Cancer cachexia is a complex syndrome characterized by progressive weight loss, anorexia, muscle loss and weakness. Muscle wasting have been emerging as one of the principal components of cancer cachexia, leading to progressive impairment of work capacity (Fearon et al., 2011) a significant hallmark of poor prognosis in cancer patients (Christensen et al., 2014). The causal understanding of the muscle wasting in cancer cachexia is complex; the pro-inflammatory scenario however, is thought to play a prominent role (Ebrahimi et al., 2004). Elevated TNF-α, IL-6 and IL-1β are associated with weight loss, hypercatabolism, reduced food intake and muscle loss in cancer patients (De Larichaudy et al., 2012; Yuan et al., 2015). Thus, elevated inflammatory interleukins and muscle wasting during tumor development can significantly modulate muscle strength and locomotor capacity in tumor-bearing mice, some of the most critical endpoints associated with cancer morbidity and mortality (Christensen et al., 2014).

Melanoma is the deadliest form of skin cancer. When diagnosed at localized stages (I and III), the five-year survival rates correspond to 98%; however, this dramatically drops to 5–19% when metastases is presented (Sandru et al., 2014; American Cancer Society, 2017). Despite early stages melanomas are not related with weight loss, the appearance of metastatic and/or solid tumor melanoma is related to cachexia development (Kawamura et al., 1999b; Das et al., 2011). Melanoma metastasis has a simultaneous lymphatic and hematogenous spread, with the potential to metastasize in any organ (Belhocine et al., 2006). Recent data has been demonstrated lung, gastrointestinal tract, extra-regional lymph nodes, liver and prostate are the principal target of metastatic melanoma; all of than with elevated rate of cachexia (40 to 80%) and mortality (von Haehling et al., 2016).

To better unveil melanoma tumor biology and tumor-host interaction, a number of murine models have been widely used in preclinical melanoma studies (Becker et al., 2010; Kuzu et al., 2015). The most widely used cell type in the melanoma model is the B16 cell line and two subclones (F1 and F10) that spontaneously form tumors after syngeneic transplantation in C57BL/6J mice (Fidler and Nicolson, 1976). The syngeneic model involves the induction of tumor cells into the same species and genetic background (Darro et al., 2005; Bobek et al., 2010). These models have important benefits over xeno-transplantation or genetically modified models since mice possess a normal immune system that opens a useful platform for new immunotherapy and adequate host response (Becker et al., 2010). However, a few number of studies have demonstrated B16 cells induces cachexia in mice following intraperitoneal inoculation through higher lipase lipoprotein activity (Kawamura et al., 1999a,b; Das et al., 2011). Indeed, there is a paucity of information dealing with tumor-host interaction in mice carrying B16F10 melanoma cells regarding cancer cachexia, specially the interaction among cancer cachexia components and skeletal muscle strength and locomotor activity. Thus, we aimed to investigate the cachexia development response to B16F10 tumor cells inoculation and it interactions with skeletal muscle adaptations (mass and strength) and mice locomotor activity. We hypothesized muscle loss and pro-inflammatory condition induced by tumor development play a pivotal role on reduced muscle strength and decreased locomotion and exploratory activity in B16F10 tumor-bearing mice.

Tumor mass was apparent only after 19 days after cell inoculation in average. The animals were monitored until euthanasia when the tumor mass reached 11.72% of total body weight. After careful analysis, we did not find any metastases in target tissues such as lung, stomach and intestines, extra-regional lymph nodes, liver, pancreas and spleen. Tumor development also caused decreased food intake over the third and fourth weeks (Figure 1), without changes in water intake.

The tissue responses of the tumor-bearing animals and the sham group are shown in Table 1. Clearly, the muscle and epididymal fat content were significantly decreased (P < 0.05) in tumor-bearing mice compared to the sham group. Spleen was 2-fold larger in tumor-bearing compared to Sham mice. Tumor-free body weight was not different, despite the P trend = 0.07 (Table 1). Cachexia index was 8.3%, higher than 5% established by as Fearon et al. (2011) as cachexia definition. Liver relative weight was not affected by tumor growth.

Figure 2 shows the data related to the systemic response of experimental groups. The tumor-bearing mice presented elevated LDH plasma levels. Plasma inflammatory cytokines were significantly elevated in the tumor bearing group; TNF-α was over two-fold and IL-6 was over eight-fold higher in tumor-bearing mice compared to Sham animals. However, there was no difference between groups in relation to IL-10.

Figure 3 presents the results of the locomotor and exploratory test as well as the gripping strength test. The overall distance and distance traveled in the center were significantly (P < 0.05) lower (40 and 56%, respectively) in tumor-bearing mice compared to sham. Tumor-bearing mice also spent significantly (P < 0.05) more time stopped than sham mice. In addition, tumor growth promoted a significantly decreased muscle gripping strength compared to sham mice.

The Pearson correlation test demonstrated reduced skeletal muscle mass and body weight loss were significantly (P < 0.05) associated to gripping strength. Elevated plasma TNF-α was inversely (P < 0.05) associated to gripping strength. Also, reduced skeletal muscle mass and elevated plasma TNF-α and IL-6 were determinant for the impaired locomotor activity parameters (Figure 4). In addition, correlation test also demonstrated negative association between decreased skeletal muscle mass and elevated plasma TNF-α (r = −0.71; P < 0.05) and IL-6 (r = −0.57; P < 0.05).

Our data demonstrated B16F10 tumor cell inoculation promoted cachexia characterized by weight loss, skeletal muscle wasting, adipose tissue loss and anorexia. Fat tissue significantly targeted presenting a 33.06% reduction in tumor-bearing mice compared to controls. The catabolic state appears to be intrinsic to the anorexia state and systemic organic pro-inflammatory response against tumor progressive growth. In addition, tumor growth-driven inflammation and muscle loss impaired voluntary muscle strength and decreased locomotor activity in tumor-bearing mice. Although previously studies have demonstrated B16F10 intraperitoneal inoculation was able to induce cancer cachexia (Kawamura et al., 1999a,b). Our data have demonstrated therefore, both muscle wasting and inflammatory condition mediated tumor growth play an important role in skeletal muscle strength loss and locomotor activity impairment.

Preclinical tumor models indicate that body and tissue wasting can impact functional parameters (Chen et al., 2015). Skeletal muscle loss appears to be the most essential component associated with poor outcome in cancer cachexia (Fearon et al., 2011). Our data highlighted reduced grip strength and decreased locomotor activity in the tumor group challenged with B16F10 cells, were associated to loss of muscle mass. These data demonstrated tumor growth promotes a critical modification in skeletal muscle that compromised animal's locomotor activity. Tumor-bearing mice inoculated with LLC cells demonstrated atrophy and lower grip strength (Chen et al., 2015). However, the skeletal muscle loss causative interaction is poorly investigated. Our results are particularly relevant since muscle loss has not been linked with lower muscle function only (Gabriel et al., 2006; Jorgensen et al., 2010). Due to complex coordination among neural, mechanic and physiological functions, muscle strength is highlighted as the most critical functional endpoint to survival and hospital discharge (Mendes et al., 2014; Joglekar et al., 2015).

Tumor-driven inflammatory factors may act as mechanisms underlying lipolysis and myolysis in wasting conditions (Der-Torossian et al., 2013; Laine et al., 2013; Inacio Pinto et al., 2015). Previously studies have demonstrated inflammatory cytokines play a critical role in muscle wasting by stimulating transcription factor nuclear factor-kB (NF-kB), which targets key skeletal muscular genes of catabolic cascade and up-regulate ubiquitin-proteasome pathway for protein catabolism (Zhou et al., 2016). In the subcutaneous tumor model, tumor-bearing mice show elevated TNF-α, IL-6 and activin A levels that up regulate ubiquitin ligases such as muscle ring finger-1 (MuRF1) and Atrogin-1 mRNA expression in gastrocnemius muscles (Matsuyama et al., 2015). Our data elucidates elevated plasma levels of TNF-α and IL-6 after 28 days of transplantation was associated to skeletal muscle loss, as demonstrated by negative association between decreased skeletal muscle mass and elevated plasma TNF-α (r = −0.71; P < 0.05) and IL-6 (r = −0.57; P < 0.05). Our study also demonstrated spleen was 2-fold larger in tumor-bearing compared to Sham mice which is a good indicator of systemic inflammation state. Indeed, chronic inflammatory condition caused by tumor development is thought to play a prominent role on muscle loss in cancer cachexia (De Larichaudy et al., 2012; Yuan et al., 2015). Although previous studies have suggested that IL-10 plays an important role in cachexia from different cancer types (Ebrahimi et al., 2004; Sun et al., 2010), our model promoted no changes in IL-10 plasma levels. In addition, taking the negative association between inflammatory interleukins and locomotor activity parameters (Figure 4), we may affirm that skeletal muscle wasting, loss of muscle strength and impaired locomotor activity imposed by B16F10 melanoma is inherent to the systemic pro-inflammatory condition promoted by tumor growth. Despite de association demonstrated among loss of muscle mass, elevated interleukin and impaired locomotion activity, is important to say that skeletal muscle tissue mass only is not the best predictor of muscle wasting. The absence of skeletal muscle cross sectional area and cardiac mass appears to be the principal limitation of the present study.

In addition to the catabolic-related inflammation scenario, a higher production of LDH is characteristic of a glycolytic phenotype reprogram. According to the Warburg effect, cancer cells undergo glycolysis rather than mitochondrial phosphorylation as a major source of energy (Vander Heiden et al., 2009), which may be directly linked with an activation of oncogenes and lower expression of tumor suppressors (Dang and Semenza, 1999; Allison et al., 2014). In melanoma patients, LDH is a stronger predictive factor of lower overall survival (Kelderman et al., 2014; Diem et al., 2015). However, the link between LDH and functional impairments in cancer is not clear. Indeed, melanoma cells favor a glycolytic phenotype and pro-inflammatory environment that may exert a key role in lipolysis, lower muscle strength and significant loss of mobility.

Anorexia is also an important component of cancer cachexia. Anorexia-related to cancer primary cause is often increased pro-inflammatory state and/or increased lactate production (Ezeoke and Morley, 2015). Administration of cytokines and lactate to rodents has been demonstrated to reduce food intake (Silberbauer et al., 2000; Patra and Arora, 2012). These two factors then modulate central nervous system neurotransmitter cascades, especially in hypothalamus, resulting in a reduction in satiation (Ezeoke and Morley, 2015). Therefore, is reasonable to affirm that elevated circulating interleukin and LDH activity demonstrated in our study may play a pivotal role in anorexia development induced by B16F10 melanoma development. Indeed, anorexia in cancer cachexia may also contribute significantly to muscle wasting and impaired locomotor activity demonstrated in our study. We cannot determine however, the significance plot of each cachexia component to locomotor activity impairment; it must be considered in future studies.

In conclusion, our data demonstrated that a subcutaneous B16F10 melanoma model promotes a catabolic state probably in response to a pro-inflammatory environment. Elevated inflammatory interleukins and muscle wasting induced by tumor development appears to play a important role on the impairment of muscle strength and decreased locomotor activity in tumor-bearing mice, some of the most critical endpoints associated with cancer morbidity and mortality. This model may be a valuable tool in testing new therapeutic approaches and future translational perspectives.

FF and RD conceived of the studies. LV, GB, and FM provided essential equipment, expertise and resources. FF, CP, MT, PC, DR, and DdO performed experiments. FV, FF, and RD analyzed and interpreted data. FV, FF, and RD wrote and edited the manuscript.