Abstract Introduction: Adult stem cell dysregulation can initiate cancer. Pancreatic ductal glands (PDGs) act as a stem cell niche for the pancreatic main duct epithelium (MD). Previous studies have shown that Tff2+ cells within the PDGs give rise to progeny that migrate to the MD epithelium during inflammation. In human IPMN, the PDGs have been shown to comprise the basal proliferative compartment of these neoplasms. Here we investigate the effect of oncogenic activation and tumor suppressor deletion within Tff2+ PDG cells. Methods: We utilized Tff2CreERT2;Rosa26mT/mG;KrasLSL-G12D;P53fl/fl mice (TTKP) to activate oncogenes in Tff2+ cells and GFP tag this lineage in vivo and ex vivo. Cre activity was induced by tamoxifen injection in TTKP experimental or Tff2CreERT2;Rosa26mT/mG (TT) control mice. Pancreata were collected at pre-determined time points post-tamoxifen with GFP tagged, Tff2 lineage cells quantified and compared between groups at each timepoint. For ex vivo experiments, ductal cells were isolated from tamoxifen-naïve TTKP pancreata via magnetic-assisted cell sorting, grown in 3D culture and induced via 4OHT. GFP+ cells were sorted, cultured, and orthotopically implanted in athymic nude mice. Tumors were collected before reaching 20mm in diameter. Spatial RNA sequencing (spRNAseq) was performed on frozen tissue sections and analyzed with R package Seurat. Cross-species analysis utilized human PDAC scRNAseq reference datasets. Results: TTKP mice developed lineage-traced PDAC eight weeks after tamoxifen induction. In vivo lineage tracing showed a significant increase of GFP+ Tff2-lineage cells in PDGs and MD between TTKP and TT mice at all analyzed time points (4-, 6- and 8-weeks post tamoxifen, ≥ 3 mice each). GFP-positive PDGs exhibited progressive PanIN-like changes before PDAC formation. Tff2-PDG cells in TT mice function as transient amplifying cells, expanding within PDGs and to the MD during inflammation and contracting upon resolution. Oncogenic activation (sans inflammation) in Tff2-PDG cells led to sustained GFP+ cell presence in PDGs and MD, indicating increased stem-like behavior preceding neoplasia. Orthotopic organoid experiments resulted in GFP+ tumor formation. SpRNAseq analysis indicated enrichment of "Ductal cell type 2" in TTKP tumors, particularly overlying the main duct epithelium. Conclusion: Our study demonstrates that dysregulation of the Tff2-lineage in PDGs can initiate PDAC. Oncogenic activation in Tff2+ cells increases their stemness as indicated by lineage expansion in PDGs and MD preceding PanIN and PDAC formation. Orthotopic organoid implantation experiments further validate the tumor-initiating capacity of Tff2+ ductal cells. Spatial transcriptomics analysis confirms the presence of similar cell types identified in human scRNAseq studies within TTKP tumors. These findings strongly support the role of PDGs as a potential niche of origin for human PDAC and provide additional insights into an under-studied disease initiating process. Citation Format: Kyle L. McAndrews, Pinaki Mondal, Dulce Maroni, Dongdong Wang, Michael A. Hollingsworth, Sarah P. Thayer. Activation of oncogenes within Tff2 expressing cells of the pancreatic ductal glands results in increased lineage stemness preceding tumorigenesis: Insights from an inducible mouse model and derived organoids [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 C055.

Among the signaling pathways that control the stem cell self-renewal and maintenance vs. acquisition of differentiated cell fates, those mediated by receptor tyrosine kinase (RTK) activation are well established as key players. CBL family ubiquitin ligases are negative regulators of RTKs but their physiological roles in regulating stem cell behaviors are unclear. While hematopoietic Cbl/Cblb knockout (KO) leads to a myeloproliferative disease due to expansion and reduced quiescence of hematopoietic stem cells, mammary epithelial KO led to stunted mammary gland development due to mammary stem cell depletion. Here, we examined the impact of inducible Cbl/Cblb double-KO (iDKO) selectively in the Lgr5-defined intestinal stem cell (ISC) compartment. Cbl/Cblb iDKO led to rapid loss of the Lgr5 Hi ISC pool with a concomitant transient expansion of the Lgr5 Lo transit amplifying population. LacZ reporter-based lineage tracing showed increased ISC commitment to differentiation, with propensity towards enterocyte and goblet cell fate at the expense of Paneth cells. Functionally, Cbl/Cblb iDKO impaired the recovery from radiation-induced intestinal epithelial injury. In vitro , Cbl/Cblb iDKO led to inability to maintain intestinal organoids. Single cell RNAseq analysis of organoids revealed Akt-mTOR pathway hyperactivation in iDKO ISCs and progeny cells, and pharmacological inhibition of the Akt-mTOR axis rescued the organoid maintenance and propagation defects. Our results demonstrate a requirement for Cbl/Cblb in the maintenance of ISCs by fine tuning the Akt-mTOR axis to balance stem cell maintenance vs. commitment to differentiation.

Supplementary Table 4 from A Novel Somatic Mouse Model to Survey Tumorigenic Potential Applied to the Hedgehog Pathway

Supplementary Figures 1-4 from A Novel Somatic Mouse Model to Survey Tumorigenic Potential Applied to the Hedgehog Pathway

Supplementary Tables 1-2, Figures 1-8 from PTEN Is a Major Tumor Suppressor in Pancreatic Ductal Adenocarcinoma and Regulates an NF-κB–Cytokine Network

Supplementary Tables 1-3 from A Novel Somatic Mouse Model to Survey Tumorigenic Potential Applied to the Hedgehog Pathway

Objective: The aim of this study was to investigate surgeon preferences for the management of patients with locally advanced pancreatic cancer (LAPC). Background: Select patients with LAPC may become candidates for curative resection following neoadjuvant therapy, and recent reports of survival are encouraging. Yet the optimal management approach remains unclear. Methods: An extensive electronic survey was systematically distributed by email to an international cohort of pancreas surgeons. Data collected included practice characteristics, management preferences, attitudes regarding contraindications to surgery, and 6 clinical vignettes of patients that ultimately received a margin negative resection (with detailed videos of post-neoadjuvant imaging) to assess propensity for surgical exploration if resection status is not known. Results: A total of 153 eligible responses were received from 4 continents. Median duration of practice is 12 years (interquartile range 6–20) and 77% work in a university setting. Most surgeons (86%) are considered high volume (>10 resections/yr), 33% offer a minimally-invasive approach, and 50% offer arterial resections in select patients. Most (72%) always recommend neoadjuvant chemotherapy, and 65% prefer FOLFIRINOX. Preferences for the duration of chemotherapy varied widely: 39% prefer ≥2 months, 43% prefer ≥4 months, and 11% prefer ≥6 months. Forty-one percent frequently recommend neoadjuvant radiotherapy, and 53% prefer 5 to 6 weeks of chemoradiation. The proportion of surgeons favoring exploration following neoadjuvant varied extensively across 5 vignettes of LAPC, from 14% to 53%. In a vignette of oligometastatic liver metastases, 31% would offer exploration if a favorable therapy response is observed. Conclusions: In an international cohort of pancreas surgeons, there is substantial variation in management preferences, perceived contraindications to surgery, and the propensity to consider exploration in LAPC. These results emphasize the importance of a robust and nuanced multidisciplinary discussion for each patient, and suggest an evolving concept of “resectability.”

OBJECTIVES Pancreatic ductal adenocarcinoma (PDAC) is characterized by early metastatic spread in more than 50% of patients. In this study, we sought to understand the migratory properties of (non)metastatic PDAC cells and determine whether the migration of cancer stem cell (CSC) populations accounts for the aggressive nature of this disease. METHODS The migratory abilities of primary and metastatic PDAC cell lines were investigated using a microfluidic device and time-lapse photography. The velocity, time of delay of mobilization, and number of migratory cells were analyzed. Cancer stem cell subpopulations were isolated by fluorescence-activated cell sorting and their migratory properties compared with their non-CSC counterparts. RESULTS Primary cancer cells exhibited higher velocities, greater number of migratory cells, and a shorter time of delay of mobilization in comparison to metastatic cell lines. Characterization of CSC populations revealed primary PDAC cell lines were composed of fewer CD133 and CD24CD44 CSC subpopulations than metastatic cells. Moreover, migratory analysis of CSC subpopulations revealed lower velocities, fewer migratory cells, and a greater time of delay of mobilization than non-CSC. CONCLUSIONS Primary cancer cells demonstrate enhanced migratory abilities in comparison to metastatic PDAC cells. Those differences may result from lower CSC subpopulations in primary cells because CSC populations demonstrated impaired migratory abilities in contrast to non-CSC.

Abstract Background: Autocrine motility factor (AMF) is secreted by tumor cells to generate autocrine and paracrine signaling and is received by its receptor, autocrine motility factor receptor (AMFR), to promote aggressive behavior in cancer. The AMF-AMFR interaction has been demonstrated to promote proliferative, anti-apoptotic, and metastatic pathways in many cancers, but its ability to promote these pathways in breast cancer is poorly defined. Our previous work has shown that AMF gene expression is higher in triple-negative breast cancer (TNBC) tumors than in luminal tumors and luminal breast cancers lose a copy of AMFR in their tumor genomes almost twice as frequently as TNBC, suggesting aggressive cancers may need the AMF-AMFR signaling pathway to promote aggressive cancer biology. While AMFR does not presently have a specific inhibitor, erythrose-4-phosphate (E4P) is an established inhibitor of extracellular AMF. Here, we demonstrate that the AMF-AMFR interaction promotes proliferation and migration in TNBC and propose that AMFR may be a potentially potent target for TNBC therapy. Methods: cBioPortal was used to investigate AMFR somatic copy-number alterations (SCNA) and gene expression in METABRIC (n = 1,904) and TCGA Pan-Cancer Atlas (n = 994) patient cohorts. TNBC cell lines MDA-MB-231 and MDA-MB-436 were transduced with lentiviral particles, selected with puromycin, and sorted for high RFP expression using flow cytometry to generate inducible AMFR-shRNA (short hairpin RNA) stable cell lines. To study the ability of AMF to promote migration, MDA-MB-231 and MDA-MB-436 in AMFR-competent and AMFR-knock down conditions were seeded onto Transwell inserts with or without E4P while extracellular AMF was present or absent in the bottom well; after 24 hours, cells were fixed, stained with crystal violet, and counted. To study the ability of AMF to promote proliferation, MDA-MB-231 cells in AMFR-competent and AMFR-knock down conditions were grown in serum-free media with or without AMF for 24 hours. Cells were fixed and incubated in propidium iodide/RNase staining solution, and cell cycle analysis was performed using flow cytometry. Results: Gene expression analysis of AMFR correlates with its copy-number status in the tumor genome. METABRIC patient cohort loss, retention, and gain of AMFR in the tumor genome resulted in median z-score mRNA levels of -0.533, -0.1424, and 0.159, respectively, and TCGA Pan-Cancer Atlas patient cohort loss, retention, and gain of AMFR in the tumor genome resulted in median z-score mRNA levels of -0.5906, -0.274, and -0.09335, respectively. TNBC frequently retains or gains AMFR in their tumor genomes, suggesting TNBC tumors have higher levels of AMFR gene expression. In the TNBC cell line MDA-MB-231, AMF increased migration by 1.4-fold while AMFR knock-down returned migration to baseline and AMF inhibition by E4P decreased migration by 2.7-fold below baseline. In the TNBC cell line MDA-MB-436, AMFR knock-down decreased migration 1.6-fold and AMF inhibition by E4P decreased migration 33-fold below baseline. This data suggest that the signaling of AMF through AMFR promotes migration in TNBC. Cell cycle analysis of MDA-MB-231 cells showed the addition of AMF to culture medium increased actively dividing cells by 8.3%, and AMFR knock-down dropped this effect to 5.9%, suggesting that the AMF-AMFR interaction promotes proliferation of TNBC cells. Conclusion: TNBC has higher AMFR gene expression than luminal tumors. AMF appears to act through AMFR to promote migration and proliferation in TNBC. Targeting AMFR may be a promising therapeutic strategy for patients with TNBC. Citation Format: Cassie Liu, Jeffrey D. Price, Sarah P. Thayer. Autocrine motility factor receptor enhances proliferative and migratory capabilities of triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-22.

Trefoil factors 1, 2, and 3 (TFFs) are a family of small secretory molecules involved in the protection and repair of the gastrointestinal tract (GI). TFFs maintain and restore epithelial structural integrity via transducing key signaling pathways for epithelial cell migration, proliferation, and invasion. In recent years, TFFs have emerged as key players in the pathogenesis of multiple diseases, especially cancer. Initially recognized as tumor suppressors, emerging evidence demonstrates their key role in tumor progression and metastasis, extending their actions beyond protection. However, to date, a comprehensive understanding of TFFs' mechanism of action in tumor initiation, progression and metastasis remains obscure. The present review discusses the structural, functional and mechanistic implications of all three TFF family members in tumor progression and metastasis. Also, we have garnered information from studies on their structure and expression status in different organs, along with lessons from their specific knockout in mouse models. In addition, we highlight the emerging potential of using TFFs as a biomarker to stratify tumors for better therapeutic intervention.

Introduction The inhibition of Hedgehog (Hh) signaling in pancreatic ductal adenocarcinoma (PDAC) reduces desmoplasia and promotes increased vascularity. In contrast to these findings, the Hh ligand Sonic Hedgehog (SHH) is a potent proangiogenic factor in non-tumor models. The aim of this study was to determine the molecular mechanisms by which SHH affects the tumor stroma and angiogenesis. Methods Mice bearing three different xenografted human PDAC ( n = 5/group) were treated with neutralizing antibodies to SHH. After treatment for 7 days, tumors were evaluated and the expression of 38 pro- and antiangiogenic factors was assessed in the tumor cells and their stroma. The effect of SHH on the regulation of pro- and antiangiogenic factors in fibroblasts and its impact on endothelial cells was then further assessed in in vitro model systems. Results Inhibition of SHH affected tumor growth, stromal content, and vascularity. Its effect on the Hh signaling pathway was restricted to the stromal compartment of the three cancers. SHH-stimulated angiogenesis indirectly through the reduction of antiangiogenic THBS2 and TIMP2 in stromal cells. An additional direct effect of SHH on endothelial cells depended on the presence of VEGF. Conclusion Inhibition of Hh signaling reduces tumor vascularity, suggesting that Hh plays a role in the maintenance or formation of the tumor vasculature. Whether the reduction in tumor growth and viability seen in the epithelium is a direct consequence of Hh pathway inhibition, or indirectly caused by its effect on the stroma and vasculature, remains to be evaluated.

4122 Background: Recent reports suggest patients with locally advanced pancreatic cancer (LAPC) may become candidates for curative resection following neoadjuvant therapy, with encouraging survival outcomes. Yet the optimal management approach for LAPC remains unclear. We sought to investigate surgeon preferences for the management of patients with LAPC. Methods: An extensive electronic survey was systematically distributed by email to an international cohort of pancreas surgeons. Data collected included surgeon practice characteristics, preferences for staging and management, and 6 clinical vignettes (with detailed videos of post-neoadjuvant arterial and venous imaging) to assess attitudes regarding eligibility for surgical exploration. Results: A total of 150 eligible responses were received from 4 continents. Median duration in practice was 12 years (IQR 6-20) and 75% respondents work in a university setting. Most (84%) are considered high volume, 33% offer a minimally-invasive approach, and 48% offer arterial resection in selected patients. A majority (70%) always recommend neoadjuvant chemotherapy, and 62% prefer FOLFIRINOX. Preferences for duration of neoadjuvant therapy varied widely: 39% prefer ≥2 months, 41% prefer ≥4 months, and 11% prefer 6 months or more. Forty-one percent frequently recommend neoadjuvant radiation, and 51% prefer standard chemoradiotherapy. Age ≥80 years and CA 19-9 of ≥1000 U/mL were commonly considered contraindications to exploration. In 5 clinical vignettes of LAPC, the proportion of respondents that would offer exploration following neoadjuvant varied extensively, from 15% to 54%. In a vignette of oligometastatic pancreatic liver metastases, 32% would offer exploration if a favorable biochemical and imaging response to therapy is observed. Conclusions: In an international cohort of high volume pancreas surgeons, there is substantial variation in attitudes regarding staging preferences and surgical management of LAPC. These results underscore the importance of coordinated multi-disciplinary care, and suggest an evolving concept of “resectability.” Patients and their oncologists should have a low threshold to consider a second opinion for the surgical management of LAPC, if desired.

We integrated clinical, genomic, and transcriptomic data from 224 primaries and 95 metastases from 289 patients to characterize progression of pancreatic ductal adenocarcinoma (PDAC). Driver gene alterations and mutational and expression-based signatures were preserved, with truncations, inversions, and translocations most conserved. Cell cycle progression (CCP) increased with sequential inactivation of tumor suppressors, yet remained higher in metastases, perhaps driven by cell cycle regulatory gene variants. Half of the cases were hypoxic by expression markers, overlapping with molecular subtypes. Paired tumor heterogeneity showed cancer cell migration by Halstedian progression. Multiple PDACs arising synchronously and metachronously in the same pancreas were actually intra-parenchymal metastases, not independent primary tumors. Established clinical co-variates dominated survival analyses, although CCP and hypoxia may inform clinical practice.

The NCCN Guidelines for Pancreatic Adenocarcinoma discuss the diagnosis and management of adenocarcinomas of the exocrine pancreas and are intended to assist with clinical decision-making. These NCCN Guidelines Insights discuss important updates to the 2019 version of the guidelines, focusing on postoperative adjuvant treatment of patients with pancreatic cancers.

The Muc-1 oncoprotein is a tumor-associated mucin often overexpressed in pancreatic cancer. We report that knockout of Muc-1 reduced the degree of pancreatic inflammation that resulted from infection with Coxsackievirus B3 (CVB3) in a mouse model. CVB3-infected Muc-1-deficient (Muc-1KO) mice had significantly reduced infiltration of macrophages into the murine pancreas. We found that Muc-1 signaling through NF-κB increased expression of ICAM-1, a pro-inflammatory mediator that recruits macrophages. Further investigation revealed that bone marrow derived macrophages (BMDM) from the Muc-1KO mice exhibited defective migration properties, in part due to low expression of the C-C motif chemokine receptor (CCR2) and the integrin Very Late Antigen 4 (VLA-4). The results presented here provide novel insight into the role of Muc-1 in regulating the inflammatory response and the cellular microenvironment in pancreatitis.

The NCCN Guidelines for Pancreatic Adenocarcinoma discuss the diagnosis and management of adenocarcinomas of the exocrine pancreas and are intended to assist with clinical decision-making. These NCCN Guidelines Insights discuss important updates to the 2019 version of the guidelines, focusing on postoperative adjuvant treatment of patients with pancreatic cancers.