NMR-based metabolic profiling of urine, serum, fecal, and pancreatic tissue samples from the Ptf1a-Cre; LSL-KrasG12D transgenic mouse model of pancreatic cancer

Abstract

Pancreatic cancer is the third leading cause of cancer deaths in the United States with more than 53,000 expected to be diagnosed with the disease in 2018. The median survival time after diagnosis is four to six months. The poor survival statistics are due in part to the fact that pancreatic cancer is typically asymptomatic until it reaches advanced stages of the disease. Although surgical resection provides the best chance of survival, pancreatic cancer is rarely detected when surgery is still possible due, in part, to lack of effective biomarkers for early detection. The goal of the research reported here was to determine if it was possible to identify metabolic biomarkers for detection of pre-cancerous pancreatic intraepithelial neoplasia (PanIN) that precede pancreatic adenocarcinoma. The transgenic Ptf1a-Cre; LSL-KrasG12D mouse strain was used as a model of pancreatic cancer progression. Nuclear magnetic resonance (NMR) spectroscopy was employed to compare metabolic profiles of urine, sera, fecal extracts, and pancreatic tissue extracts collected from control and study mice aged 5, 11, and 15 months, including 47 mice with tumors. We were able to identify the following potential biomarkers: decreased 3-indoxylsulfate, benzoate and citrate in urine, decreased glucose, choline, and lactate in blood, and decreased phenylalanine and benzoate and increased acetoin in fecal extracts. Potential biomarkers were validated by p-values, PLS-DA VIP scores, and accuracies based on area under ROC curve analyses. Essentially, all of the metabolic profiling changes could be explained as being associated with the consequences of bicarbonate wasting caused by a complete substitution of the normal pancreatic acinar tissue by tissue entirely composed of PanIN. Given the nature of the mouse model used here, our results indicate that it may be possible to use NMR-based metabolic profiling to identify biomarkers for detection of precancerous PanIN that immediately precede pancreatic cancer.

Influence of Drying Method on NMR-Based Metabolic Profiling of Human Cell Lines

“…To be considered significant, a bucket had to have either a p -value <0.05 or an AUC >0.70 [58]. In MiaPaCa-2 cells, 53 (Table S1) metabolites out of a total of 74 metabolites identified (Table S2) were present at significantly different abundances depending on drying technique.…”

“…One-dimensional 1 H Carr-Purcell-Meiboom-Gill (CPMG) NMR spectra were collected using a spectral width of 20.0 ppm and 32K points resulting in 1.21 s acquisition time, 512 scans, 4 dummy scans, 1.80 s recycle delay and 1 ms mixing time. Two-dimensional 1 H- 1 H TOCSY NMR experiments were performed using a spectral width of 10.0 ppm and 2K points, 1.5 s recycle time and 60 ms mixing time [14,17,19,20,21,58,59,60,61]. Two-dimensional 1 H- 13 C HSQC NMR experiments were performed using non-uniform sampling in the indirect carbon dimension [62,63].…”

The Effects of Rhein and Honokiol on Metabolic Profiles in a Mouse Model of Acute Pancreatitis

“…In addition, the association between linoleic acid metabolism and the severity of AP has been demonstrated [ 32 ]. Pentose and glucuronate interconversions are an insulin-relevant tissue-specific signaling pathway [ 33 ], which is the pathway associated with metabolites in fecal samples of patients with pancreatic cancer [ 34 ]. The pathological changes observed in the pancreatic tissues of the AP group were in line with the injuries induced by cerulein.…”

Spatial variations in gut permeability are linked to type 1 diabetes development in non-obese diabetic mice

“…D16-00100). Surgical procedures were carried out as previously described 22–27. All authors completed online and in-class animal training prior to animal work.…”

“…All dissections and intestinal histologies were performed as described previously 22–29. Details regarding the histological procedures can be found in online supplementary material.…”