Introduction

Cancer-induced cachexia accounts for approximately 20% of all cancer deaths [1]. In pancreatic cancer, the syndrome affects nearly 80% of patients [2, 3]. The uncontrollable extreme weight loss due to cachexia results in a particularly poor quality of life causing profound weakness, listlessness, and an inability to function. In pancreatic cancer especially, where resection is not possible for a majority of patients, palliation with chemotherapy is the only option of prolonging life, and cachexia results in lower tolerance to chemotherapy. Cachectic patients experience a wide range of symptoms affecting the function of several organs such as, muscle, liver, brain, and heart, which decrease the quality of life and worsens prognosis. A major characteristic of cachexia is accelerated skeletal muscle and storage fat wasting causing nutrient mobilization both directly as lipid and amino acids and indirectly as ketone bodies and glucose derived from liver keto- and gluconeogenesis with systemic distribution including to the tumor through the bloodstream [4]. Patients with cachexia develop extensive metabolic stress from increased proteins and fat tissue burning resulting in increased energy expenditure. Previously, we have reported the initial characterization of a myoblast optical imaging reporter that allowed real-time longitudinal monitoring of the early onset of cancer induced wasting and measured plasma metabolic changes associated with PDAC-induced cachexia [5]. Here, for the first time, we have performed high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of brain tissue obtained from normal mice and mice with human PDAC xenografts that induce cachexia (Pa04C) or are noncachectic (Panc1). Profound alterations in 1H MRS derived metabolite profiles specific to the brains of cachectic mice were detected.

Methods

Human pancreatic cancer cell lines, Panc1 and Pa04C were obtained from ATCC or provided by Dr. Maitra [6] respectively. Generation of the dual fluorescence myoblast cell line has been described in our previous report [5]. Male severe combined immunodeficient (SCID) mice were inoculated in the right flank with cancer cells (2 × 106) and in the right hind leg muscle with reporter myoblasts (2×106). Once the mice were sacrificed, brains were harvested, freeze clamped and stored in -80°C until 1H MRS analysis. Dual phase solvent extraction was performed on brain tissues. The water phase was separated, freeze dried, reconstituted in D2O PBS and transferred to a 5mm NMR tube for spectral acquisition. All 1H MR spectra were acquired on an Avance III 750 MHz (17.6T) Bruker NMR spectrometer equipped with a 5 mm broadband inverse (BBI) probe. 1H MR spectra with water suppression were acquired using a single pulse sequence with the following experimental parameters: spectral width of 15495.86 Hz, data points of 64 K, 90° flip angle, relaxation delay 10 sec, acquisition time 2.11 sec, 64 scans with 8 dummy scans, receiver gain 80.6. Spectral acquisition, processing and quantification were performed using TOPSPIN 2.1 software.

Results and Discussion

As anticipated, mice with cachexia-inducing Pa04C tumors showed significant weight loss over time. No significant change in average brain weights was observed between the normal, cachectic and noncachectic groups. For the first time, we found that brains from Pa04C tumor bearing mice exhibited a profound reduction in water soluble metabolites as compared to Panc1 and nontumor bearing normal mice. Representative spectra from normal, Panc1 and Pa04C are displayed in Figure 1. Significant decreases in neurotransmitters: g-aminobutyric acid (GABA), N-acetyl aspartate (NAA), and taurine as well as lactate, myo-inositol, phosphocholine, glycerophosphocholine (GPC), creatine, formate, and essential amino acids: Leu, ILe, Val, and Phe were observed in brains from cachectic (Pa04C) mice compared to noncachectic (Panc1) and healthy Normal mice (Figure 2). A heat map display of brain metabolites from normal mice and Panc1, and Pa04C tumor bearing mice is presented in Figure 3. Non-cachexia inducing Panc1 tumors also induced a significant decrease of brain GABA, glutamate, aspartate, total choline and tyrosine compared to normal brains. Although this decrease was not as profound as in the cachectic mice, it highlighted the potential for systemic changes induced in the brain by cancers. These results provide new insights into profound changes in brain metabolism during cachexia that are likely indicative of compromised CNS function and may be a major contributing factor to the systemic control of cachectic wasting. These data provide strong evidence to support investigating new metabolic interventions to reverse CNS injury and cachexia as well as providing new CNS targets for early detection using in-vivo MRS techniques.

Acknowledgements

This work was supported by NIH R01 CA193365, NIH R35CA209960, and NIH P30CA06973.