Introduction

Cachexia is an underexplored and yet devastating consequence of cancer that is the cause of 20% of all cancer related deaths [1]. Cachexia inducing tumors cause a ‘wasting away’ of the body. The condition is associated with poor treatment outcome [2], fatigue, and extremely poor quality of life [2, 3]. Because of the multi-factorial characteristics of this condition, it has been difficult to understand the mechanisms driving the impact of the tumor on body organs and the sequence of events that leads to this lethal condition. Tissue wasting is one of the characteristics associated with cachexia syndrome. Here we have used 1H MRS to characterize the metabolic profile of skeletal muscle and heart muscle obtained from normal mice and noncachexia (Panc1) and cachexia inducing (Pa04C) tumors bearing mice to further understand the cascade of events that occur during cachexia.

Methods

The human pancreatic cancer cell line, Panc1, was obtained from ATCC. The human pancreatic cancer cell line, Pa04C, was provided by Dr. Maitra [4]. 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) [5]. Once the mice were sacrificed, the heart and skeletal muscle were harvested, freeze clamped and stored in -80°C until 1H MRS analysis. Dual phase solvent extraction was performed on the 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 and statistical analysis was performed using The UnscramblerX 10.

Results and Discussion

Representative 1H MR spectra of skeletal muscle obtained from normal mice, and from mice bearing noncachexia inducing Panc1 tumors, and cachexia inducing Pa04C tumors are shown in Figure 1. Quantitative changes in the 1H MRS detected metabolites in muscle are shown in Figure 2. Representative 1H MR spectra of the heart from normal mice, and from mice bearing noncachexia inducing Panc1 tumors, and cachexia inducing Pa04C tumors are displayed in Figure 3. Quantitative changes in 1H MRS detected metabolites in the heart are shown in Figure 4. Leucine, creatine, lactate and glucose were significantly lower, and acetate and formate were significantly higher, in the muscle of cachectic mice compared to non-cachetic mice. Cachectic mice had significantly lower alanine, succinate, glycine, lipid and PUFA (Polyunsaturated fatty acids), and significantly higher acetate, pyruvate, and formate compared to normal mice. Non-cachectic mice had significantly lower alanine, succinate, phosphocreatine, and glycine, and significantly higher leucine, isoleucine, valine, pyruvate, creatine, taurine and glucose compared to normal mice. In the heart, leucine, isoleucine, valine, aspartate, glucose, lipid and PUFA were significantly lower, and glutamine was significantly higher, in cachectic mice compared to non-cachetic mice. Cachectic mice had significantly lower leucine, isoleucine, valine, lactate, alanine, glutamate, and PUFA, and significantly higher glutamine and glucose compared to normal mice. Non-cachectic mice had significantly lower alanine, and significantly higher glucose and lipid compared to normal mice. These data highlight, for the first time, the profound metabolic changes that occur in skeletal muscle and the heart with cachexia, identifying potential in vivo 1H MRS indices to detect the onset of cachexia from changes in branched chain amino acids, glucose, and PUFAs. Our data also provide new insights into the effects of cachexia as well as noncachexia inducing tumors on skeletal muscle and heart metabolism that may lead to metabolic interventions in ‘metabolotheranostic’ strategies to reduce the morbidity associated with cancer and cachexia.

Acknowledgements

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