Cancer-induced cachexia is a significant cause of morbidity and mortality.^1,2 In pancreatic cancer, especially, the syndrome affects approximately 80% of patients.² Noninvasive biomarkers that identify precachectic patients who will progress to refractory cachexia are an urgent and unmet requirement.³ We report on the initial characterization of a myoblast optical imaging reporter that allowed real-time longitudinal monitoring of the early onset of cancer induced wasting. The reporter myoblasts were engineered with cachexia inducible red fluorescence protein (tdTomato: tdT) expression. The myoblast reporter was validated in mice with human pancreatic cancer xenografts that induced weight loss. As a first step to holistically identifying the sequence of metabolic changes that occur with onset, quantitative ¹H magnetic resonance spectroscopy (MRS) of plasma identified a depletion of total lipids, total cholesterol, triacyglycerides (TAG), and valine levels along with alanine in muscle of tumor bearing cachectic mice relative to tumor bearing weight gaining and non-tumor bearing control mice.The human pancreatic cancer cell line, Panc1, and rat L6 myoblasts were from ATCC. The human pancreatic cancer cell line, Pa04C, was provided by Dr. Maitra.³ The dual fluorescence vector construct was generated using standard molecular biology protocols. The cachexia inducing promoter (tTMuRF1) driving tdT expression was a triple tandem (tT) glucocorticoid binding element /FOXO1 binding element sequence from the proximal promoter of the human MuRF1 gene⁴ fused to its core promoter sequence. Post transfection, pure stable reporter myoblast clones exhibiting dual fluorescence were obtained by FACS. Six to 8 week old male severe combined immunodeficient mice were inoculated in the right flank with cancer cells (5 × 10⁶) and in the right hind leg muscle with reporter myoblasts (2 × 10⁶). Live animal optical imaging was done using a Xenogen IVIS® Spectrum (PerkinElmer) optical scanner. Quantitative fluorescence intensities were acquired using Xenogen Living Image® 4.2 software package. All ¹H MR spectra were acquired on an Avance III 750 MHz (17.6 T) Bruker MR spectrometer equipped with a 5 mm broad band inverse (BBI) probe. Plasma MR spectra with water suppression were acquired using a Carr-Purcell-Meiboom-Gill pulse sequence (CPMG: [D₁-90-(δ-180-δ)_n-aq]) with the following experimental parameters: spectral width of 15495.86 Hz, data points of 64 K, 90^o flip angle, relaxation delay 6 sec, acquisition time 2.11 sec, 32 scans with 8 dummy scans, receiver gain 64 and spin echo time of 15ms (δ = 15μs and n = 100). For muscle extracts, spectra were acquired using a single pulse sequence with 64 and 32 scans respectively at a receiver gain of 128; all other acquisition parameters were kept the same. All spectral acquisition, processing and quantification were performed using TOPSPIN 2.1 software.

A schematic representation of pGL3b-tTMuRF1-tdT:EF1α-eGFP is shown in Figure 1. Representative optical imaging of mice bearing tumors of similar sizes are shown in Figure 2A. Weight losing Pa04C mice exhibited strong tdT signals while little or no tdT signals were detected from weight gaining Panc1 mice. Figure 2B shows tdT/GFP signal ratios plotted against percent weight change. Weight gain (Panc1 mice) was associated with low tdT/GFP ratios while weight loss (Pa04C mice) was associated with high tdT/GFP ratios. Moreover, we observed tdT/GFP ratios above the background of ~0.25 at 8 and 10 days post Pa04C tumor cell inoculation at which time small weight losses in the range of 1-2% were observed, indicative of early onset of cachexia. Representative ¹H MR spectra of plasma obtained from Panc1, Pa04C, and normal mice are shown in Figure 3A. Differential changes in the lipid, triacylglycerol (TAG), valine, and cholesterol signals from these three groups are evident. Quantification of the ¹H MRS plasma metabolites detected a significant reduction in Pa04C tumor bearing mice plasma compared to plasma of Panc1 tumor bearing mice and normal plasma. A significant decrease of alanine in the muscle of Pa04C mice relative to the muscle obtained from Panc1 tumor bearing mice and normal mice was observed as shown in Figure 3B.

The myoblast reporter system described here has the ability to noninvasively detect early onset of weight loss in living mice which, in combination with ¹H MRS, results provides new insights into metabolic changes that can be assessed clinically.