Background Hyperinsulinemia is independently associated with increased risk and mortality of pancreatic cancer. We recently reported that genetically reduced insulin production resulted in ~ 50% suppression of pancreatic intraepithelial neoplasia (PanIN) precancerous lesions in mice. However, only female mice remained normoglycemic, and only the gene dosage of the rodent-specific Ins1 alleles was tested in our previous model. Moreover, we did not delve into the molecular and cellular mechanisms associated with modulating hyperinsulinemia. Methods We studied how reduced Ins2 gene dosage affects PanIN lesion development in both male and female Ptf1aCreER;KrasLSL-G12D mice lacking the rodent-specific Ins1 gene (Ins1-/-). We generated control mice having two alleles of the wild-type Ins2 gene (Ptf1aCreER;KrasLSL-G12D;Ins1-/-;Ins2+/+) and experimental mice having one allele of Ins2 gene (Ptf1aCreER;KrasLSL-G12D;Ins1-/-;Ins2+/-). We then performed thorough histopathological analyses and single-cell transcriptomics for both genotypes and sexes. Results High-fat diet–induced hyperinsulinemia was transiently or modestly reduced in female and male mice, respectively, with only one allele of Ins2. This occurred without dramatically affecting glucose tolerance. Genetic reduction of insulin production resulted in mice with a tendency for less PanIN and acinar-to-ductal metaplasia (ADM) lesions. Using single-cell transcriptomics, we found hyperinsulinemia affected multiple cell types in the pancreas, with the most statistically significant effects on local immune cell types that were highly represented in our sampled cell population. Specifically, hyperinsulinemia modulated pathways associated with protein translation, MAPK-ERK signaling, and PI3K-AKT signaling, which were changed in epithelial cells and subsets of immune cells. Conclusions These data suggest a potential role for the immune microenvironment in hyperinsulinemia-driven PanIN development. Together with our previous work, we propose that mild suppression of insulin levels may be useful in preventing pancreatic cancer by acting on multiple cell types.

The rising incidence of pancreatic cancer is largely driven by increased prevalence of obesity and type 2 diabetes (T2D). Hyperinsulinemia is a cardinal feature of obesity and T2D, and is associated with increased cancer incidence and mortality. Genetic reduction of insulin production suppresses formation of pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in mice with mutant Kras. However, it remained unclear whether hyperinsulinemia exerts its tumorigenic effects directly on the cells that give rise to PanINs or indirectly on the tumor microenvironment. Here, we tested whether diet-induced hyperinsulinemia contributes to pancreatic cancer directly through insulin receptor (Insr) signaling in KrasG12D-expressing pancreatic acinar cells. Loss of insulin receptors in wild-type or KrasG12D-expressing acinar cells did not significantly influence fasting glucose or insulin. Strikingly, mice lacking Insr in KrasG12D-expressing acinar cells and their progeny had a significant reduction in PanIN plus tumor area in males (2.7-fold) and females (5.3-fold) compared to control mice. Mechanistically, proteome analyses implicated a reduction in digestive enzymes among altered protein networks in mice protected from PanINs, and together with phospho-proteome analysis, linked the spliceosome, ribosome, and secretory pathway to insulin signaling in context of pancreatic cancer initiation. Collectively, these data demonstrate that insulin receptor signaling in acinar cells promotes PanIN initiation in the context of obesity.

A central goal of physiological research is the understanding of cell-specific roles of disease-associated genes. Cre-mediated recombineering is the tool of choice for cell type-specific analysis of gene function in pre-clinical models. In the type 1 diabetes research field, multiple lines of NOD mice have been engineered to express Cre recombinase in pancreatic β-cells using insulin promoter fragments, but tissue promiscuity remains a concern. Constitutive Ins1tm1.1(cre)Thor (Ins1Cre) mice on the C57/bl6-J background has high β-cell specificity and with no reported off-target effects. We explored if NOD:Ins1Cre mice could be used to investigate β-cell gene deletion in type 1 diabetes disease modeling. We studied wildtype (Ins1WT/WT), Ins1 heterozygous (Ins1Cre/WT or Ins1Neo/WT), and Ins1 null (Ins1Cre/Neo) littermates on a NOD background. Female Ins1Neo/WT mice exhibited significant protection from diabetes, with further near-complete protection in Ins1Cre/WT mice. The effects of combined neomycin and Cre knock-in in Ins1Neo/Cre mice were not additive to the Cre knock-in alone. In Ins1Neo/Cre mice, protection from diabetes was associated with reduced insulitis at 12 weeks of age. Collectively, these data confirm previous reports that loss of Ins1 alleles protects NOD mice from diabetes development and demonstrates, for the first time, that Cre itself may have additional protective effects. This has significant implications for the experimental design and interpretation of pre-clinical type 1 diabetes studies using β-cell-specific Cre in NOD mice.

505 Background: Complete resection followed by adjuvant chemotherapy is standard of care for patients with localized cholangiocarcinoma (CC) or gallbladder cancer (GBC) but is not always feasible and recurrence rates remain high. Understanding the exact proportions and reasons for treatment failure is important to design new approaches, data regarding this information remain scarce. Methods: We performed a retrospective population-based review of patients with GBC or CC (intrahepatic (ICC) or extrahepatic (ECC)) resected between 2005-2019 using the BC Cancer provincial database. Chart review was conducted to characterize demographics, treatments and outcomes. Results: 594 patients were identified of whom 416 (70%) had disease recurrence. Baseline characteristics and treatments received are shown in the table. Most GBCs (96%) were diagnosed incidentally. Repeat oncologic resection was performed for 55% of these, the most common reason for not proceeding was interval disease progression between initial cholecystectomy and planned re-resection (24%). Adjuvant chemotherapy was received by 51% of 163 patients after 2017 and consisted of capecitabine (86%), gemcitabine (4%), cisplatin and gemcitabine (5%) or chemoradiation (4%). Common reasons for not receiving adjuvant therapy were post op complications or comorbidities (18 and 24%), progression (17%) and patient’s preference (17%). Of those receiving adjuvant therapy, 31% did not complete all planned cycles due to progression (45%) or intolerance (55%). Median overall survival (OS) after resection was 31.6 and 18.0 months respectively for R0 and R1 resection (HR 0.43, 95% CI 0.35-0.53), 29.4 and 19.0 months with and without reresection for GBC (HR 0.55, 95% CI 0.41-0.73), and 29.4 and 25.9 months with and without adjuvant therapy (HR 0.79, 95% CI 0.61-1.02). Stage, R0 resection, re-resection for GBC and adjuvant chemotherapy remained associated with improved OS in multivariate analysis. Taken together, only 25% of patients in the more contemporary cohort of 2017-2019 had complete (R0) resection and completed adjuvant chemotherapy. Conclusions: Complete resection, including reresection for incidentally diagnosed GBCs, and adjuvant chemotherapy were associated with improved outcomes in this retrospective cohort, yet many patients were not able to complete these treatments. Neoadjuvant strategies may improve treatment delivery and ultimately, outcomes. [Table: see text]

522 Background: While only accounting for 3% of gastrointestinal malignancies, cholangiocarcinoma (CC) has poor prognosis. New targeted therapies are on the horizon and understanding the potential size of the population eligible for such therapies is of importance. Contemporary incidence and outcomes in Canada have not been studied. Methods: Patients diagnosed with CC between 2005-2019 in British Columbia were identified using the BC Cancer Registry. Patient characteristics, treatments received and outcomes were collected and analyzed. Results: A total of 1520 patients were identified. Median age at diagnosis was 69 (interquartile 61-76) and 52% were female, 594 patients presented with localized disease (39%) of whom 416 had disease recurrence. The proportion of intrahepatic CC (ICC) rose from 17% in 2005-2009 to 44% in 2015-2019 while the incidence of extrahepatic (ECC) and gallbladder (GB) cancers followed populational trends. More patients with ICC had stage IV cancer at the time of diagnosis (64% vs 43% for ECC and 45% for GB, p<0.01). Treatments received are shown in the table. Among 1086 patients with recurrent or metastatic disease, 54% and 14% respectively received first- and second-line chemotherapy. Overall survival (OS) was 13.7 months [HR 0.43 (95% CI 0.37-0.49)] with 1 line of treatment and 21.3 months with 2 lines [HR 0.30 (95% CI 0.24-0.37)], compared to 4.8 months with best supportive care. On multivariate analysis, ECOG, first and second-line treatment and liver-directed therapy remained associated with improved outcomes while tumor site or time period did not. Conclusions: The incidence of CC is rising and most patients present with advanced disease. Treatment attrition is high and few patients receive second line therapies. The outcomes of patients receiving first line, second line and liver-directed therapies are significantly better than those receiving supportive care only. Understanding contemporary trends in presentation and treatment patterns is essential as we consider strategies to improve implementation and access of emerging novel therapies in this setting. [Table: see text]