Abstract
Background/Aim: Cancer cachexia encompasses several deleterious physiological alterations associated with functional impairments, poor quality of life, and increased mortality. The aim of this study was to examine the effects of chronic moderate intensity exercise training on markers of cachexia. Materials and Methods: Balb/c mice were randomly assigned to sedentary (SED) or exercise (EX) groups and EX mice were further randomly assigned to one of three exercise modalities (aerobic, resistance, combined). Results: Cachexia was induced in SED animals inoculated with C26 cells, as evidenced by significant changes in numerous markers. All cachexia-related perturbations were significantly attenuated in EX versus SED animals. Systemic inflammation was significantly decreased in all EX groups, as evident by a normalization of spleen mass and plasma IL-6. Conclusion: Multiple moderate intensity exercise modalities can provide significant benefits in cachectic mice, and this may be due, at least in part, to decreased systemic inflammation.
Cancer cachexia is most readily identified by loss of muscle mass and declines in muscle function. Studies of cachexia have identified layers of underlying mechanisms that are related to energy intake, energy expenditure, metabolic dysregulation, and systemic inflammation (1-3). Although significant resources are currently being directed toward the development of pharmacological treatments for cachexia, sarcopenia, and muscle wasting (4-6), the development of highly effective therapies to treat these syndromes has proven to be difficult and no standardized treatment has been established. Although a strong rationale exists for the utilization of exercise, it is still viewed as a novel approach in the treatment of cancer cachexia (5). At this time, there is relatively little empirical evidence to determine the safety and efficacy of exercise interventions in patients with cancer cachexia (7), and questions remain as to the motivation or ability of cachectic patients to engage in exercise (8). A number of preclinical studies have been conducted that support the use of exercise in this context, yet much remains unclear regarding the effects of exercise on muscle mass and function in models of cachexia, particularly the effects of varying exercise modalities.
The American College of Sports Medicine’s Roundtable on Exercise and Cancer Prevention and Control recently released their updated recommendations for cancer survivors, and these recommendations include the incorporation of multiple exercise modalities (9). Others, including our group, have built upon these recommendations and provided more specific exercise intervention guidelines that include both aerobic and resistance exercise (10). Alone, both aerobic (11, 12) and resistance (13) exercise have been shown to attenuate the effects of cancer-induced cachexia in preclinical models. Rodent treadmill running studies have suggested that aerobic exercise may preserve muscle mass or function in cachectic models through a number of potential pathways including mitochondrial regulatory proteins (e.g., PCG-1α, Mfn1) (14, 15), increased ribosomal biogenesis and protein translation (e.g., mTOR) (16, 17), the ubiquitin-proteasome pathway (12), or myofiber degeneration/regeneration processes (18). While overlap in the protective mechanisms provided by these two training modalities could exist, there may be unique protective pathways provided by resistance training including myogenic regulators (e.g., myogenin, IGF-I) (14) and oxidative stress (13).
A common theme with clinically diagnosed cancer cachexia, as well as in preclinical models, is systemic inflammation, which can be regulated by interleukin-6 (IL-6). In models of cancer cachexia, this pleiotropic cytokine has been linked to the onset of cachexia (2, 19), it is related to the degree of muscle protein degradation (20), its over-expression accelerates the loss of muscle and fat (21), and its inhibition prevents muscle wasting (22, 23). In contrast to these actions, IL-6 also has anti-inflammatory effects (24) and has been shown to play a role in counteracting muscle wasting in cancer cachexia. Reports suggest that IL-6 stimulates muscle satellite cell proliferation, fusion, and differentiation (25, 26), and in IL-6 knockout mice there is a blunted hypertrophic response to muscular overload (27). Many questions remain as to the duality of IL-6 function in models of cachexia and exercise training.
Currently, there is limited information comparing the individual and collective effects of aerobic and resistance exercise in the setting of cancer cachexia. Therefore, the aim of this investigation was to examine the effects of treadmill running, resistance training, or a combination of the two on a range of muscle characteristics in a well-established model of cancer cachexia and to determine if any observed protective effects were associated with changes in markers of systemic inflammation.
Materials and Methods
Ethical approval. Experiments described in this study were completed at the University of Northern Colorado Animal Research Facility. All procedures were approved by the University of Northern Colorado Institutional Animal Care and Use Committee (IACUC) and were in compliance with the Animal Welfare Act guidelines.
Subjects. Sixty male Balb/c mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA; stock #000651) and housed individually in a temperature-controlled facility with a 12:12-h light-dark cycle. Mice were given two weeks to acclimate to the facility prior to beginning any experiments. Standard chow and water were provided ad libitum throughout the study.
Study design. At six weeks of age (n=60), Balb/c mice were randomly assigned to sedentary (SED) or exercise (EX) groups. SED mice were housed individually and were restricted to cage activity alone. Mice selected to EX groups were further randomized into a treadmill running (TM, n=12), resistance training (RT, n=12), or combination exercise (TM+RT, n=12) group. After five weeks of exercise (11 weeks of age), all mice in EX groups were inoculated with C26 carcinoma cells. SED mice were randomly assigned to control (SED+CON, n=12) or tumor-bearing groups (SED+C26 n=12). SED+CON mice received a sham injection of 0.9% saline and SED+C26 mice were inoculated with C26 carcinoma cells. Three weeks after inoculation or sham injection, mice were sacrificed (14 weeks of age). At the time of sacrifice, the gastrocnemius was harvested, weighed, and prepared for histology, while the spleen was harvested and weighed. Blood was collected via left ventricular puncture, centrifuged for the collection of plasma, and stored at –80°C until analysis. Investigators conducting grip strength trials, histology fiber typing, and plasma IL-6 assays were blinded to the animal group assignments.
Exercise training protocol. Mice in the RT group were placed in cages that allow for a progressive elevation of the cage lid, requiring animals to rise to a bipedal stance each time they eat or drink. Cage height was incrementally raised using specially designed plastic spacers positioned between the standard cage and the standard cage lid. Placement of the spacers between the cage and the lid raises food and water to the desired height.This model has been used in rodents and has been shown to increase muscle mass, tibial cortical bone, and peak twitch force, while decreasing muscular fatigue (28-30). Mice were initially housed in standard mouse cages with a cage height of 12.5 cm. On day one of the exercise protocol, cage height was raised to 15.5 cm, where it remained for one week. Following the first week of the RT protocol, cage height was then increased by 2.5 cm, resulting in a total cage height of 18 cm. This final cage height was maintained for the remainder of the RT intervention. Food consumption, water consumption, and body mass were monitored daily to ensure animals were able reach both food and water.
Mice in the TM group were housed in standard cages for the entirety of the eight-week intervention. Animals were acclimated to the treadmill for 3 days prior to the beginning of the intervention. Acclimatization consisted of running on the treadmill at gradually increased speeds ranging between 10-15 m/min for 20 min. The running protocol for the intervention consisted of a brief 5-min warm-up at a speed of 10 m/min and 5% grade, followed by 55-min of running at 15 m/min and 5% grade. Mice ran during their dark cycle, under red lights, 5 days/week, for a total of 8 weeks.
The combination group (TM+RT) concurrently completed both the RT and TM protocols as described above. Mice in the combination group were continuously housed in resistance training cages for the entire 8-week intervention and were removed from their cages for treadmill running 5 days/week during this same 8-week intervention.
Colon-26 cell culture and inoculation. Colon-26 cells (DCTD Tumor Repository, National Cancer Institute; Frederick, MD, USA) were grown in Roswell Park Memorial Institute (RPMI) 1640 complete growth medium, supplemented with 10% FB Essence, 1% L-glutamine, and 1% streptomycin/penicillin. Cells were maintained in an incubator at 37°C in a humidified atmosphere of 5% CO2. Cells were counted using a commercially available cell counter (Invitrogen, Carlsbad, CA, USA). On the day of implantation, adherent cells were dissociated with a trypsin-EDTA solution and total of 1×106 cells were resuspended in sterile phosphate buffered saline (PBS) for tumor cell inoculation. Prior to injection, mice were anesthetized using isoflurane by inhalation, and maintained under anesthesia during the time necessary to perform the tumor inoculation. Cells were injected subcutaneously, dorsally, between the scapulae (31-33). Animals and tumors were monitored daily for end point criteria, including relative tumor mass, significant loss in body mass, body score condition, and tumor ulceration.
In vivo skeletal muscle function. Since muscular weakness is a hallmark of cachexia, in vivo skeletal muscle function was measured using a commercially available grip strength meter (Columbus Instruments, Columbus, OH, USA) as described by others (34, 35). Forelimb grip strength was measured as mice grasped on to a metal grid attached to a force transducer. After attaining a firm, secure grasp on the grid, the mouse was gently pulled away, by the distal end of the tail, in a slow and steady manner until the mouse released its grip on the metal grid. All forelimb grip trials were performed by the same researcher. Grip strength was quantified as the force produced at the time of release from the grid for each trial. After each trial, mice were returned to their cage for a 5-min recovery period. Each mouse completed a total of five trials. Grip strength for each animal was calculated as the mean score of the five trials.
Tissue collection. At the time of sacrifice, animals were euthanized via lethal injection using heparinized (500 U) sodium pentobarbital (50 mg/kg), followed by cervical dislocation. Tumor and spleen tissues were immediately excised and weighed prior to preparation and storage. The gastrocnemius was removed and placed in optimal cutting temperature compound, then frozen in isopentane cooled in liquid nitrogen. Tumor and spleen samples were flash frozen in liquid nitrogen and stored at –80°C until further biochemical analysis.
Histochemical analyses. Transverse sections from the mid-belly region of the gastrocnemius were sectioned at 10 μm on a cryostat (Leica; CM1950) at –20°C. Hematoxylin and eosin staining was completed on muscle sections to measure cross-sectional area (CSA). Digital images were obtained (Olympus DP21) and imaging software (ImageJ, NIH, Bethesda, MD, USA) was used to trace muscle fiber borders. The number of surface area pixels traced was converted to μm2 based on a calibrated known standardized area. Immunofluorescence was used to determine skeletal muscle fiber type via identification of myosin heavy chain IIA (SC-71) and IIB (BF-F3) expression. Myosin heavy chain (MHC) primary antibodies were purchased from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA, USA). Muscle sections were air dried and placed in a blocking solution containing 1% bovine serum albumin, 1% dry milk, 1% Triton-X, and 10% natural goat serum for one hour at room temperature and then incubated with MHC primary antibodies (IIa, 1:600; IIb, 1:100) for two hours at room temperature. Muscle sections were then rinsed in PBS 3×5 min and then incubated with a secondary antibody containing Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA, USA; 1:500) for one hour at room temperature. Digital images (Leica Microsystems DM1RB) at 40× magnification were taken and MHC types IIA and IIB were visualized and measured using imaging software (Image J; NIH).
Biochemical analysis. At the time of sacrifice, blood was quickly collected via left ventricular puncture and centrifuged at 1,000×g for 10 min at 4°C. Plasma was aliquoted and stored at -80°C until further analysis. Plasma IL-6 was analyzed via enzyme-linked immunosorbent assay according to the manufacturer’s instructions (Abcam, ab100712, Cambridge, MA, USA).
Statistical analyses. Statistical analyses were completed using GraphPad Prism Software 8 (La Jolla, CA, USA). All data are presented as means±standard deviation (M±SD). For body mass and gastrocnemius wet mass a one-way analysis of variation (ANOVA) was performed to identify differences between SED, SED+C26, TM+C26, RT+C26, and TM+RT+C26 groups. For CSA, a one-way ANOVA was performed to identify differences in gastrocnemius myofiber size between groups. Additionally, a one-way ANOVA was performed to identify any differences in IL-6 concentrations between SED and EX groups. For ANOVA analyses detecting significant differences (p<0.05), a Tukey’s post hoc test was performed to identify where significant differences occurred. The alpha level was set at 0.05.
Results
General observations. All animals acclimated well to treadmill running prior to onset of the endurance training protocol and no animals were removed from the study for exercise noncompliance. Chow consumption was measured to ensure that mice were able to reach food in the raised lid cages, and to ensure that body/muscle wasting did not occur due to reduced food intake. All RT animals were able to reach food and water throughout the study, and no significant differences in food consumption were observed between any of the experimental groups (data not shown). All animals inoculated with C26 carcinoma cells developed tumors and none of the exercise interventions significantly affected tumor mass when compared to SED+C26 (Table I), which is consistent with other published reports (32). Heart and liver masses were significantly reduced in SED+C26 versus SED+CON (p<0.05), while no significant changes existed in soleus mass across all groups.
Body mass changes.
Skeletal muscle function. Grip strength was measured in all animals prior to sacrifice in order to assess any loss of muscle function that may be attributable to cachexia and any potential for exercise to attenuate that loss (Figure 1). At the time of sacrifice, SED+C26 grip strength was significantly lower (p<0.05) when compared to SED+CON. Grip strength for TM+C26 and TM+RT+C26 was not significantly different when compared to SED+CON, suggesting an attenuation of grip strength loss in these groups. Furthermore, RT+C26 grip strength was significantly higher than SED+C26 (p<0.05) and was not significantly different (p>0.05) when compared to SED+CON. Thus, sedentary C26 tumor-bearing animals displayed muscular weakness indicative of cachexia and moderate exercise interventions attenuated this decline in muscle function.
Grip strength. SED: Sedentary; CON: control; TM: treadmill trained; RT: resistance trained. Data are presented as means±standard deviation. *p<0.01 versus SED+CON; **p<0.01 versus SED+C26; †p<0.05 versus TM+C26, TM+RT+C26.
Gastrocnemius mass and cross-sectional area. Relative gastrocnemius mass was significantly lower (p<0.05) in SED+C26 when compared to SED+CON (Figure 2), while no differences were observed between SED+CON and all EX groups (TM+C26, RT+C26, TM+RT+C26). Histological analysis revealed that gastrocnemius CSA was also significantly lower (p<0.01) in SED+C26 when compared to SED+CON. Similar to what was observed with muscle mass, muscle CSA for all EX groups (TM+C26, RT+C26, and TM+RT+C26) was not significantly different from SED+CON. Furthermore, for all EX groups (TM+C26, RT+C26, and TM+RT+C26), gastrocnemius CSA was significantly larger (p<0.01) than SED+C26. Fiber type analysis demonstrated that there was an equal distribution of MCHIIa and MCHIIb CSA in the gastrocnemius of SED+CON, and that there was a significant reduction (p<0.05) in CSA for both fiber types in C26 tumor-bearing mice (Table II). All exercise interventions prevented atrophy in both fiber types, and CSA was significantly larger (p<0.05) in TM+C26, RT+C26, and TM+RT+C26 when compared to SED+CON. Collectively, these data indicate that tumor burden resulted in a significant decrease in gastrocnemius muscle mass, total gastrocnemius CSA, and type IIa/IIb CSA, and that all of the exercise interventions were able to attenuate these declines.
Gastrocnemius mass and CSA. CSA: Cross sectional area; SED: sedentary; CON: control; TM: treadmill trained; RT: resistance trained. Data are presented as means±standard deviation. (A) Relative gastrocnemius mass; *p<0.05 versus SED+CON. (B) Gastrocnemius CSA; *p<0.01 versus SED+CON, **p<0.01 versus SED+C26.
Cross sectional area of myosin heavy chain IIa and IIb fibers.
Systemic inflammation. During infection and other inflammatory challenges, including cancer, there is widespread systemic inflammation, and this is accompanied by splenomegaly and an increase in systemic IL-6 (36). Both spleen mass and plasma IL-6 followed the same general response under each of the experimental conditions – a significant increase in sedentary tumor bearing animals and normalization of these variables with all exercise interventions (Figure 3). Furthermore, mean values from all EX groups were significantly lower (p<0.01) than mean values from SED+C26, and not significantly different from SED+CON. These data indicate that C26 tumor burden is associated with significant systemic inflammation and that multiple moderate exercise modalities are capable of attenuating this response.
Indicators of systemic inflammation. SED: Sedentary; CON: control; TM: treadmill trained; RT: resistance trained. Data are presented as means±standard deviation. (A) Splenomegaly and (B) plasma IL-6 concentration. *p<0.01 versus SED+CON; **p<0.01 versus SED+C26.
Discussion
The complex nature of cancer cachexia is well recognized, and the criteria for defining this condition continues to evolve and expand (37). The critical need to establish effective strategies in the treatment of cancer cachexia is highlighted by data showing that it reduces tolerance to therapies, it is associated with a poor prognosis, and it correlates with increased rates of morbidity and mortality (27). Cancer cachexia has been reported to occur in 50-80% patients with advanced cancers (38), and conservative estimates are that approximately 23% of cancer patients will have cachexia at some time after their cancer diagnosis (39). Considering the diversity of mechanisms that are involved with the onset and progression of cachexia, many clinicians and researchers support a multimodal treatment strategy that includes medications and physical activity (40, 41). Unlike many targeted pharmacological interventions, chronic exercise provides a broad range of effects that reach far into multiple regulatory pathways, including those regulatory pathways of muscle mass and systemic inflammation. Current exercise recommendations for cancer survivors include both aerobic and resistance activities as key components for individualized prescribed interventions (9, 10), and this study sought to determine if such combined exercise training provides any protective benefits against cancer cachexia.
Mice bearing the C26 carcinoma represent a well-established model of muscle wasting conditions that has been used to investigate the effects of exercise on cancer cachexia (17). In the present study, cancer-induced cachexia was indeed confirmed in sedentary mice inoculated with C26 cells as evidenced by significant losses in body mass, gastrocnemius mass, gastrocnemius cross-sectional area, and forelimb grip strength, along with significant increases in spleen mass and circulating IL-6. In contrast, there were no significant differences in any of these variables when comparing SED+CON with all of the exercise groups (TM+C26, RT+C26, TM+RT+C26), indicating that exercise attenuated the effects of cachexia on skeletal muscle and systemic inflammation. Except for grip strength, there were no differences in the degree of protection provided by each of the exercise interventions, which suggests that equivalent benefits are provided by aerobic exercise, resistance exercise, and combined aerobic and resistance exercise in this model. It is possible that the similarities in the exercise responses are related to the fact that each of the exercise interventions could be categorized as moderate intensity.
Muscle wasting that occurs in the C26 model has long been believed to occur primarily through a hypercatabolic state that causes a reduction in muscle mass and cross-sectional area (42-44). The C26 model is associated with sarcomere misalignment, poor definition of contractile filaments, and a reduction in fiber cross-sectional area in both glycolytic and oxidative muscle fibers (45). The mouse gastrocnemius muscle has a mixed phenotype, yet over 80% of the fibers are classified as MHC type II (IIa+IIb) (46), which are typically larger, faster contracting fibers that are highly dependent on glycolytic pathways for energy production. We show here that atrophy of both IIa and IIb fibers in the gastrocnemius occurs in C26 tumor-bearing mice. Furthermore, aerobic exercise alone, resistance exercise alone, and a combination of aerobic and resistance exercise all protected against this atrophic response with the greatest effect observed as a result of combined exercise (TM+RT+C26>RT+C26>TM+C26). It is unlikely that these observations are affected by any type I→II fiber shifting since gastrocnemius muscles in mice show no shifting between type I and type II fibers as a result of C26 tumor cell inoculation (46) or as a result of chronic activity (47). Collectively, these data suggest that there was no preferential wasting of either fiber type and that multiple moderate exercise modalities are capable of preventing cachexia-induced atrophy of both MHCIIa and MHCIIb fibers in gastrocnemius muscle (48).
A generalized systemic inflammatory state is also a characteristic of cancer cachexia, which relies on complex signaling pathways of multiple cytokines. Of these cytokines, IL-6 has received a bulk of the attention due to its strong connection to the regulation of body mass and its value as a prognostic indicator of weight loss (49). IL-6 has been described as a “bridge” between chronic inflammation and malignant tissues, which maintains a protumorigenic environment that facilitates tumor growth and development (50). In contrast, chronic physical activity induces an anti-inflammatory response, and this is believed to be the result of muscle-derived IL-6, which favorably regulates protein metabolism, inhibits the production of proinflammatory cytokines such as IL-1 and tumor necrosis factor α, and stimulates the production of anti-inflammatory cytokines such as IL-1 receptor agonist and IL-10. It has recently been demonstrated that exercise-induced lactate production directly activates endogenous proteases that in turn facilitate the release of IL-6 from intramyocellular vesicles (51). These studies showed a high correlation between lactate and IL-6 across a wide range of lactate concentrations. Furthermore, both moderate aerobic (52) and resistance exercise (53) bouts have been shown to significantly increase plasma lactate in mice, supporting the possibility that the exercise interventions we used here provided adequate stimuli for IL-6 release. However, others have shown that even short-term low-intensity aerobic exercise training conducted below the lactate threshold (~2 mM), can still confer protective benefits against cancer cachexia by suppressing the ubiquitin-proteasome pathway, increasing hypoxia-inducible factor-1α, and phosphorylating AMPK independently of suppression of proinflammatory cytokines (12). Key distinctions between pro- and anti-inflammatory actions of IL-6 are likely peak circulating concentrations and the time course of release. A primary feature of IL-6 in the development of cancer cachexia is its chronic elevation under resting conditions that can be maintained for weeks or months, whereas exercise-induced IL-6 release is intermittent and peak values are much higher. In the C26 model we show that all of the experimental exercise interventions significantly attenuated resting levels of plasma IL-6 and spleen mass, which are indicative of a blunting of the systemic inflammatory response associated with cancer cachexia.
To our knowledge, only one other study has examined the effects of combined aerobic and resistance exercise in a mouse 4T1 tumor model of cancer cachexia (54). In their studies, combined training attenuated the decline in gastrocnemius mass, preserved grip strength, and reduced the degree of splenomegaly. These protective attributes of exercise were associated with a reduction in muscle autophagy and improved mitochondrial function. Other studies in tumor-bearing models have also shown that autophagy is down-regulated with resistance and endurance training in skeletal (55) and cardiac muscle (56). Metabolic dysfunction related to mitochondrial degeneration has been attributed to cancer cachexia, and it appears that this dysfunction occurs prior to the onset of cachectic muscle loss (57). While combined aerobic and resistance exercise did not upregulate the expression of indicators of mitochondrial biogenesis (PGC-1α) or abundance (cytochrome c), it was shown to attenuate the reduction in mitochondrial enzymatic activity (54), suggesting a preservation of mitochondrial function. Our results compliment these findings by showing that combined exercise interventions can also significantly attenuate chronic systemic inflammation and that there is uniform atrophy and preservation across fiber types in a type II dominant muscle. From these, and other studies, it is clear that there are a spectrum of potential benefits provided by physical activity in combating the deleterious effects of cancer cachexia.
In a study comparing the effects of aerobic versus resistance training in 4T1 tumor-bearing mice, Khamoui et al. reported that there was no convincing evidence to confirm superior benefits of either exercise modality individually (17). Data presented here are in support of their observation that both aerobic and resistance exercise performed at a moderate intensity provide roughly equivalent benefits, and that combining these exercise modalities does not provide additive or synergistic benefits at least in terms of indicators of cancer cachexia. It is important to note that these experiments focused solely on cancer cachexia, whereas in most clinical scenarios these effects on skeletal muscle are likely exacerbated by standard cancer treatments (58). Overall, our data further support a growing body of evidence indicating that combined exercise interventions should be a primary component of cancer care for all survivors, including those with cancer cachexia.
Footnotes
Authors’ Contributions
NRW, JMH, and RH contributed to the conception and design of the research; NRW, JG, AM acquired data; NRW, JMH, and RH analyzed and interpreted data as well as drafted the manuscript. All Authors contributed to editing and revising the manuscript.
Conflicts of Interest
The Authors report no competing interests in relation to this study.
- Received October 27, 2021.
- Revision received November 17, 2021.
- Accepted November 18, 2021.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
