Richard M. Walsh, Austin E. Eades, Michael N. VanSaun. Clec4f is expressed in a subset of primary PDAC cells and confers invasive behavior [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 A110.

Abstract C059: Hub gene analysis identifies CAD as a potential mediator for drug resistance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a complex disease pathobiology and poor treatment options, emphasizing the need for novel therapeutic targets. Hub genes have high connectivity (i.e., exceptionally many interaction partners), for example, certain proteases with numerous interaction partners. Thereby, hub genes are often central biological regulators and dysregulated proteolysis is indeed a key trait of malignant tissues. Here, we aimed to identify novel protease-related hub genes with potential involvement in pancreatic cancer. We applied an integrative computational approach to compartment-specific multiOMIC profiles of laser-capture micro-dissected human PDAC tissues (n = 32) and queried associations of identified candidate genes with clinical outcomes in early (n = 186) and advanced PDAC patients (n = 253). This identified 20 protease-related genes with ≥ 10 physically verified interaction partners in pancreatic cancer. Among these, the pyrimidine biosynthesis regulator gene CAD emerged as a potentially druggable target in pancreatic cancer. CAD expression was nearly restricted to the malignant PDAC epithelium. Furthermore, higher CAD expression was associated with significantly worse overall survival in patients with advanced but not resectable disease, suggesting a role in treatment response rather than disease progression. Moreover, patients with higher CAD expression specifically demonstrated poor response to mFFX but not Gemcitabine-nab-Paclitaxel treatment, which is in line with previous reports on CAD expression being protective against 5-Fluouracil, a component of FFX. Moving forward, we will interrogate the role of CAD in PDAC tissue biology and scrutinize its involvement in FFX resistance, using patient-derived organoids. Citation Format: Foram Vyas, Barbara Grünwald, Kazeera Aliar, Gun Ho Jang, Grainne O'Kane, Thomas Kislinger, Steven Gallinger, Benjamin Haibe-Kains, Rama Khokha. Hub gene analysis identifies CAD as a potential mediator for drug resistance in pancreatic cancer [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 C059.

Abstract A102: Secreted frizzled related-protein 2 is prognostic for survival in pancreatic cancer

Abstract INTRODUCTION: Secreted frizzled-related protein 2 (SFRP2) is overexpressed in several tumor types and blocks tumor apoptosis, increases angiogenesis, and promotes T-cell exhaustion. It has also been shown to be associated with fibrosis in non-neoplastic pathologies. We hypothesize SFRP2 is prognostic for survival and associated with PDAC fibrosis and mesenchymal transformation. METHODS: SFRP2 gene expression and protein levels in patients with PDAC were analyzed using NCI’s The Cancer Genome Atlas (TCGA) data and data from resected tumors analyzed for protein levels by western blot. Disease free survival (DFS) and overall survival (OS) were analyzed using Kaplan Meier test. The association of stroma content and SFPR2 RNA expression in human PDAC was studied using Spearman correlation between the ESTIMATE algorithm stromal score and TCGA data. The GEPIA tool was used to compare SFRP2 mRNA levels between PDAC and normal pancreas tissue. Immunohistochemistry (IHC) for SFRP2 in KPC PDAC tumors were correlated with fibrosis stains Picrosirus red and Massone trichome. The role of SFRP2 in mesenchymal transformation was assessed by western blot in fibroblasts. RESULTS: TCGA RNAseq analysis of SFRP2 expression showed highest SFRP2 mRNA levels in pancreatic cancer out of 32 cancer types. GEPIA analysis of PDAC (n=179) and normal pancreas (n=177) mRNA data showed higher levels of SFRP2 mRNA in PDAC compared to normal pancreas (p<.01). High SFRP2 mRNA levels were associated with decreased disease-free survival hazard ratio = 2.6; p=0.0097). PDAC tissue showed higher SFRP2 protein compared to matched normal-adjacent tissue (n=24, p=0.0003). High SFRP2 expression was associated with decreased OS (p=0.0158. The ESTIMATE stromal score correlated with SFRP2 expression in human PDAC (Spearman correlation coefficient 0.81). Fibrosis levels in KPC PDAC tumors correlated with SFRP2 IHC (Spearman correlation coefficient 0.75). Fibroblasts treated with SFRP2 and TGFβ showed higher mesenchymal markers compared to untreated control. CONCLUSIONS: SFRP2 is increased in PDAC, associated with tumor fibrosis, and prognostic for survival. SFRP2 is a novel therapeutic target in PDAC. Citation Format: Lillian Hsu, Julie Siegel, Patrick Nasarre, Rupak Mukherjee, Meghan Gormley, Bailey Richardson, Jordan Morningstar, Imran Khan, Eleanor Hilliard, John O’Bryan, Krist Helke, Laura Spruill, Nathan Dolloff, Nancy Klauber-Demore. Secreted frizzled related-protein 2 is prognostic for survival in pancreatic cancer [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 A102.

Abstract A106: Single cell RNA sequencing of EPCAM+ cells reveals unique malignant biology

Abstract Pancreatic ductal adenocarcinoma (PDAC) is notable due to its complex and dense stroma which comprises a high proportion of non-malignant cells. Studying PDAC epithelial cells at the single cell level may yield insights which have not been appreciated through bulk tissue analysis. We analysed a novel single cell RNA sequencing (scRNA seq) dataset of 11000 epithelial (EPCAM+) cells from 7 resected PDAC samples and used a validation cohort of 43 samples from two published scRNA seq datasets. Malignant epithelial cells were identified by: gene expression, changes in copy number, and by the presence of known PDAC single nucleotide variants. This confirmed that sorting for EPCAM+ cells prior to scRNA seq resulted in an enriched proportion of malignant vs non-malignant tumor epithelial cells. This dataset of enriched single cell dataset was therefore useful for studying the influence of intrinsic malignant cell factors on PDAC biology. We observed that malignant cells do not segregate into classical or basal-like signatures, despite these being clinically validated bulk transcriptomic tumor subtypes. Indeed, classical and basal-like signatures module scores in PDAC in individual malignant cells were positively correlated, suggesting they are not divergent programs in malignant cells. Bulk transcriptomic signature subtypes are therefore most likely to reflect differences in PDAC tissue stroma, rather than in intrinsic malignant cell factors. Moreover, we sort to investigate intra- and inter- sample heterogeneity to understand the intrinsic malignant cell factors which may influence the biology of PDAC. We analysed single cell velocity to understand the dynamics of malignant cell states. This showed heterogeneity in expression of known pan-cancer malignant cell meta-programs which identified a population of cells with high expression of cell cycle genes, suggesting greater proliferation. We then explored expression of immune modulating genes to determine how malignant cells influence the immunosuppressive microenvironment. For example, we identified heterogeneity in HLA class 1 and antigen presentation gene expression which may be a mechanism of immune suppression in PDAC. Together these data demonstrate how studying scRNA seq of EPCAM+ cells reveals intrinsic malignant cell factors which cannot be appreciated through bulk RNA seq. Citation Format: Edward H. Arbe-Barnes, Ashwin Jainarayanan, Enas Abu-Shah, Rachael J.M. Bashford-Rogers, Shivan Sivakumar. Single cell RNA sequencing of EPCAM+ cells reveals unique malignant biology [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 A106.

Abstract B006: Targeting BARD1 in human pancreatic ductal adenocarcinoma: How to expand the target population of vulnerable tumors?

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the deadliest malignancy of the pancreas, with an overall five-year survival rate of only 12%. Despite therapeutic advances, effective therapies are still limited. It is now known that many of the DNA damage repair (DDR) genes that are mutated in PDAC are crucial for the proper functioning of the homologous recombination repair (HRR) pathway. Loss-of-function alterations in DDR genes such as BRCA1/2 mutations render PDAC cells vulnerable to certain DNA damage agents, for example, poly (ADP-ribose) polymerase inhibitors (PARPi) and platinum-based agents. Since this treatment strategy is currently limited to only 5-10% of PDAC patients, it underscores a dire need to find new therapeutic avenues and expand the PDAC patient population that could benefit from these therapies. We have recently published that BARD1 (BRCA1- Associated- Ring- Domain- 1), an obligate binding partner of BRCA1 in the process of HRR, is upregulated in PDAC cells exposed to PARPi/platinums. PDAC tissue microarray stained for BARD1 also shows higher BARD1 expression in PDAC tissues compared to normal pancreas. Here, we explored the effects of targeting BARD1 on PDAC cells. For this, we created genetic models of BARD1 inhibition and utilized these model systems to study if loss of BARD1 slows PDAC growth, invasion, and chemo-sensitizes PDAC cells to PARPis and DNA damage agents. Doxycycline inducible shRNA and BARD1 CRISPR KO models were created to inhibit gene expression of BARD1 in PDAC cells. Loss of BARD1 protein and mRNA expression was confirmed by western blot and qRT-PCR analyses. Using colony formation assays, Pico green cell survival assays, and Boyden matrigel invasion assays, we found that inhibition of BARD1 resulted in slower growth and decrease in invasion of pancreatic cancer cells in vitro. The expression levels of EMT markers such as zeb-1 and vimentin, were also decreased. These studies were recapitulated in a mouse xenograft subcutaneous model, where BARD1 CRISPR KO (+/- and -/-) and WT PDAC cells were injected subcutaneously on flanks of mice and tumor growth was monitored over time. Kaplan Meier curves were calculated and plotted using Log-rank test. We found that inhibiting BARD1 significantly (P=0.026) delayed PDAC tumor growth in vivo and extended survival (P<0.001). CRISPR KO of BARD1 also enhanced the efficacies of PARP inhibitors like olaparib, rucaparib and veliparib, and lowered their IC50s (fold changes >10). Our work indicates that targeting BARD1 affects key hallmarks of cancer (growth and invasion), such that the window of therapeutic opportunity is extended, which further allows chemo-sensitization of PDAC cells to DNA damaging agents. Our ongoing studies will explore if BARD1 loss is a predictive marker of PDAC sensitivity to other DNA-damaging agents besides PARPi and allow the development of therapeutic strategies to target BARD1 in PDAC. Citation Format: Aditi Jain, Rutuj P. Kusurkar, Eleanor Jenkins, Avinoam Nevler, Wei Jiang, Charles J. Yeo. Targeting BARD1 in human pancreatic ductal adenocarcinoma: How to expand the target population of vulnerable tumors? [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B006.

The importance of spatial transcriptomics in translational research: identification of druggable targets in PDAC tissue samples.

The importance of spatial transcriptomics in translational research: identification of druggable targets in PDAC tissue samples.

Morphine Analgesia, Cannabinoid Receptor 2, and Opioid Growth Factor Receptor Cancer Tissue Expression Improve Survival after Pancreatic Cancer Surgery.

Pancreatic cancer (PDAC) has a poor prognosis despite surgical removal and adjuvant therapy. Additionally, the effects of postoperative analgesia with morphine and piritramide on survival among PDAC patients are unknown, as are their interactions with opioid/cannabinoid receptor gene expressions in PDAC tissue. Cancer-specific survival data for 71 PDAC patients who underwent radical surgery followed by postoperative analgesia with morphine (n = 48) or piritramide (n = 23) were therefore analyzed in conjunction with opioid/cannabinoid receptor gene expressions in the patients' tumors. Receptor gene expressions were determined using the quantitative real-time polymerase chain reaction. Patients receiving morphine had significantly longer cancer-specific survival (CSS) than those receiving piritramide postoperative analgesia (median 22.4 vs. 15 months; p = 0.038). This finding was supported by multivariate modelling (p < 0.001). The morphine and piritramide groups had similar morphine equipotent doses, receptor expression, and baseline characteristics. The opioid/cannabinoid receptor gene expression was analyzed in a group of 130 pancreatic cancer patients. Of the studied receptors, high cannabinoid receptor 2 (CB2) and opioid growth factor receptor (OGFR) gene expressions have a positive influence on the length of overall survival (OS; p = 0.029, resp. p = 0.01). Conversely, high delta opioid receptor gene expression shortened OS (p = 0.043). Multivariate modelling indicated that high CB2 and OGFR expression improved OS (HR = 0.538, p = 0.011, resp. HR = 0.435, p = 0.001), while high OPRD receptor expression shortened OS (HR = 2.264, p = 0.002). Morphine analgesia, CB2, and OGFR cancer tissue gene expression thus improved CSS resp. OS after radical PDAC surgery, whereas delta opioid receptor expression shortened OS.

PITX2 in pancreatic stellate cells promotes EMT in pancreatic cancer cells via the Wnt/β-catenin pathway.

Since the prognosis of patients with pancreatic cancer is very poor and there is a lack of treatment methods, this study is performed to investigate the function of PITX2 in pancreatic stellate cells (PSCs) in the progression of pancreatic cancer. Scientific hypotheses are proposed according to bioinformatics analysis and tissue microarray analysis. Stable knockdown of PITX2 in PSCs is achieved through lentiviral infection. The relative expressions of PITX2, α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are measured in wild-type PSCs and PITX2-knockdown PSCs. Proliferative capacity is measured by EdU assay. After coculture with PSCs, the proliferation, invasion and migration capacity of pancreatic cancer cells are tested. EMT and Wnt/β-catenin downstream genes of pancreatic cancer cells are investigated to reveal the potential mechanism. Bioinformatics analysis reveals that the PITX2 gene is highly expressed in stromal cells in pancreatic cancer and is correlated with squamous-type PDAC. Analysis of PDAC tissue microarray further demonstrates that high PITX2 level in stromal cells is correlated with poor prognosis in PDAC. After stable knockdown of PITX2 in PSCs, the relative protein levels of α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are decreased, and the proliferative capacity of PSCs is also decreased. After coculture with PSCs, in which PITX2 expression is downregulated, the proliferation, invasion and migration capacities of pancreatic cancer cells are inhibited. Thus, our results show that PITX2-silenced PSCs inhibit the growth, migration and invasion of pancreatic cancer cells via reduced EMT and Wnt/β-catenin signaling.

Abstract 3821: Berberine and emodin synergistically suppress the EGFR signaling cascade by targeting LAMB3 in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic cancer is one of the most devastating malignancies due to the development of intrinsic chemoresistance following chemotherapy. Extracellular matrix (ECM) proteins are intimately linked to cellular proliferation, invasion, and acquisition of chemoresistance in PDAC cells, making them promising therapeutic targets in this malignancy. Naturally occurring dietary botanicals, including berberine (BER) and emodin (EMO), have been shown to suppress ECM as one of the mechanism(s) for their anti-tumorigenic activity, along with their time-tested safety and cost-effectiveness. In addition, both BER and EMO are also known to induce apoptosis by modulating different pathways and regulating pro-apoptotic genes. Herein, we hypothesized that combined treatment with BER and EMO might exhibit synergistic anticancer efficacy by targeting the ECM and apoptotic pathways in PDAC cells. Methods: We undertook genomewide transcriptomic profiling analysis to identify critical ECM-related genes differentially expressed in PDAC. Subsequently, the TCGA dataset was analyzed to identify the prognostic significance of ECM-associated genes with overall survival (OS) and disease-free survival (DFS) in PDAC. A series of cell culture experiments were performed using PDAC cells, followed by their validation in patient-derived organoids to examine the synergistic anti-proliferative and chemopreventive effects of BER and EMO against PDAC. Results: Transcriptomic profiling identified that LAMB3 expression was significantly upregulated in PDAC tissue (P &amp;lt; 0.01) and was significantly associated with poor OS and DFS in PDAC patients (P &amp;lt; 0.01). The combination of BER and EMO displayed superior synergistic anti-tumor potential in PDAC cells vs. individual compounds, as revealed by cell proliferation, clonogenicity, migration, and invasion assays. The combination of BER and EMO also altered the expression of key proteins involved in cellular apoptosis, epithelial-mesenchymal-transition, and EGFR/ERK//AKT growth factor signaling pathways. Finally, these findings were successfully validated in PDAC patient-derived 3D organoids. Conclusions: We provide the first evidence that combined treatment with berberine and emodin exerts synergistic anti-cancer activity in PDAC, primarily regulated through the LAMB3-mediated interaction with other members of the EGFR-signaling pathway. Citation Format: CAIMING XU, SILEI SUI, Keisuke Okuno, Silvia Pascual-Sabater, Cristina Fillat, Ajay Goel. Berberine and emodin synergistically suppress the EGFR signaling cascade by targeting LAMB3 in pancreatic ductal 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 3821.

Abstract 1729: MG53 suppresses the tumor growth via transcriptional inhibiting the expression of KIF11 in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with limited therapeutic options and a dismal long-term survival. The upregulation of KIF11, a motor protein that regulates cell mitosis, has been associated with the aggressive proliferation of pancreatic cancer cells. A molecular strategy to counteract the hyperactivity of KIF11 may represent a potential means to treat PDAC. We previously identified MG53, a member of the tripartite motif family protein (TRIM72), as an important component of tissue repair, and revealed its potential application in regenerative medicine. So far, most published studies have focused on the cytosolic and vesicle-trafficking function of MG53, little is known on the nuclear action of MG53 in pancreatic tumor suppression. In the present study, our confocal imaging revealed frequent appearance of MG53 in the nucleus of human pancreatic cancer cells, and subcellular fractionation assay showed that a majority of MG53 protein was present in the nuclear fraction. Decreased MG53 and increased KIF11 protein expression were observed in both pancreatic cancer cell lines (PANC-1 and mPanc96) and PDAC tissue from surgically resected tumors. Overexpressing MG53 inhibited colony formation and proliferation of PANC-1 cells with significant reduction of KIF11 expression. Furthermore, we demonstrated that MG53-mediated control of KIF11 expression occurs at the gene transcription level. Chromatin immunoprecipitation demonstrated an interaction between MG53 and the promoter of KIF11 in PANC-1 cells. Using a luciferase cell reporter driven by the KIF11 promoter, we found that overexpressing MG53 led to downregulation of the KIF11 transcription activity, and induced cell cycle arrest at the G2/M phase. We also conducted syngeneic orthotopic transplantation of a mouse pancreatic cancer cell line, KPC cells derived from pancreatic tumor in KRasG12D/Trp53-/-/Pdx-1-Cre (KPC) mice, into the pancreas of wild type (WT) and mg53-/- (MG53-KO) mice, which showed aggressive pancreatic tumor growth in MG53-KO mice compared with WT mice after 3-week of transplantation. Western blots revealed that a considerable amount of MG53 protein was found in the pancreatic tumors derived from KPCs in WT mice vs. those in MG53-KO mice, indicating the accumulation of circulating MG53 during tumor growth. Overall, these findings demonstrated that MG53 functions as a tumor suppressor to inhibit cancer cell proliferation and the tumor growth through transcriptional down-regulating the expression of KIF11 in pancreatic cancer, and provided a new therapeutic target for the treatment of patients with PDAC. Citation Format: Xiao-Liang Wang, Xiangfei He, Zobeida Cruz-Monserrate, Allen Tsung, Jianjie Ma, Chuanxi Cai. MG53 suppresses the tumor growth via transcriptional inhibiting the expression of KIF11 in pancreatic cancer [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 1729.

Abstract 2148: Robust detection of somatic genetic alterations in pancreatic cancer ascites

Abstract Introduction: Regardless of the stage at diagnosis most patients with pancreatic ductal adenocarcinoma develop peritoneal disease and some malignant ascites (MA) as well. Prior studies have shown that MA negatively affects overall treatment efficacy and survival. Despite the clinical significance of MA it has not been studied to any great extent. Methods: We collected MA and matched normal tissue samples at autopsy from 20 PDAC patients who were initially diagnosed at stages IIB to IV. Whole exome or targeted sequencing was previously performed on each PDAC. Each MA sample was centrifuged twice at 4000 RPM first and then at 15000 RPM to separate the cell pellet (CP) from the cell-free ascites fluid. We next extracted DNA from the CP, matched normal tissue, and the cell-free DNA (cfDNA) from the ascites fluid, and all were submitted to the Genomics Core for MSK-IMPACT, a targeted cancer gene panel representing 505 genes. Results: Results of the first five patients are complete and the remaining are in process. Comparison of the CPs and/or cfDNA to the matched tumor samples indicated 100% concordance for detected variants. However, the somatic alterations of the CP specifically versus the matched cfDNA were divergent in all patients analyzed thus far. Virtually all copy number alterations in all patients were deep deletions (range 66 to 187 cancer genes deleted) affecting multiple DNA repair pathways including homologous recombination deficiency and microsatellite repair. Conclusions: Samples of MA, when both the cell pellet and cfDNA are sequenced, accurately represent the genetic features of the matched PDAC tissue and may serve as an alternative mode of sampling for precision medicine. Differences in the genetics of the CP versus the cfDNA suggest polyclonality in the peritoneal space. Moreover, the finding of deep deletions in targetable DNA repair pathways suggest a therapeutic vulnerability for exploration. Given that paracentesis is often performed in the palliative setting and may be performed multiple times over the course of a patients’ management, it also offers an opportunity to determine how clonal dynamics in the peritoneal space change over time. Patients with MA have poor overall survival compared to patients without MA so these patients may benefit from this type of tracking which could potentially help with their treatment. Citation Format: Rajya L. Kappagantula, Alvin P. Makohon-Moore, Shigeaki Umeda, Elias-Ramzey R. Karnoub, Jerry P. Melchor, Laura D. Wood, Christine A. Iacobuzio-Donahue. Robust detection of somatic genetic alterations in pancreatic cancer ascites [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 2148.

Abstract 5530: A community led approach to addressing disparities in pancreatic cancer care for Native Americans in Oregon

Abstract Pancreatic adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States (US). Due to unique barriers facing Native Americans (NA) patients, this underrepresented population has worse outcomes and increased mortality. In Oregon, NAs have nearly double the PDAC incidence rate compared to the general US population. Oregon Health &amp; Science University (OHSU) is a leader in PDAC care and research, yet NAs remain underrepresented in pancreatic cancer research at OHSU, and across the country. This highlights the need for a focused and comprehensive research program that collaborates with local tribal communities to identify specific issues and barriers that can be addressed to improve patient care in the NA population. We hypothesize that if we establish a workflow that strengthens existing relationships with tribal communities and leverages established OHSU infrastructure and expertise, we can directly address the unmet need of understanding, preventing and treating PDAC for NA individuals in our local communities. This includes increasing enrollment of NAs in PDAC clinical trials and developing a genetic registry that could help both depict genetic underpinnings that predispose NA individuals to PDAC and potentially guide therapeutic approaches. To build trust within NA communities, we initiated collaborations with Oregon tribes to develop culturally appropriate programs guided by NA community members. Through OHSU’s Northwest Native American Center of Excellence, we developed a close relationship with the Confederated Tribes of Warm Springs. Together with our institution’s Community Outreach, Research, and Engagement (CORE) team, we are working with the Confederated Tribes of Warm Springs to better understand provider and NA community member attitudes towards and barriers to clinical trial participation. We will incorporate these perceptions into a tribe-specific program that describes the multi-faceted facilitators and barriers that inform decision-making. Moreover, we are implementing a patient navigator program to help NA patients overcome clinical trial barriers by providing culturally appropriate patient education, reimbursement for travel-associated expenses, local administration of chemotherapy, and telehealth capabilities. Future work includes co-implementing strategies for tribes to review incoming research requests with the goal of implementing a system to promote awareness and enrollment of NAs in PDAC clinical research. This system will be then be utilized to establish the first NA PDAC tissue registry to identifying gene-environment interactions and unique genetic alterations that may predispose NA individuals to PDAC. Overall, this collaborative work will create a roadmap for engagement and a conscientious process to provide awareness and the best in class treatment for NA individuals with PDAC. Citation Format: Claymore Kills First, Grace A. McCarthy, Charles D. Lopez, Brett C. Sheppard, Jackilen Shannon, Jonathan R. Brody. A community led approach to addressing disparities in pancreatic cancer care for Native Americans in Oregon. [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 5530.

IL-17A-producing CD8+ T cells promote PDAC via induction of inflammatory cancer-associated fibroblasts

ObjectivePancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic stroma composed of cancer-associated fibroblasts (CAF) and interspersed immune cells. A non-canonical CD8+ T-cell subpopulation producing IL-17A (Tc17) promotes autoimmunity...

Comprehensive immunoprofile analysis of prognostic markers in pancreaticobiliary tract cancers.

Pancreaticobiliary tract cancer has a poor prognosis with unmet needs in a new target treatment. Some studies have reported that an enhancement of T-cell immunity is associated with a good prognosis. The aim of this study is to investigate the immunoprofile as a prognostic marker of pancreaticobiliary tract cancers. Unresectable pancreatic ductal adenocarcinoma (PDAC, n=80) and biliary tract cancer (BTC, n=74) diagnosed between January 2012 and December 2018 in Samsung Medical Center were analyzed. Expression levels of CD8, FOXP3, PD-1, PD-L1, and CXCL13 in PDAC and BTC tissue samples were examined with immunohistochemical staining, which was evaluated with various clinical factors. In PDAC, higher degree of PD-L1 expression was significantly associated with shorter overall survival (OS) (p=0.0095). On the other hand, higher infiltrations of PD-1+ immune cells (p=0.0002) and CD8+ T cells (p=0.0067) were associated with longer OS. In BTC, higher FOXP3+ (p=0.0343) and CD8+ (p=0.0028) cell infiltrations were associated with better survival. Low infiltration of CD8+ (p=0.0148), FOXP3+ (p=0.0208), PD-1+ (p=0.0318) cells in PDAC, and FOXP3+ cells (p=0.005) in BTC were considerably related to metastasis. In a combined evaluation of clinical factors and immunoprofiles, univariate analysis revealed that operation after chemotherapy (p < 0.0001), mass size (p=0.0004), metastasis (p=0.006), PD-L1 (p < 0.0001), PD-1 (p=0.003) and CD8 (p=0.0063) was significantly associated with OS in PDAC, and CD8 (p=0.007) was statistically related to OS in BTC. In multivariate analysis, prognostic factors were operation after chemotherapy (p=0.021) in PDAC and CD8 (p=0.037) in BTC. Therefore, immunoprofile analysis of cells expressing CD8, FOXP3, PD-1, and PD-L1 might have prognostic values in patients with pancreaticobiliary tract cancers.

Abstract 3648: Hedgehog represses angiogenesis in PDAC through a paracrine cascade mediated by Wif1

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by an extremely heterogeneous tumor microenvironment (TME) paired with a sparse and compromised vascular network. These features of the TME shape drug responses and contribute to PDAC’s distinctive chemotherapeutic resistance. Prior work demonstrated that inhibition of the Hedgehog (Hh) signaling pathway using Smoothened (Smo) inhibitors led to increased angiogenesis. However, the precise mechanisms through which the Hh pathway regulates angiogenesis has remained elusive and subsequent studies with different agents and timepoints yielded conflicting data. Frustratingly, the phenotype could not be recreated in simple co-culture models. In order to maintain the cellular heterogeneity and spatial organization that characterizes PDAC, we developed a novel tumor explant system for short-term cultures of both murine and human PDAC. Briefly, we cultured intact 300 μm slices of PDAC tissue on a modified gelatin platform system with a novel culture medium informed by the metabolic composition of PDAC tumor interstitial fluid. This system replicates some of the nutrient/waste gradient of PDAC and enables tumors to be cultured up to one week maintaining the representation of multiple cell types. Studies in explants were complemented by work in the Kras-LSL.G12D/+, p53-LSL.R172H/+, Pdx1-Cre (KPC) genetically engineered model of PDAC. Comparison of short versus long-term Smo inhibition in KPC mouse pancreatic tumors revealed a robust increase in vessel density in short-term treatment that is lost at later timepoints. Studies in murine and human PDAC explants employing different Smo inhibitors demonstrated increased endothelial tip cell formation upon two days of treatment, resulting in increased vessel density after four days. Computational analyses of human PDAC single cell RNAseq data identified candidate cellular subsets potentially involved in the signaling cascade. RNAseq of pre- and on-treatment tumor samples from KPC mice identified Wif1 as a candidate Hh pathway target gene that effected these programs. Mechanistic studies in tumor explants reveal a complex, multi-step mechanism involving several cell types that led to suppression of angiogenesis in PDAC. These studies both provide a candidate mechanism for Hh-mediated angiosuppression and also demonstrated how the tumor explant platform can elucidate complex intercellular signaling cascades in both murine and human PDAC. Citation Format: Marie C. Hasselluhn, Amanda R. Decker, Alvaro Curiel Garcia, Carlo Maurer, Dafydd H. Thomas, Kenneth P. Olive. Hedgehog represses angiogenesis in PDAC through a paracrine cascade mediated by Wif1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3648.

Drug screening and genome editing in human pancreatic cancer organoids identifies drug-gene interactions and candidates for off-label therapy

Pancreatic cancer (PDAC) is a highly aggressive malignancy for which the identification of novel therapies is urgently needed. Here, we establish a human PDAC organoid biobank from 31 genetically distinct lines, covering a representative range of tumor subtypes, and demonstrate that these reflect the molecular and phenotypic heterogeneity of primary PDAC tissue. We use CRISPR-Cas9 genome editing and drug screening to characterize drug-gene interactions with ARID1A and BRCA2. We find that missense- but not frameshift mutations in the PDAC driver gene ARID1A are associated with increased sensitivity to the kinase inhibitors dasatinib (p < 0.0001) and VE-821 (p < 0.0001). We conduct an automated drug-repurposing screen with 1,172 FDA-approved compounds, identifying 26 compounds that effectively kill PDAC organoids, including 19 chemotherapy drugs currently approved for other cancer types. We validate the activity of these compounds in vitro and in vivo. The in vivo validated hits include emetine and ouabain, compounds which are approved for non-cancer indications and which perturb the ability of PDAC organoids to respond to hypoxia. Our study provides proof-of-concept for advancing precision oncology and identifying candidates for drug repurposing via genome editing and drug screening in tumor organoid biobanks.

Potential Metabolite Biomarkers for Early Detection of Stage-I Pancreatic Ductal Adenocarcinoma.

Background & ObjectivesPancreatic ductal adenocarcinoma remains an extremely malignant tumor having a poor prognosis. The 5-year survival rate of PDAC is related to its stage (about 80% for stage I vs 20% for other stages). However, detection of PDAC in an early stage is difficult due to the lack of effective screening methods. In this study, we aimed to construct a novel metabolic model for stage-I PDAC detection, using both serum and tissue samples.MethodsWe employed an untargeted technique, UHPLC-Q-TOF-MS, to identify the potential metabolite, and then used a targeted technique, GC-TOF-MS, to quantitatively validate. Multivariate and univariate statistics were performed to analyze the metabolomic profiles between stage-I PDAC and healthy controls, including 90 serum and 53 tissue samples. 28 patients with stage-I PDAC and 62 healthy controls were included in this study.ResultsA total of 10 potential metabolites presented the same expression levels both in serum and in tissue. Among them, a 2-metabolites-model (isoleucine and adrenic acid) for stage-I PDAC was constructed. The area under the curve (AUC) value was 0.93 in the discovery set and 0.90 in the independent validation set. Especially, the serum metabolite model had a better diagnostic performance than CA19-9 (AUC = 0.79). Pathway analysis revealed 11 altered pathways in both serum and tissue of stage-I PDAC.ConclusionsThis study developed a novel serum metabolites model that could early separate stage-I PDAC from healthy controls.