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).