Regulators of ECM Remodeling are Altered during Cachexia Development
In female mice, MMP-2 mRNA abundance was ~1.1-fold higher in the 1 wk post-LLC injection group compared to the LT or HT groups (p<0.05; Figure 3A). MMP-9 mRNA abundance was ~2.0-3.8-fold higher in the HT group compared to all the other groups (p<0.05; Figure 3B). Male mice saw no changes in MMP-2 mRNA abundance with LLC-injection (p>0.05; 3C); however, the 4 wk post-LLC injection group had an ~9.6-fold higher MMP-9 mRNA abundance compared to all other groups (p<0.05; Figure 3D).

TGF-β-regulated Fibrosis during Cancer Cachexia Development

In female mice, TGF-β1 mRNA abundance was ~2.3-fold higher in the 1wk post-LLC injection group compared to all other groups (p<0.05; Figure 4A). We found no differences in SMAD 2 and 3 mRNA abundance (p>0.05; Figure 4B & 4C). In male mice, there was an ~1.6-fold higher TGF-β1 mRNA abundance in the 4 wk post-LLC injection group compared to the PBS and 1 wk post-LLC groups (p<0.05; Figure 4D). There were no differences detected in mRNA abundance of SMAD 3 (p>0.05; Figure 4E); however, the 4 wk post-LLC group had a ~0.5-fold higher SMAD 2 mRNA abundance compared to the 1 wk post-LLC injection groups (p<0.05; Figure 4G).

In vitro Analysis of Skeletal Muscle-ECM Dynamics in a Cachectic Environment

Myotube diameter was ~20% lower following treatment with LCM compared to Vehicle (p<0.05; Figure 5A & 5B). There was a main effect of myotubes to have ~4-fold less Collagen 1 mRNA abundance compared to 3T3 fibroblasts cells (p<0.05; Figure 6A). There was main effect of LCM treatment to reduce Collagen 1 mRNA abundance independent of cell type (p<0.05; Figure 6A). There was a main effect of 3T3 fibroblasts cells to have lower Collagen 3 mRNA abundance to C2C12 myotubes independent of treatment (p<0.05; Figure 6B). There were main effects for myotubes to have a greater Collagen 3:1 ratio independent of treatment (p<0.05: Figure 6C). Additionally, there was a man effect for groups treated with LCM to have a higher Collagen 3:1 ratio (p<0.05; Figure 6C). There were no differences in mRNA abundance of MMP-2 in either myotubes or 3T3 cells (p>0.05; Figure 6D), however mRNA abundance of MMP-9 was ~450-fold greater in 3T3 cells than in myotubes and LCM treatment reduced MMP-9 mRNA ~200-fold in 3T3 cells compared to Vehicle (p<0.05; Figure 6E). There was a main effect for 3T3 cells to have lower mRNA abundance of TIMP-1 independent of treatment (p<0.05; Figure 6F). No differences were observed in C2C12 or 3T3 cells for protein content of β-catenin (p>0.05; Supplemental Figure 1C).