Development of skeletal muscle fibrosis in a rodent model of cancer cachexia

Abstract

Cachexia is characterized by losses in lean body mass and its progression results in worsened quality of life and exacerbated outcomes in cancer patients. However, the role and impact of fibrosis during the early stages and development of cachexia in under-investigated. The purpose of this study was to determine if fibrosis occurs during cachexia development, and to evaluate this in both sexes. Female and male C57BL6/J mice were injected with phosphate-buffered saline or Lewis Lung Carcinoma (LLC) at 8-week of age, and tumors were allowed to develop for 1, 2, 3, or 4 weeks. 3wk and 4wk female tumor-bearing mice displayed a dichotomy in tumor growth and were reassigned to high tumor (HT) and low tumor (LT) groups. In vitro analyses were also performed on cocultured C2C12 and 3T3 cells exposed to LLC conditioned media. Immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis were used to investigate fibrosis and fibrosis-related signaling in skeletal muscle. Collagen deposition in skeletal muscle was increased in the 1wk, LT, and HT groups in female mice. However, collagen deposition was only increased in the 4wk group in male mice. In general, female mice displayed earlier alterations in extracellular matrix (ECM)-related genes beginning at 1wk post-LLC injection. Whereas this was not seen in males. While overall tumor burden is tightly correlated to cachexia development in both sexes, fibrotic development is not. Male mice did not exhibit early-stage alterations in ECM-related genes contrary to what was noted in female mice.

Keywords: biological sex; cancer cachexia; extracellular matrix; fibrosis; muscle wasting.

FIGURE 1

Collagen deposition within skeletal muscle measured via Picrosirius Red staining during the progression of cancer cachexia in the (A) plantaris (female) and (B) tibialis anterior muscle (male). N of 4–5 per group. Different letters denote statistical significance at an alpha set a p ≤ .05. 1wk, 2wk, 3wk, 4wk, denotes 1, 2, 3, and 4 weeks of cancer progression, respectively; LT and HT denote low tumor and high tumor, respectively; PBS, phosphate-buffered saline. Different letters denote statistical significance at an alpha set a p ≤ .05.

FIGURE 2

Collagen 1 and 3 mRNA abundance during the progression of cancer cachexia in females (left) and males (right). (A and B) Collagen 1 and 3 mRNA abundance in female tibialis anterior muscle. (C) Collagen 3:1 mRNA abundance female tibialis anterior muscle. (D and E) Collagen I and III mRNA abundance in male tibialis anterior muscle. (F) Collagen 3:1 mRNA abundance male tibialis anterior muscle. For females, N of 10–14 per group were used. For males, N of 7–8 per group were used. 1wk, 2wk, 3wk, 4wk, denotes 1, 2, 3, and 4 weeks of cancer progression, respectively; LT and HT denote low tumor and high tumor, respectively; PBS, phosphate-buffered saline. Different letters denote statistical significance at an alpha set a p ≤ .05. HT, high tumor; LT, low tumor; mRNA, messenger RNA.

FIGURE 3

mRNA abundance of markers of extracellular matrix remodeling and turnover during the progression of cancer cachexia in females (left) and males (right). (A and B) Mmp2 and Mmp9 mRNA abundance in female tibialis anterior muscle. (C and D) Mmp2 and Mmp9 mRNA abundance in male tibialis anterior muscle. For females, N of 10–14 per group was used. For males, N of 7–8 per group were used. 1wk, 2wk, 3wk, 4wk, denotes 1, 2, 3, and 4 weeks of cancer progression, respectively; LT and HT denote low tumor and high tumor, respectively; PBS, phosphate-buffered saline. Different letters denote statistical significance at an alpha set a p ≤ .05. HT, high tumor; LT, low tumor; mRNA, messenger RNA.

FIGURE 4

mRNA abundance of the TGF-β/SMAD signaling pathway components during the progression of cancer cachexia. (A–C) Relative mRNA abundance of Tgfb1, Smad 2, and 3 in female tibialis anterior muscle. (D and F) Relative mRNA abundance of Tgfb1, Smad 2, and 3 in male tibialis anterior muscle. For females, N of 10–14 per group were used. For males, N of 7–8 per group were used. 1wk, 2wk, 3wk, 4wk, denotes 1, 2, 3, and 4 weeks of cancer progression, respectively; LT and HT denote low tumor and high tumor, respectively; PBS, phosphate-buffered saline. Different letters denote statistical significance at an alpha set a p ≤ .05. HT, high tumor; LT, low tumor; mRNA, messenger RNA.

FIGURE 5

Effect of LCM treated media on C2C12 myotube size. (A) Representative images of C2C12 myotubes in media treated with vehicle or LCM. (B) Myotube diameter on C2C12 myotubes treated with either Vehicle (Veh) or LLC conditioned media (LCM). Different letters denote statistical significance at an alpha set a p ≤ .05.

FIGURE 6

Effect of LCM treated media and coculture on ECM regulation in C2C12 myotubes. (A-C) mRNA abundance of collagen 1, 3, and the collagen 3–1 ratio. (D–F). mRNA abundance of ECM remodeling and turnover markers Mmp2, 9, and Timp1. Different letters denote statistical significance at an alpha set a p ≤ .05. ECM, extracellular matrix; LCM, LLC conditioned media; mRNA, messenger RNA.

FIGURE 7

Effect of LCM treated media and coculture on Smad mRNA abundance in C2C12 myotubes or 3T3 fibroblasts treated with vehicle (Veh) or LLC conditioned media (LCM) treated. Different letters denote statistical significance at an alpha set a p ≤ .05.