Discussion

Herein, we are among the first to evaluate fibrosis and associated markers during the early stages of development of cancer cachexia, specifically with regards to dimorphisms across biological sex. Our analysis consisted of measuring major ECM components such as collagens as well as modulators of the ECM (i.e., MMPs). We further examined a key signaling pathway (i.e., TGF-β/SMAD) associated with fibrosis and its downstream effectors. These key markers of fibrosis were analyzedin vivo and in vitro to create a comprehensive approach to understanding if the ECM is altered during the development of cancer cachexia, and if so, what are the likely contributors. While no direct statistical comparisons were made between males and females, we observed clear differences in fibrosis progression during the development of cancer cachexia across sex. Female mice exhibited alterations in collagen content, as well as mRNA of collagen and TGF-β as early as 1-week post-LLC injection. Male mice on the other hand displayed no significant changes in fibrosis or fibrosis-related markers until 4-weeks post-LLC injection. Specifically, the collagen deposition in females was mild compared to males (5% vs. 15%). In vitroco-culturing of myotubes and fibroblasts in a cachectic environment yielded interesting findings, albeit in the absence of sex distinctions, but with the added distinction of isolating muscle and ECM associated cell lines. Of note, both C2C12 and 3T3L1 cells had altered mRNA abundance in certain collagens, MMPs, and TIMP-1 relative to one another. Addition of LCM did indeed reduce myotube diameter significantly as well as reduce Collagen 1 mRNA and increased Collagen 3:1 mRNA in both cell types. Our findings support the growing theme across the literature for divergent responses to myopathologies between male and female sexes as well as reinforce the need for these sex-based comparisons in future studies 8, 31, 32.
As we reported 27, the dichotomization of tumor burden observed in the female mice supports the correlation of overall tumor burden to the development of a cachectic phenotype. However, with regards to incidence of fibrosis, we observed increased collagen content and ECM dysregulation as early as 1wk post-LLC injection in female mice. This observation indicates that while overall tumor burden is tightly correlated to cancer cachexia in both sexes, the fibrotic development as it pertains to cancer cachexia is not as tightly correlated to overall tumor burden. In contrast, we did not observe the alterations in male mice 1-week post-LLC injection seen in female mice suggesting fibrotic development is not correlated to overall tumor burden in male mice. Male mice did not display the same early stages alterations in fibrosis markers. These data suggest both males and females experience alterations in their ECM as early as 1wk post-injection with LLC cells, but not with complete consistency across the associated regulators in MMPs and TGF-β. Recent studies report reductions in TGF-β mRNA in cardiac muscle in a colon adenocarcinoma cells 26 model of cancer cachexia 33 and increased TGF-β mRNA in adipose and skeletal muscle tissue in models of pancreatic cancer cachexia13, 34, but all studies exhibit increased fibrosis. TGF-β then appears to be a clear and consistent regulator of fibrosis in cancer cachexia, and our findings suggest its role can even precede development, at least in female mice.
The role of TGF-β in fibrosis across pathologies has been well documented 35-37. The canonical pathway of TGF-β is to activate its downstream regulators in SMAD 2 and 3, which in turn can induce expression of collagens including the Collagen 1 and 3 which are highly expressed in the muscle belly 36. Dysregulations in TGF-β lead to improper deposition of ECM components via SMAD 2 and 3, ultimately resulting in fibrosis. However, here we report no significant alterations in SMAD 2 or 3 mRNA content in either male or female mice or even in vitro following addition of LCM. Further examination by measurement of β-catenin content is needed as crosstalk between TGF-β and the Wnt/β-catenin pathway has been noted in previous literature 38-41. The lack of SMAD 2 and 3 induction appears to suggest a non-canonical pathway is involved in regulation of the ECM and its components in this model of cancer cachexia. However, what pathway(s) is/are involved remains unclear and requires further study.
The remodeling of the ECM is one of the most important features of cancer progression. Therefore, understanding how fibroblasts respond to a cachectic environment as well as how they may influence skeletal muscle is important to understand. Therefore, we co-cultured C2C12 myotubes with 3T3L1 fibroblasts and then created a cachectic environment via addition of LCM. We then analyzed the same key components and regulators of the ECM and fibrotic pathways that were targeted in thein vivo approach. We demonstrated a reduction in myotube diameter with the addition of LCM media suggesting a cachectic phenotype. We observed fibroblasts exposed to a cachectic environment reduced their gene expression of MMP-9 significantly. As collagens are a major target of MMP-9, reduced expression could lead to increased collagen deposition. This was a phenotype observed in the in vivoexperiments. Additionally, we observed that myotubes co-cultured with fibroblasts and exposed to this cachectic environment altered their expression of collagen 3 relative to collagen 1. This suggests the cachectic environment not only induces fibroblasts to alter expression of MMP-9 but influences skeletal muscle cells to alter their expression of collagens. These findings could explain the increased collagen deposition noted in this study and the ECM remodeling noted in several other studies during cachexia.
In summary, this study set out to obtain a better understanding of fibrosis during development of cancer cachexia across biological sex. Our findings support the recent findings that females and males to display divergent responses to pathologies with females being somewhat protected from cancer cachexia 10. While both sexes displayed increased fibrosis following the onset of canonical cachexia, females exhibited alterations in markers associated with fibrosis and ECM regulation as early as 1wk post-injection with LLC. With the noted protection females exhibit to cachexia these alterations could point to a compensatory response. Overall, increased fibrosis is involved with the development of cancer cachexia and males and females seemingly differ in the timing of alterations in the relevant fibrosis-related markers. However, more research is necessary to ascertain the molecular regulators involved in the elevated collagen deposition and fibrosis shown in our results as TGF-β was elevated but the traditional downstream effectors in SMADs 2 and 3 and β-catenin were unchanged. A greater understanding of these pathways involved with fibrosis development during the pre- and early-stage development of cancer cachexia could point to potential therapeutic avenues and improve overall patient outcomes.