Methods

Animals and Interventions

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Arkansas, Fayetteville. Male and female C57BL/6J mice were purchased from Jackson Laboratories. We have previously reported on the phenotype and aspects of mitochondrial and protein metabolism in these animals27-29. The mice were kept on a 12:12 h light–dark cycle with ad libitum  access to normal rodent chow and water. LLC cells (1 × 106) suspended in 100 μL sterile PBS were implanted to the hind flank of mice at 8 weeks of age as previously described 28. The tumor was then allowed to develop for 1, 2, 3, or 4 weeks in separate cohorts. For sham control, one group of mice received a bolus injection of 100 μL sterile PBS. PBS controls were age-matched to the most cachectic group (4 weeks post-implantation, 12 weeks of age at tissue collection). In the female mice, there was a clear dichotomization between the 3 and 4 weeks groups to cluster into smaller or larger tumor sizes. These 2 groups were then isolated and split into low tumor (LT; ≤ 1.2 g) and high tumor (HT; ≥ 2 g) groups based on this dichotomization of tumor size as described previously27. Animal tissues, organs, and blood plasma were quickly collected under isoflurane anesthesia prior to euthanasia. Tissues were weighed and snap-frozen in liquid nitrogen for further processing and stored at −80°C. The tibialis anterior muscle from males (TA) and plantaris muscle from females were submerged in optimum cutting temperature compound (OCT) and then placed in liquid nitrogen cooled isopentane. OCT mounted tissue was then stored at -80°C for histological analysis.

Lewis Lung Carcinoma and Implantation

LLC cells were prepared as described previously 28. LLC cells (ATCC CRL-1642) were plated at passage 2. Cells were cultured in 250 ml culture flasks in DMEM supplemented with 10% fetal bovine serum supplemented with 1% penicillin and streptomycin. Once cells reached confluence, they were counted, trypsinized and diluted in PBS for implantation. Mice were anesthetized with isoflurane and hair was removed from the right hind flank. LLC cells (1x106) suspended in 100 µl sterile PBS and injected subcutaneously into the hind flank of mice at 8 weeks of age as previously described28. Tumors developed for up to 4 weeks. Experimental endpoints were adjusted for signs of distress and veterinary recommendation for humane care.

Myoblast and Fibroblast Co-Culture

C2C12 myoblast cells (ATCC CRL-1772) and 3T3 fibroblast cells (ATCC CRL-1658) were co-cultured for an in vitro analysis of fibroblasts to skeletal muscle interaction. C2C12 cells were grown until they reached ~80% confluency at passage 2 where they were then transferred to a 6-well plate and differentiated for 5 days. 3T3 fibroblast cells were then added via a transwell-insert, which possesses a semi-permeable membrane, into the 6-well plate. LLC conditioned media (LCM) or control media (Veh) were added for 24hr. Both myotubes and 3T3 fibroblasts cells were harvested for subsequent RT-qPCR and Western Blot analysis.

RNA Isolation, cDNA synthesis, and Quantitative Real-Time PCR

RNA isolation, cDNA synthesis, and quantitative real-time PCR were performed as we have previously described 29, 30. All targets were assayed using Taqman probes including: 18S (Mm03928990_g1), Collagen 1 (Mm00801666_g1), Collagen 3 (Mm00802305_g1), MMP-2 (Mm00439498_m1), MMP-9 (Mm00439498_m1), Timp-1 (Mm01341361_m1), Timp-2 (Mm00441825_m1), TGF-β1 (Mm01178820_m1), SMAD 2 (Mm00487530_m1), SMAD 3 (Mm01170760_m1). Taqman probes were purchased from Applied Biosystems. RT-qPCR measured cycle threshold (Ct) and the ΔCt value was calculated as the difference between the Ct value and the 18S Ct value. Final quantification of mRNA abundance was calculated using the ΔΔCT method Ct = [ΔCt (calibrator) – ΔCt (sample)]. Relative quantifications were then calculated as 2-ΔΔCt. 18S Ct values were confirmed to not differ between experimental conditions for any comparison.

Histology

TA and plantaris skeletal muscle stored in OCT were cryosectioned into 10μm thick sections on polarized microscope slides. Muscle sections were then histologically stained with Picro Sirius Red for quantification and analysis of overall collagen content within the muscle. Images were analyzed using Nikon NIS Elements BR software package.

Statistical Analysis

Normality of data was established via a Shapiro Wilk test prior to any parametric analysis. A one-way ANOVA was employed as the global analysis for each dependent variable in both experiments. All statistics were performed across experimental groups within each sex. Where significance was detected, differences among means were determined by a Student–Newman–Keuls post hoc test. A Student’s t-test was utilized for the myotube diameter experiment. For all other cell culture experiments, a two-way ANOVA was employed to test for main effects of treatment (Veh or LCM) and cell type (C2C12 or 3T3) and whether any interactions existed between the dependent variables. For all experiments, statistical significance was set a P ≤ 0.05. All data were analyzed using the Statistical Analysis System (SAS) and figures were compiled using GraphPad Prism and data expressed as mean ± standard error of the mean (SEM).