Invasive Pancreatic Ductal Adenocarcinoma Research Articles

Semaphorin 3D promotes pancreatic ductal adenocarcinoma progression and metastasis through macrophage reprogramming.

Axon guidance molecules are frequently altered in pancreatic ductal adenocarcinoma (PDA) and influence PDA progression. However, the molecular mechanism remained unclear. Using genetically engineered mouse models to examine semaphorin 3D (SEMA3D), we identified a dual role for tumor- and nerve-derived SEMA3D in the malignant transformation of pancreatic epithelial cells and invasive PDA development. Pancreatic-specific knockout of the SEMA3D gene from the KRASG12D and TP53R172H mutation knock-in, PDX1-Cre(KPC) mouse model demonstrated delayed tumor initiation, prolonged survival, absence of metastasis, and reduced M2 macrophage expression. Mechanistically, tumor- and nerve-derived SEMA3D indirectly reprograms macrophages through KRASMUT-dependent ARF6 signaling in PDA cells, resulting in increased lactate production, which is sensed by GPCR132 on macrophages to stimulate protumorigenic M2 polarization. Multiplex immunohistochemistry demonstrated increased M2-polarized macrophages proximal to nerves in SEMA3D-expressing human PDA tissue. This study suggests that altered SEMA3D expression leads to an acquisition of cancer-promoting functions, and nerve-derived SEMA3D is "hijacked" by PDA cells to support growth and metastasis in a KRASMUT-dependent manner.

Cancer-Associated Fibroblasts: From Spectators to Protagonists in Pancreatic Cancer Progression.

Our knowledge of the origins, heterogeneity, and functions of cancer-associated fibroblasts (CAF) in pancreatic ductal adenocarcinoma (PDAC) has exponentially increased over the last two decades. This has been facilitated by the implementation of new models and single-cell technologies. However, a few key studies preceded the current exciting times in CAF research and were fundamental in initiating the investigation of CAFs and of their roles in PDAC. With their study published in Cancer Research in 2008, Hwang and colleagues have been first to successfully isolate and immortalize human pancreatic stellate cells (HPSC) from PDAC tissues. This new tool allowed them to probe the roles of CAFs in PDAC as never done before. By performing complementary in vitro and in vivo analyses, the authors demonstrated the involvement of HPSCs in PDAC malignant cell proliferation, invasion, and therapy resistance. Here, we leverage that seminal study as a framework to discuss the advances made over the last 16 years in understanding the complexity and central roles of CAFs in PDAC progression. See related article by Hwang and colleagues, Cancer Res 2008;68:918-26.

Abstract PR-13: Disruption of sulfatide metabolism by targeting UGT8 is an actionable metabolic vulnerability for pancreatic cancer early interception

Abstract The existence of asymptomatic precursor lesions that are known to predate invasive pancreatic ductal adenocarcinoma (PDAC) by years provides a compelling rationale for cancer interception efforts. One such precursor is intraductal papillary mucinous neoplasm (IPMN). Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) imaging of metabolites in tissue sections from resected patient IPMN cases (N=23) and from a KrasG12D/GnasR201C mouse model of IPMN, we have made a discovery of long chain hydroxylated sulfatide species as being selectively enriched in IPMN. Spatial transcriptomics analysis of serial sections from the same IPMN tissues confirmed cognate transcripts, including ceramide galactosyltransferase (CGT, also known as UGT8) and galactose-3-O-sulfotransferase (CST, also known as Gal3St1) enzymes that, respectively, catalyze the addition of galactose from UDP-galactose to ceramide and the O-sulfation of the galactose residue on galactosylceramide (GalCer), to also be highly enriched in IPMN. Functional studies using CRISPR/cas9 UGT8 knockout Kras;Gnas IPMN cells demonstrated that loss of UGT8, the key enzyme that produces the sulfatide precursor GalCer, results in attenuation of sulfatide biosynthesis and impaired mitochondrial function, as evidenced by reduced mitochondrial respiration and ATP production. These changes were met with concomitant decreases in proliferation and invasiveness, and increased susceptibility to intrinsic apoptosis. Targeting of sulfatide metabolism using a selective small molecule inhibitor of UGT8 suppressed sulfatide biosynthesis, induced ceramide-mediated compensatory mitophagy, and subsequent apoptosis in Kras;Gnas IPMN cells. In vivo, UGT8 inhibition suppressed tumor growth in an IPMN allograft model. In conclusion, our work identifies enhanced sulfatide metabolism as an early metabolic alteration in cystic pre-cancerous lesions of the pancreas that persists through invasive neoplasia and represents an actionable vulnerability for PDAC early interception. Citation Format: Riccardo Ballarò, Yihui Chen, Fredrik I Thege, Rongzhang Dou, Jimin Min, Michele Yip-Schneider, Jianjun Zhang, Ranran Wu, Ehsan Irajizad, Yuki Makino, Kimal I Rajapakshe, Mark W Hurd, Ricardo A León-Letelier, Edwin Ostrin, Jody Vykoukal, Jennifer B Dennison, Kim-Anh Do, Samir Hanash, Robert A Wolff, Paola A Guerrero, Michael P Kim, C Max Schmidt, Anirban Maitra, Johannes F Fahrmann. Disruption of sulfatide metabolism by targeting UGT8 is an actionable metabolic vulnerability for pancreatic cancer early interception [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-13.

Study Protocol for a Prospective Self-Controlled Trial on Success in Meeting Comprehensive Genomic Profiling Analysis Criteria for Specimens Obtained by Endoscopic Ultrasound-Guided Tissue Acquisition Using a 19G Needle from Primary and Metastatic Lesions in Pancreatic Cancer with Metastatic Lesions: The PRIMATE Study.

EUS-TA in unresectable pancreatic cancer requires not only a tissue diagnosis but also tissue collection in anticipation of comprehensive genomic profiling. However, the optimal puncture target remains controversial. Therefore, the Primary and Metastatic Lesions in Pancreatic Cancer (PRIMATE) study was designed to clarify the optimal target by comparing the success rates for meeting OncoGuide NCC Oncopanel (NOP) analysis criteria on pre-check primary and metastatic lesion specimens obtained during the same EUS-TA session in patients with invasive pancreatic ductal adenocarcinoma. In this ongoing prospective study, two specimens, each from primary and metastatic lesions, are obtained by EUS-TA (typically using a 19G fine-needle biopsy needle) in patients with invasive pancreatic ductal adenocarcinoma. The primary endpoint is the proportion of EUS-TA specimens that meet NOP analysis criteria during pre-check (i.e., tumor cellularity of ≥20% and a tissue area of ≥4 mm2), which are then compared between primary and metastatic lesions. This study has been approved by the National Cancer Center Institutional Review Board (Research No. 2022-168). The results of this study will be reported at an international conference and published in an international peer-reviewed journal. The trial registration number is UMIN 000048966.

PLEK2 promotes migration and invasion in pancreatic ductal adenocarcinoma by MMP1 through IL-17 pathway.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis primarily due to metastasis. Accumulating evidence suggests that PLEK2 acts as an oncogene in various tumors. This study aimed to investigate the effects of PLEK2 on PDAC. Expression analysis of PLEK2 was conducted using qRT-PCR, Western blot, and immunohistochemistry in PDAC. Wound healing and transwell assays were performed to evaluate the impact of PLEK2 on cell migration and invasion. A xenograft tumor model was employed to assess the in vivo proliferation of PLEK2. Additionally, the downstream pathway of PLEK2 was analyzed through RNA-seq and confirmed by Western blot analysis. The results demonstrated the upregulation of PLEK2 expression in tumor specimens. High PLEK2 expression was significantly associated with poor overall survival and advanced TNM stages. Correlation analyses revealed positive correlations between PLEK2 and TGF-β, EGFR, and MMP1. Wound healing and transwell assays demonstrated that PLEK2 promoted PDAC cell migration and invasion, potentially through the activation of the epithelial-to-mesenchymal transition process. The in vivo experiment further confirmed that PLEK2 knockdown suppressed tumor growth. RNA-seq analysis revealed PLEK2's regulation of MMP1 and activation of p-ERK and p-STAT3, which were verified by Western blot analysis. Overall, the present study suggests that PLEK2 may play a tumor-promoting role in PDAC. These findings provide valuable insights into the molecular mechanisms of pancreatic cancer and highlight the potential of PLEK2 as a therapeutic target.

Efficacy of color Doppler ultrasound and contrast-enhanced ultrasound in identifying vascular invasion in pancreatic ductal adenocarcinoma

ObjectivesTo compare color Doppler ultrasound and contrast-enhanced ultrasound (CEUS) in evaluating vascular invasion in pancreatic ductal adenocarcinoma (PDAC).Materials and methodsThis retrospective study included 210 patients with PDAC who were evaluated by color Doppler ultrasound, CEUS, and contrast-enhanced computed tomography (CECT) at our institution between January 2017 and December 2020. Pathologic results were used as the gold standard in patients who underwent surgical and intraoperative exploration. For nonsurgical patients, CECT results were used as the reference standard. The vessels evaluated included those in the peripancreatic arterial system and venous system. The diagnostic performances of color Doppler ultrasound and CEUS for vascular invasion were compared.ResultsIn 51 patients who underwent surgery and intraoperative exploration, color Doppler ultrasound and CEUS differed only in assessing venous system invasion in patients with PDAC of the pancreatic body and tail, with the former being superior to the latter. In 159 nonsurgical patients, there was no difference between CEUS and color Doppler ultrasound in assessing superior mesenteric arteriovenous invasion. CEUS was superior to color Doppler ultrasound in evaluating the celiac artery and its branches, with an accuracy of up to 97.8% for some vessels. Color Doppler ultrasound was ideal for evaluating the splenic and portal veins.ConclusionCEUS is more suitable for the evaluation of peripancreatic arteries than color Doppler. CEUS combined with color Doppler ultrasound can be used as a potential supplement to CECT and is also expected to be used to evaluate vascular invasion of PDAC after chemotherapy.Critical relevance statementContrast-enhanced US and color Doppler in the assessment of vascular invasion in pancreatic ductal adenocarcinoma have their respective advantages, through standardized ultrasound processes are expected to improve the efficiency of inspection.Key PointsContrast-enhanced US has unique advantages in assessing pancreatic ductal adenocarcinoma invasion of the celiac artery.Doppler imaging is of high value in assessing venous system invasion.Standardization of ultrasound imaging procedures for pancreatic ductal adenocarcinoma is expected to improve efficiency.Graphical

Scribble Deficiency Promotes Pancreatic Ductal Adenocarcinoma Development and Metastasis.

Perturbation of cell polarity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) progression. Scribble (SCRIB) is a well-characterized polarity regulator that has diverse roles in the pathogenesis of human neoplasms. To investigate the impact of SCRIB deficiency in PDAC development and progression, Scrib expression was genetically ablated in well-established mouse models of PDAC. Scrib loss in combination with KrasG12D did not influence development of pancreatic intraepithelial neoplasms in mice. However, Scrib deletion cooperated with KrasG12D and concomitant Trp53 heterozygous deletion to promote invasive PDAC and metastatic dissemination, leading to reduced overall survival. Immunohistochemical and transcriptome analyses revealed that Scrib-null tumors display a pronounced reduction of collagen content and an abundance of cancer-associated fibroblasts (CAF). Mechanistically, IL1α levels were reduced in Scrib-deficient tumors, and Scrib knockdown downregulated IL1α in mouse PDAC organoids (mPDO), which impaired CAF activation. Furthermore, Scrib loss increased YAP activation in mPDOs and established PDAC cell lines, enhancing cell survival. Clinically, SCRIB expression was decreased in human PDAC, and SCRIB mislocalization was associated with poorer patient outcome. These results indicate that SCRIB deficiency enhances cancer cell survival and remodels the tumor microenvironment to accelerate PDAC development and progression, establishing the tumor suppressor function of SCRIB in advanced pancreatic cancer. Significance: SCRIB loss promotes invasive pancreatic cancer development via both cell-autonomous and non-cell-autonomous processes and is associated with poorer outcomes, denoting SCRIB as a tumor suppressor and potential biomarker for the prediction of recurrence.

Survival benefit associated with screening of patients at elevated risk for pancreatic cancer.

All patients enrolled in screening at a High-Risk Pancreatic Cancer Clinic (HRC) from July 2013 to June 2020 were identified from a prospectively maintained institutional database and compared to patients evaluated at a Surgical Oncology Clinic (SOC) at the same institution during the same period. Clinical outcomes of patients selected for surgical resection, particularly clinicopathologic stage and overall survival, were compared. Among 826 HRC patients followed for a median (IQR) of 2.3 (0.8-4.2) years, 128 were selected for surgical resection and compared to 402 SOC patients selected for resection. Overall survival was significantly longer among HRC patients (median survival: not reached vs. 2.6 years, p < 0.001). Among 31 HRC and 217 SOC patients with a diagnosis of pancreatic ductal adenocarcinoma(PDAC), the majority of HRC patients were diagnosed with stage 0 disease (carcinoma in situ), while the majority of SOC patients were diagnosed with stage II disease (p < 0.001). Overall survival after resection of invasive PDAC was also significantly longer among HRC patients compared to SOC patients (median survival 5.5 vs. 1.6 years, p = 0.002). Patients at increased risk for PDAC and followed with guideline-based screening exhibited downstaging of disease and improved survival from PDAC in comparison to patients who were not screened.

Combined effects of matrix stiffness and obesity-associated signaling directs progressive phenotype in PANC-1 pancreatic cancer cells in vitro.

Obesity is a leading risk factor of pancreatic ductal adenocarcinoma (PDAC) that contributes to poor disease prognosis and outcomes. Retrospective studies have identified this link, but interactions surrounding obesity and PDAC are still unclear. Research has shifted to contributions of fibrosis (desmoplasia) on malignancy, which involves increased deposition of collagens and other extracellular matrix (ECM) molecules and increased ECM crosslinking, all of which contribute to increased tissue stiffening. However, fibrotic stiffening is underrepresented as a model feature in current PDAC models. Fibrosis is shared between PDAC and obesity, and can be leveraged for in vitro model design, as current animal obesity models of PDAC are limited in their ability to isolate individual components of fibrosis to study cell behavior. In the current study, methacrylated type I collagen (PhotoCol®) was photo-crosslinked to pathological stiffness levels to recapitulate fibrotic ECM stiffening. PANC-1 cells were encapsulated within PhotoCol®, and the tumor-tissue constructs were prepared to represent normal (healthy) (∼600 Pa) and pathological (∼2000 Pa) tissues. Separately, human mesenchymal stem cells were differentiated into adipocytes representing lean (2D differentiation) and obese fat tissue (3D collagen matrix differentiation), and conditioned media was applied to PANC-1 tumor-tissue constructs. Conditioned media from obese adipocytes showed increased vimentin expression, a hallmark of invasiveness and progression, that was not seen after exposure to media from lean adipocytes or control media. Characterization of the obese adipocyte secretome suggested that some PANC-1 differences may arise from increased interleukin-8 and -10 compared to lean adipocytes. Additionally, high matrix stiffness associated induced an amoeboid morphology in PANC-1 cells that was not present at low stiffness. Amoeboid morphology is an accessory to epithelial-to-mesenchymal transition and is used to navigate complex ECM environments. This plasticity has greater implications for treatment efficacy of metastatic cancers. Overall, this work 1) highlights the importance of investigating PDAC-obesity interactions to study the effects on disease progression and persistence, 2) establishes PhotoCol® as a matrix material that can be leveraged to study amoeboid morphology and invasion in PDAC, and 3) emphasizes the importance of integrating both biophysical and biochemical interactions associated within both pathologies for in vitro PDAC models.

A phase I/II open label study to assess safety and preliminary evidence of a therapeutic effect of azeliragon in patients refractory to first-line treatment of metastatic pancreatic cancer.

TPS4212 Background: Azeliragon is an orally administered small molecule, administered once daily, that inhibits RAGE interactions with its natural ligands, including HMGB1 and S100 proteins. Azeliragon was previously under development for Alzheimer’s disease and failed to show efficacy in phase 3 trials. Clinical safety data from these trials, with over 1500 individuals dosed for periods up to 18 months, indicated that azeliragon has a high level of safety and tolerability, virtually indistinguishable from placebo. RAGE, the receptor for advanced glycation end products, is highly expressed in pancreatic ductal adenocarcinoma (PDAC). RAGE interaction with its ligands, including S100 proteins and HMGB1 released from PDAC cells, promotes PDAC invasion, metastasis, and resistance to 5 FU and Gemcitabine. PDAC-secreted S100 proteins can also stimulate fibroblasts surrounding PDAC cells since RAGE is positively expressed in fibroblasts, promoting the desmoplastic response. RAGE has also been implicated in the muscle wasting associated with cancer and with aging, as well as in neuropathic pain, suggesting a potential supportive role in PDAC treatment. Methods: This is an open label study to determine the safety and preliminary evidence of a therapeutic effect of azeliragon in patients refractory to the standard of care treatment of metastatic pancreatic cancer. Azeliragon is administered at three dose levels in a rolling dose escalation strategy with 6 patients at each dose level: 5 mg/day (level 1), 10 mg/day (level 2) and 20 mg/day (level 3). These dose levels have an initial loading dose for 6 days at 15 mg daily, 15 mg twice a day and 30 mg twice a day, respectively. The primary objective is to determine the RP2D, defined as the dose for which &lt;33% patients experience a dose limiting toxicity (DLT) within 28 days from initiation of dosing. Secondary endpoints include disease control, overall survival, changes in pain as determined by the Brief Pain Inventory, and changes in ECOG performance status, weight, and serum albumin. The study received regulatory approval and accrual started in June 2023. Clinical trial information: NCT05766748 .

Oncological Outcomes and Patterns of Recurrence after the Surgical Resection of an Invasive Intraductal Papillary Mucinous Neoplasm versus Primary Pancreatic Ductal Adenocarcinoma: An Analysis from the German Cancer Registry Group of the Society of German Tumor Centers.

Intraductal papillary mucinous neoplasms (IPMNs) are premalignant cystic neoplasms of the pancreas (CNPs), which can progress to invasive IPMN and pancreatic cancer. The available literature has shown controversial results regarding prognosis and clinical outcomes after the resection of invasive IPMN. This study aims to characterize the oncologic outcomes and metastatic progression pattern after the resection of non-metastatic invasive IPMN. Data were obtained from 24 clinical cancer registries participating in the German Cancer Registry Group of the Society of German Tumor Centers (ADT). Patients with invasive IPMN (n = 217) as well as PDAC (n = 5794) between 2000 and 2021 were included and compared regarding oncological outcomes. Invasive IPMN was significantly smaller in size (p < 0.001) and of a lower tumor grade (p < 0.001), with fewer lymph node metastases (p < 0.001), lymphangiosis (p < 0.001), and consequently a higher R0 resection rate (88 vs. 74%) compared to PDAC. Moreover, invasive IPMN was associated with fewer local (11 vs. 15%) and distant recurrences (29 vs. 46%) and metastasized more frequently in the lungs only (26% vs. 14%). Invasive IPMN was associated with a longer median OS (29 vs. 19 months) and DFS (31 vs. 15 months) compared to PDAC and stayed independently prognostic in multivariable analyses. These survival differences were most pronounced in early tumor stages. Interestingly, postoperative chemotherapy was not associated with improved overall survival in surgically resected invasive IPMN. Invasive IPMN is a rare pancreatic entity with increasing incidence in Germany. It is associated with favorable histopathological features at the time of resection and longer OS and DFS compared to PDAC, particularly before the locoregional spread has occurred. Invasive IPMNs are associated with lung-only metastasis. The benefit of postoperative chemotherapy after the resection of invasive IPMN remains uncertain.

CT-based fully automated artificial intelligence model to predict extrapancreatic perineural invasion in pancreatic ductal adenocarcinoma

Background: Extrapancreatic perineural invasion (EPNI) increases the risk of postoperative recurrence in pancreatic ductal adenocarcinoma (PDAC). This study aimed to develop and validate a computed tomography (CT)-based, fully automated preoperative artificial intelligence (AI) model to predict EPNI in patients with PDAC. Methods: We retrospectively enrolled 1065 patients from two Shanghai hospitals between June 2014 and April 2023. Patients were split into training (n=497), internal validation (n=212), internal test (n=180), and external test (n=176) sets. The AI model used perivascular space and tumor contact for EPNI detection. We evaluated the AI model’s performance based on its discrimination. Kaplan-Meier curves, log-rank tests, and Cox regression were used for survival analysis. Results: The AI model demonstrated superior diagnostic performance for EPNI with 1-pixel expansion. The area under the curve in the training, validation, internal test, and external test sets were 0.87, 0.88, 0.82, and 0.83, respectively. The log-rank test revealed a significantly longer survival in the AI-predicted EPNI-negative group than the AI-predicted EPNI-positive group in the training, validation, and internal test sets (P&lt;0.05). Moreover, the AI model exhibited exceptional prognostic stratification in early PDAC and improved assessment of neoadjuvant therapy’s effectiveness. Conclusion: The AI model presents a robust modality for EPNI diagnosis, risk stratification, and neoadjuvant treatment guidance in PDAC, and can be applied to guide personalized precision therapy.

CHES1 modulated tumorigenesis and senescence of pancreas cancer cells through repressing AKR1B10

Pancreatic ductal adenocarcinoma (PDAC), is characteristic by a heterogeneous tumor microenvironment and gene mutations, conveys a dismal prognosis and low response to chemotherapy and immunotherapy. Here, we found that checkpoint suppressor 1 (CHES1) served as a tumor repressor in PDAC and was associated with patient prognosis. Functional experiments indicated that CHES1 suppressed the proliferation and invasion of PDAC by modulating cellular senescence. To further identify the downstream factor of CHES1 in PDAC, label-free quantitative proteomics analysis was conducted, which showed that the oncogenic Aldo-keto reductase 1B10 (AKR1B10) was transcriptionally repressed by CHES1 in PDAC. And AKR1B10 facilitated the malignant activity and repressed senescent phenotype of PDAC cells. Moreover, pharmaceutical inhibition of AKR1B10 with Oleanolic acid (OA) significantly induced tumor regression and sensitized PDAC cells to gemcitabine, and this combined therapy did not cause obvious side effects. Rescued experiments revealed that CHES1 regulated the tumorigenesis and gemcitabine sensitivity through AKR1B10-mediated senescence in PDAC. In summary, this study revealed that the CHES1/AKR1B10 axis modulated the progression and cellular senescence in PDAC, which might provide revenues for drug-targeting and senescence-inducing therapies for PDAC.

Effect of Risk Factor Score on Early Recurrence After Pancreatectomy for Invasive Pancreatic Ductal Adenocarcinoma.

This study aimed to identify the risk factors for early recurrence (ER) after pancreatic ductal adenocarcinoma (PDAC) resection to create a novel scoring system for ER and analyze their effect on the recurrence pattern. Sixty patients with PDAC who underwent pancreatectomy were included. The predicted risk factors for ER were analyzed. A new score defining ER was created and analyzed for recurrence pattern and prognosis. Independent predictors included high CA 19-9 (≥147 U/ml), high lymph node ratio (LNR of ≥0.1277), and no adjuvant chemotherapy (AC). The 5-year overall survival rates with a score of 0, 1, and 2 were 55.8%, 11.0%, and 0%, respectively. In the moderate- risk score group, prognosis was improved by induction of AC within 58 days. Preoperative high CA19-9, high LNR, and no AC could be ER predictors. Induction of postoperative chemotherapy within 58 days may improve prognosis.

Abstract 1206: Three-dimensional immune atlas of pancreatic cancer precursor lesions reveals large inter- and intra-lesion heterogeneity

Abstract Immunotherapies are generally ineffective in pancreatic ductal adenocarcinoma (PDAC). Paradoxically, inflammation is believed to play a key role in PDAC development and invasion: patients suffering from chronic pancreatitis have a 13-fold increase in risk of developing PDAC. PDAC cells are surrounded by a dense network of fibrotic tissue containing immunosuppressive cells such as regulatory T cells, tumor-associated macrophages, and cancer-associated fibroblasts. Better understanding of the role of inflammation in PDAC could lead to the design of effective immunotherapies. Pancreatic intraepithelial neoplasia (PanIN) is a precursor to PDAC. While most of us will develop PanINs, few of these lesions will progress to invasive cancer. Efforts to determine which PanINs are likely to progress have shown that the size, incidence, and genetic variation of these lesions is high. Here, we add to these efforts through in-depth quantitative analysis of the pancreatic immune microenvironment. CODA is a novel 3D imaging technique used extensively to study the structure, transcriptomic signatures, and genetic heterogeneity of PanINs. In serial histological samples, CODA integrates image registration, cell detection, and segmentation of nine pancreatic structures: PanIN, normal ducts, islets, acini, nerves, vasculature, fat, stroma, and immune aggregates. Through integration with CD45, CD3, and FoxP3 labelled images, we explored immune infiltration in 48 large (cm3) samples of human pancreas tissues containing &amp;gt;1,000 PanIN lesions to describe spatial correlations between local immune hotspots, 3D tumor morphology, and the surrounding pancreas structures. We quantified inter- and intra-sample heterogeneity in immune response to pancreatic cancer precursor lesions. We determined that this cohort varied greatly in both the number of PanIN (range 3 - 92 per cm3) and overall immune cell density (range 3,000 - 40,000 immune cells/mm3). We determined that, while PanIN size is not related to local immune cell density, overall PanIN burden is highly correlated to global immune cell density. This suggests that PanIN content does not have a short-range relationship to inflammation, but does have a long-range relationship with pancreatic structures and immune infiltration. As chronic inflammation in the pancreas shows correlation with development of pancreatic cancer, increased knowledge of the heterogeneous, local immune cell environment in precancerous samples will help us determine the role of inflammation in reacting to and controlling malignant progression. Citation Format: Ashley L. Kiemen, Cristina Almagro-Pérez, Valentina Matos-Romero, Alicia M. Braxton, Jaanvi Mahesh-Babu, Elizabeth D. Thompson, Toby C. Cornish, PeiHsun Wu, Laura D. Wood, Arrate Muñoz-Barrutia, Ralph H. Hruban, Denis Wirtz. Three-dimensional immune atlas of pancreatic cancer precursor lesions reveals large inter- and intra-lesion heterogeneity [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 1206.

Abstract 4277: CREB-ROCK driven extracellular matrix remodeling exasperates pancreatic cancer progression

Abstract Background: Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by a heightened oncogenic mutational burden, an immunosuppressive tumor microenvironment, and a dense desmoplastic stroma. During the tumor progression from in situ carcinoma to intraductal neoplasia, cancer cells manage to evade the stromal compartment and infiltrate into the ducts. This invasive tumor progression is often associated with the overexpression of extracellular matrix (ECM) modulating genes. Despite this correlation, there are currently no effective and specific inhibitors of ECM-altering pathways. By understanding the signaling mechanism, the ECM remodeling pathways may be intercepted to prevent invasive tumor progression. This study aims to elucidate the role of cAMP-response element binding protein 1 (CREB1) and its downstream effectors in regulating PDAC ECM remodeling. Methods: The Cancer Genome Atlas (TCGA) was quired for CREB1 expression and other ECM modulators in PDAC patients. In vitro, CRISPR/CAS9-based genomic editing was utilized to knockout CREB1 in the murine KPC cell line, on which RNA-seq analysis was performed. To investigate the effects of CREB in PDAC progression, a novel conditional CREB1 knockout (CREBfl/fl) was created in LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice (KPCC-/-). Tumors were extracted and histologically evaluated. Immunoblotting and staining were conducted on both in vitro and in vivo samples. Chromatin immunoprecipitation sequencing (ChIP-seq) and qPCR were performed on the KPC CREBKO. Eukaryotic Promoter Database (EPD) and ENCODE databases were explored to evaluate the transcriptional role of CREB in regulating ECM genes. Results: Patient PDAC samples data from TCGA revealed elevated levels of CREB1, ROCK1, and ROCK2 compared to normal pancreatic tissues. When comparing high CREB expression to low CREB expression, upregulation of several ECM genes was noted, including ROCK1 and ROCK2 in patients with high CREB expression. In CREBKO, RNA-seq analysis revealed the downregulation of key ECM genes, including Mmp3, Mmp10, Lama3, and Fn1 compared to CREB wildtype. In our novel murine conditional knockout model, a substantial reduction in fibrosis was observed. In both in vitro and in vivo, immunoblot and staining demonstrated a marked reduction in the expression of ECM remodeling proteins upon CREB deletion. Through ChIP-seq, the direct involvement of CREB1 in the transcriptional regulation of Rock1 and Rock2 was established. EPD and ENCODE were utilized to confirm and validate our CREB regulation experimental findings. Conclusion: These findings demonstrate the role of CREB in PDAC progression, which may provide a potential target to intercept ECM remodeling via inhibition of the CREB-ROCK axis to suppress PDAC tumor progression and invasion. Citation Format: Sudhakar Jinka, Siddharth Mehra, Varunkumar Krishnamoorthy, Anna Bianchi, Karthik Rajkumar, Andrew Adams, Haleh Amirian, Samara Singh, Edmond W. Box, Erin Dickey, Nivelo L. Alberto, Ban Yuguang, Jashodeep Datta, Nipun Merchant, Nagaraj Nagathihalli. CREB-ROCK driven extracellular matrix remodeling exasperates pancreatic cancer progression [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 4277.

Abstract 286: Multiplex imaging of human pancreatic tumors reveal novel dynamics of dysplastic stem cells, remodeling of associated fibroblasts and cell-to-cell spatial dependencies

Abstract This study aims to investigate human normal pancreatic tissue, pancreatic precursor lesions, and invasive pancreatic ductal adenocarcinoma to provide the most extensive spatial characterization as disease progress by mapping dysplastic stem cells (DSCs) - a reservoir of cancer cells - and fibroblast populations which modulate the tumor microenvironment and participate in disease progression. We performed 12 cycles of multiplex immunofluorescence using 26 validated antibodies covering fibroblasts [CXCL12, αSMA, FAP, CD74, CD105, PDGFRA, COL1A1], DSCs diversity [CD44v9, CD133, CD166, SPP1, S100P], and immune populations, onto tissue samples from 75 patients screened for normal, low grade, high grade dysplasia, and invasive cancer. We used our codes in Python, R, and QuPath 0.4.3. The pipeline includes shading correction, whole slide image registration (at subcellular level), region of interest and stromal mask annotations by a pathologist [normal duct, acinar to ductal metaplasia (ADM), low grade, high grade, invasive], automatic cell detection, and signal extraction. We developed a novel Python code for cell-to-cell spatial dependency analysis by using the x,y centroid positions of one cell from the tumor to other cells from the stroma mask based on pre-defined distances. For this abstract, we interrogated 264,732 cells and observed SPP1+, S100P+ DSCs expansion from low grade to high grade dysplasia, whereas CD133+ and CD166+ populations contract (p&amp;lt;0.0001). S100P+ DSCs gradually expand as disease progress reaching 74.13% of invasive cells being positive. CD44v9 follows dynamic changes variations, being expressed in 59.6% of cells undergoing ADM, dropping to 5.4% in dysplasia, and re-increasing to 25.4% in invasive cancer (all p&amp;lt;0.0001). Focusing on stromal composition, CXCL12+, CD105+, αSMA+, CD74+ fibroblasts expand whereas FAP+ fibroblasts contract from low- to high grade (p-values &amp;lt;0.0001). COL1A1 expression increases at each step of the normal duct - ADM - low grade - high grade - invasive cancer sequence (all p&amp;lt;0.0001). The pattern of CD44v9 tumor population showed dynamic alterations with the CD105 fibroblast population, as CD44v9 and CD105 are highly expressed in coordination during ADM. However, in dysplastic lesions and invasive cancer, mutual exclusivity between the tumor CD44v9 populations and the underlying CD105 fibroblast populations is observed, confirmed by our spatial algorithm within 50 μm of cell-to-cells distance (p&amp;lt;0.0001). In tumors, when CD44v9 tumor cell populations are expressed, the adjacent fibroblasts were CD105 negative. Multiplex imaging reveals dysplastic stem cells and fibroblasts dynamics in low- to high grade transition. The high-throughput capabilities of this method lead to the discovery of the CD44v9 tumor and CD105 fibroblast dependencies in pancreatic tumors. Citation Format: Maëlle Batardière, Shoukari Ayman, Camille Beaussier, Jumanah Baig, Zean Ghanmeh, Melisa Farias-Gonzalez, Jahg Wong, Alexandre Archambault-Marsan, Louise Rousseau, Simon Turcotte, Anna Means, Marcus Tan, Kathleen DelGiorno, Elham Dianati, Vincent Quoc-Huy Trinh. Multiplex imaging of human pancreatic tumors reveal novel dynamics of dysplastic stem cells, remodeling of associated fibroblasts and cell-to-cell spatial dependencies [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 286.

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.

Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency.

The generation of induced pluripotent stem cells (iPSCs) using transcription factors has been achieved from almost any differentiated cell type and has proved highly valuable for research and clinical applications. Interestingly, iPSC reprogramming of cancer cells, such as pancreatic ductal adenocarcinoma (PDAC), has been shown to revert the invasive PDAC phenotype and override the cancer epigenome. The differentiation of PDAC-derived iPSCs can recapitulate PDAC progression from its early pancreatic intraepithelial neoplasia (PanIN) precursor, revealing the molecular and cellular changes that occur early during PDAC progression. Therefore, PDAC-derived iPSCs can be used to model the earliest stages of PDAC for the discovery of early-detection diagnostic markers. This is particularly important for PDAC patients, who are typically diagnosed at the late metastatic stages due to a lack of reliable biomarkers for the earlier PanIN stages. However, reprogramming cancer cell lines, including PDAC, into pluripotency remains challenging, labor-intensive, and highly variable between different lines. Here, we describe a more consistent protocol for generating iPSCs from various human PDAC cell lines using bicistronic lentiviral vectors. The resulting iPSC lines are stable, showing no dependence on the exogenous expression of reprogramming factors or inducible drugs. Overall, this protocol facilitates the generation of a wide range of PDAC-derived iPSCs, which is essential for discovering early biomarkers that are more specific and representative of PDAC cases.

Single-cell RNA sequencing highlights epithelial and microenvironmental heterogeneity in malignant progression of pancreatic ductal adenocarcinoma

Intraductal papillary mucinous neoplasms (IPMNs) of the pancreas are bona fide precursor lesions of pancreatic ductal adenocarcinoma (PDAC). Single-cell transcriptomics provides a unique perspective for dissecting the epithelial and microenvironmental heterogeneity that accompanies progression from benign IPMNs to invasive PDAC. Single-cell RNA sequencing was performed through droplet-based sequencing on 35 693 cells from three high-grade IPMNs and two IPMN-derived PDACs (all surgically resected). Analysis of single-cell transcriptomes revealed heterogeneous alterations within the epithelium and the tumor microenvironment during the progression of noninvasive dysplasia to invasive cancer. For epithelial cells, we identified acinar–ductal cells and isthmus–pit cells enriched in IPMN lesions and profiled three types of PDAC-unique ductal cells. Notably, a proinflammatory immune component was distinctly observed in IPMNs, comprising CD4+ T cells, CD8+ T cells, and B cells, whereas M2 macrophages were significantly accumulated in PDAC. Through the analysis of cellular communication, the osteopontin gene (SPP1)–CD44 pathway between macrophages and epithelial cells were particularly strengthened in the PDAC group. Further prognostic analysis revealed that SPP1 is a biomarker of IPMN carcinogenesis for surveillance. This study demonstrates the ability to perform high-resolution profiling of single cellular transcriptomes during the progression of high-grade IPMNs to cancer. Notably, single-cell analysis provides an unparalleled insight into both epithelial and microenvironmental heterogeneity associated with early cancer pathogenesis and provides practical markers for surveillance and targets for cancer interception.