Tumor-Specific Delivery of 5-Fluorouracil-Incorporated Epidermal Growth Factor Receptor-Targeted Aptamers as an Efficient Treatment in Pancreatic Ductal Adenocarcinoma Models.

BACKGROUNDS & AIMS: Fluoropyrimidine c (5-fluorouracil [5FU]) increasingly represents the chemotherapeutic backbone for neoadjuvant, adjuvant, and palliative treatment of pancreatic ductal adenocarcinoma (PDAC). Even in combination with other agents, 5FU efficacy remains transient and limited. One explanation for the inadequate response is insufficient and nonspecific delivery of 5FU to the tumor. METHODS: We designed, generated, and characterized 5FU-incorporated systematic evolution of ligands by exponential enrichment (SELEX)-selected epidermal growth factor receptor (EGFR)-targeted aptamers for tumor-specific delivery of 5FU to PDAC cells and tested their therapeutic efficacy in vitro and in vivo. RESULTS: 5FU-EGFR aptamers reduced proliferation in a concentration-dependent manner in mouse and human pancreatic cancer cell lines. Time-lapsed live imaging showed EGFR-specific uptake of aptamers via clathrin-dependent endocytosis. The 5FU-aptamer treatment was equally effective in 5FU-sensitive and 5FU-refractory PDAC cell lines. Biweekly treatment with 5FU-EGFR aptamers reduced tumor burden in a syngeneic orthotopic transplantation model of PDAC, in an autochthonously growing genetically engineered PDAC model (LSL-KrasG12D/+;LSL-Trp53flox/+;Ptf1a-Cre [KPC]), in an orthotopic cell line-derived xenograft model using human PDAC cells in athymic mice (CDX; Crl:NU-Foxn1nu), and in patient-derived organoids. Tumor growth was significantly attenuated during 5FU-EGFR aptamer treatment in the course of follow-up. CONCLUSIONS: Tumor-specific targeted delivery of 5FU using EGFR aptamers as the carrier achieved high target specificity; overcame 5FU resistance; and proved to be effective in a syngeneic orthotopic transplantation model, in KPC mice, in a CDX model, and in patient-derived organoids and, therefore, represents a promising backbone for pancreatic cancer chemotherapy in patients. Furthermore, our approach has the potential to target virtually any cancer entity sensitive to 5FU treatment by incorporating 5FU into cancer cell-targeting aptamers as the delivery platform.

Common variants in the CLDN2-MORC4 and PRSS1-PRSS2 loci confer susceptibility to acute pancreatitis.

BACKGROUND/OBJECTIVES: Acute pancreatitis (AP) is one of the most common gastrointestinal disorders often requiring hospitalization. Frequent aetiologies are gallstones and alcohol abuse. In contrast to chronic pancreatitis (CP) few robust genetic associations have been described. Here we analysed whether common variants in the CLDN2-MORC4 and the PRSS1-PRSS2 locus that increase recurrent AP and CP risk associate with AP. METHODS: We screened 1462 AP patients and 3999 controls with melting curve analysis for SNPs rs10273639 (PRSS1-PRSS2), rs7057398 (RIPPLY), and rs12688220 (MORC4). Calculations were performed for the overall group, aetiology, and gender sub-groups. To examine genotype-phenotype relationships we performed several meta-analyses. RESULTS: Meta-analyses of all AP patients depicted significant (p-value < 0.05) associations for rs10273639 (odds ratio (OR) 0.88, 95% confidence interval (CI) 0.81-0.97, p-value 0.01), rs7057398 (OR 1.27, 95% CI 1.07-1.5, p-value 0.005), and rs12688220 (OR 1.32, 95% CI 1.12-1.56, p-value 0.001). For the different aetiology groups a significant association was shown for rs10273639 (OR 0.76, 95% CI 0.63-0.92, p-value 0.005), rs7057398 (OR 1.43, 95% CI 1.07-1.92, p-value 0.02), and rs12688220 (OR 1.44, 95% CI 1.07-1.93, p-value 0.02) in the alcoholic sub-group only. CONCLUSIONS: The association of CP risk variants with different AP aetiologies, which is strongest in the alcoholic AP group, might implicate common pathomechanisms most likely between alcoholic AP and CP.

Genetic Analysis of the ATG16L1 c.898A>G (p.T300A) Variant in Acute and Chronic Pancreatitis.

OBJECTIVES: Human and animal studies suggest an important role of autophagy in the pathogenesis of pancreatitis. ATG16L1 (autophagy-related 16 like 1) is part of a protein complex that is involved in the formation of autophagosomes. The c.898A > G (p.T300A) variant of ATG16L1 is associated with Crohn disease. In this study, we analyzed ATG16L1 c.898A > G (p.T300A) for an association with pancreatitis. METHODS: We genotyped 777 patients and 551 control subjects of German origin by melting curve analysis using fluorescence resonance energy transfer probes. The patient group included 429 patients with nonalcoholic chronic pancreatitis (CP), 141 patients with alcoholic CP, and 207 patients with acute pancreatitis (AP). We classified AP by severity according to the Atlanta symposium 1992. RESULTS: Allele and genotype frequencies of ATG16L1 c.898A > G (p.T300A) did not differ significantly between patients and controls (G allele frequencies: nonalcoholic CP, 49.9%; alcoholic CP, 48.2%; AP, 49.5%; controls, 52.7%). We found no significant association with the severity of AP either. CONCLUSIONS: Our data do not support a role of ATG16L1 c.898A > G (p.T300A) in the pathogenesis of AP or CP or an influence on the severity of AP.

Cathepsin D Expression and Gemcitabine Resistance in Pancreatic Cancer.

BACKGROUND: Cathepsin-D (CatD), owing to its dual role as a proteolytic enzyme and as a ligand, has been implicated in cancer progression. The role of CatD in pancreatic ductal adenocarcinoma is unknown. METHODS: CatD expression quantified by immunohistochemistry of tumor-tissue microarrays of 403 resected pancreatic cancer patients from the ESPAC-Tplus trial, a translational study within the ESPAC (European Study Group for Pancreatic Cancer) trials, was dichotomously distributed to low and high H scores (cut off 22.35) for survival and multivariable analysis. The validation cohort (n = 69) was recruited based on the hazard ratio of CatD from ESPAC-Tplus. 5-fluorouracil-, and gemcitabine-resistant pancreatic cancer cell lines were employed for mechanistic experiments. All statistical tests were two-sided. RESULTS: Median overall survival was 23.75 months and median overall survival for patients with high CatD expression was 21.09 (95% confidence interval [CI] = 17.31 to 24.80) months vs 27.20 (95% CI = 23.75 to 31.90) months for low CatD expression (χ2 LR, 1DF = 4.00; P = .04). Multivariable analysis revealed CatD expression as a predictive marker in gemcitabine-treated (z stat = 2.33; P = .02) but not in 5-fluorouracil-treated (z stat = 0.21; P = .82) patients. An independent validation cohort confirmed CatD as a negative predictive marker for survival (χ2 LR, 1DF = 6.80; P = .009) and as an independent predictive marker in gemcitabine-treated patients with a hazard ratio of 3.38 (95% CI = 1.36 to 8.38, P = .008). Overexpression of CatD was associated with a concomitant suppression of the acid sphingomyelinase, and silencing of CatD resulted in upregulation of acid sphingomyelinase with rescue of gemcitabine resistance. CONCLUSIONS: Adjuvant gemcitabine is less effective in pancreatic ductal adenocarcinoma with high CatD expression, and thus CatD could serve as a marker for biomarker-driven therapy.