Introduction

Cancer cachexia is a progressive and involuntary wasting condition of lean body mass that afflicts approximately 80 % of all cancer patients and can be attributed to 20-40% of cancer-associated deaths1, 2. Cancer cachexia is defined as losses in body mass >5 %, primarily due to loss of muscle mass which can be accompanied with or without losses in adipose tissue3, 4. To date, no singular method of treatment has shown effective at eliminating cancer cachexia development due to the systemic and complex nature of its underlying mechanisms5-7. Further complicating research into effective treatments for cancer cachexia is the emerging role of sexual dimorphism on cancer cachexia progression, as recent studies have shown that males and females can differentially respond to cancer cachexia8-10. Metabolic and inflammatory instabilities are known contributors to cancer cachexia progression and have been greatly examined 11, 12, however the role of fibrosis on the development of cancer cachexia remains incompletely understood.
Excessive fibrosis of healthy skeletal muscle worsens overall outcomes in cancer patients 13. Moreover, unlike metabolic and inflammatory abnormalities that can improve following cancer cessation, this fibrosis of vital and functional lean tissue is largely irreversible and can affect patients throughout their lifespan. The extracellular matrix (ECM) is composed primarily of collagen 1 and 314. The ECM is critical for force transduction, growth factor secretion, etc. The matrix metalloproteinases (MMPs) are responsible for the degradation of collagens, and in skeletal muscle MMP2 and MMP9 are typically the most abundant15. Skeletal muscle MMP expression is altered in cancer cachexia16 which may contribute to the excessive fibrosis associated with cancer cachexia. Transforming growth factor-β (TGF-β) is a transcriptional activator of many cellular processes including the activation of satellite cells as well as synthesis, degradation, and remodeling of the ECM that surrounds skeletal muscle17, 18. Upregulation of TGF-β can result in excessive ECM dysregulation and deposition of ECM components ultimately resulting in the replacement of healthy skeletal muscle with non-contractile proteins such as collagens 19, 20. This upregulation of TGF-β can be induced via its upstream regulator in Interleukin-6 (IL-6), an inflammatory cytokine which is commonly elevated in cachectic cancer patients 21. Taken together, these data suggest fibrosis in cancer patients is altered via TGF-β, and evaluation of TGF-β and its downstream effectors could provide insights on fibrosis as it pertains to cancer cachexia progression.
Males and females exhibit sexual dimorphism in skeletal muscle properties such as fiber type composition within the same muscle, mitochondrial content, and number of satellite cells innately, and this dimorphism is documented in some muscle conditions such as disuse atrophy 22-24. Differences in the relative amounts of circulating hormones like testosterone and estrogen only adds to the complexity of the male and female phenotypic makeup within skeletal muscle. For example, testosterone is typically higher in males and promotes muscle protein synthesis and regeneration, while estrogen is typically higher in females and has a greater impact on reducing inflammation 25, 26. Therefore, female mice could possess a greater protective effect from inflammatory-based muscle wasting conditions such as cancer cachexia when compared to males10. Innate differences between sexes can therefore greatly affect overall skeletal muscle health and even skeletal muscle health in pathologies such as cancer cachexia 8, 10.
This study is, to our knowledge, the first to evaluate the role of fibrosis during the development of cancer cachexia across both sexes. Implantation of Lewis Lung Carcinoma (LLC) cells allows for the controlled development of cancer cachexia, and thorough analysis of fibrotic pathways during the progression of cachexia. Evaluation of these fibrotic markers could elucidate potential avenues for therapeutic interventions aimed towards the prevention of fibrosis and its contributions to cancer cachexia development. Therefore, the purpose of this study was to utilize the LLC allograft model of cachexia to evaluate key regulators of the ECM during the controlled development of cancer cachexia. We hypothesized the ECM and signaling pathways involved in its modulation would be altered prior to cachexia. Herein we provide novel evidence of altered fibrotic signaling coinciding with cancer cachexia development, however this varied between sexes. These data highlight the emerging role of fibrosis on cancer cachexia and further strengthens the growing need for research in both males and females as sex continues to create divergent responses to cancer cachexia and its underlying mechanisms.