Loss of skeletal muscle mass and function (cachexia) is severe in patients with colorectal liver metastases because of the large increase in resting energy expenditure but remains understudied because of a lack of suitable preclinical models. Our aim was to characterize a novel preclinical model of cachexia in colorectal liver metastases. We tested the hypothesis that mice with colorectal liver metastases would exhibit cachexia, as evidenced by a reduction in liver-free body mass, muscle mass, and physiological impairment. Twelve-week-old male CBA mice received an intrasplenic injection of Ringer solution (sham) or murine colorectal cancer cells (MoCR) to induce colorectal liver metastases. At end-point (20–29 days), the livers of MoCR mice were infiltrated completely with metastases, and MoCR mice had reduced liver-free body mass, muscle mass, and epididymal fat mass compared with sham controls (P < 0.03). MoCR mice exhibited impaired rotarod performance and grip strength (P < 0.03). Histochemical analyses of tibialis anterior muscles from MoCR mice revealed muscle fiber atrophy and reduced oxidative enzyme activity (P < 0.001). Adipose tissue remodeling was evident in MoCR mice, with reduced adipocyte diameter and greater infiltration of nonadipocyte tissue (P < 0.05). These findings reveal the MoCR mouse model exhibits significant cachexia and is a suitable preclinical model of cachexia in colorectal liver metastases. This model should be used for identifying effective treatments for cachexia to improve quality of life and reduce mortality in patients with colorectal liver metastases.
- muscle wasting
- cancer cachexia
- muscle weakness
- colorectal liver metastases
cancer cachexia is defined as a multifactorial syndrome characterized by an ongoing loss of skeletal muscle mass (with or without loss of fat mass) that cannot be fully reversed by conventional nutritional support, leading to progressive functional impairment (8). Cachexia occurs in up to 80% of patients with advanced cancers of the gastrointestinal tract, colon, lung, breast, sarcoma, and prostate (33). It is also present early in the progression of gastrointestinal, pancreatic, and lung cancers (33). Cachexia reduces mobility and functional independence and causes severe fatigue, which together reduce overall quality of life (26). Cachexia can also increase the risk of postoperative complications, impair the response to antineoplastic treatments, and result in the eventual failure of respiratory and cardiac muscle function that causes 20–30% of all cancer-related deaths (33). Unfortunately, there is no Food and Drug Administration-approved treatment for cancer cachexia, and this is due, at least in part, to a lack of suitable preclinical models that closely mimic the human condition for maximizing translational outcomes (24).
Colorectal cancer is the third most common cancer worldwide and is the fourth-most common cause of cancer-related death (9). The most frequent complication of colorectal cancer is the development of liver metastases, which occurs in 70% of cases and is the main cause of death in colorectal cancer patients (30). Only a small minority (∼10%) of patients with colorectal liver metastases are considered candidates for resection, which may offer a possible cure (30). For the remaining ∼90% of patients, liver metastases are unresectable, and chemotherapy is the main alternative. Cachexia is particularly prevalent and severe in patients with colorectal liver metastases due to the very high-energy demands of the liver. In the healthy state, liver metabolism represents ∼20% of whole body resting energy expenditure (REE) (7). However, REE increases in proportion to liver size and the large increase in liver mass due to metastases results in a greatly elevated REE (19). A prospective study of patients with colorectal liver metastases found that a 1-kg increase in liver mass (including metastases) resulted in a 343-kcal increase in REE (19). The study also showed an accelerated loss of skeletal muscle and adipose tissue mass and an accelerated gain of liver mass in patients with colorectal liver metastases (19).
Treatments are needed urgently to counteract cachexia in patients with unresectable colorectal liver metastases, to improve their quality of life, enhance their response to chemotherapy, and to reduce mortality. Potential treatments need to be tested first in preclinical models that closely mimic the human condition but despite the availability of well-characterized preclinical models of nonmetastatic colorectal cancer, such as the colon-26 (C-26) tumor-bearing mouse (24), no preclinical model of cachexia in colorectal liver metastases has been described. It is imperative that a model of cachexia in metastatic colorectal cancer is identified, as the etiology and response to potential treatments are likely to be different to that in models of nonmetastatic colorectal cancer. The aim of this study was to characterize the cachexia in our well-described mouse model of colorectal liver metastases (17, 18, 27). We tested the hypothesis that mice with colorectal liver metastases would exhibit cachexia, with reductions in liver-free body mass and muscle mass and impairments in whole body function, and, therefore, represent the first validated preclinical model of cachexia in colorectal liver metastases.
MATERIALS AND METHODS
All experiments were approved by the Animal Ethics Committee of The University of Melbourne and conducted in accordance with the Australian Code of Practice for the care and use of animals for scientific purposes, as stipulated by the National Health and Medical Research Council (Australia). Twelve-week-old male CBA mice (Laboratory Animal Services, The University of Adelaide, South Australia) were allocated randomly into one of two experimental groups: a sham control group (sham; n = 12) or a colorectal liver metastases group (MoCR; n = 12). All mice were housed in the Biological Research Facility at The University of Melbourne under a 12:12-h light-dark cycle. Water was available ad libitum, and both water and standard laboratory chow were provided, changed, and monitored daily. The amount of food consumed per mouse per day was determined and expressed as cumulative food intake, and the amount of water consumed per mouse per day was determined and expressed as cumulative water intake.
Mouse model of colorectal liver metastases.
The mouse model of colorectal liver metastases was that described by Kuruppu et al. (17). A dimethyl-hydrazine-induced primary colon carcinoma was maintained by in vivo serial passages in the flanks of 10–12-wk-old male CBA mice. Tumors were removed from passage mice and used to make a tumor cell suspension (1 × 106 cells/ml in Ringer solution with 0.1% glucose). Three cohorts [n = 4/cohort for a total of n = 12 murine colorectal cancer cell (MoCR) mice] of 12-wk-old mice were injected with MoCR cells that had been passaged once (cohort 1), twice (cohort 2), or four times (cohort 3). For tumor induction, mice were anesthetized with an intraperitoneal injection of a mixture of ketamine (100 mg/kg body mass) and xylazine (10 mg/kg; VM Supplies, Chelsea Heights, Victoria, Australia), such that they were unresponsive to tactile stimuli. Preoperative carprofen (5 mg/kg; Lyppard Australia, Keysborough, Victoria, Australia) was injected subcutaneously in the nape for pain relief. Mice were shaved on their side, and the spleen was exteriorized through a subcostal incision. Two hemostatic clips were applied adjacent to each other across the center of the spleen. Tumor cell suspension (0.05 ml) was slowly injected into the lateral portion of the spleen using a 25-gauge needle over a period of 1 min. The needle was retracted, and even pressure was applied to the spleen for 2 min. A hemostatic clip was then applied across the splenic vessels supplying the section of spleen injected, and a portable cautery was used to cauterize the splenic vessel following which a partial splenectomy was performed. The muscle and skin were sutured, and mice were given a subcutaneous injection of atipamezole (Antisedan; 1 mg/kg; VM Supplies) to partially reverse the effects of xylazine and to promote more rapid recovery from sedation. Mice recovered on a heated pad until fully conscious. This model has been characterized previously, and results in metastases exclusively confined to the liver (17). Liver angiogenesis is established by day 10, followed by an exponential growth of tumors between days 10 and 16, and a plateau from days 19 to 22. Sham mice had a 0.05-ml injection of Ringer solution with 0.1% glucose and a partial splenectomy.
Criteria for humane end point.
End point analyses were conducted if any of the following criteria were met: a decrease of >20% weight loss from the start of the experiment; a body condition score (BCS) of <2 as described previously (10); infection of the wound; or changes in respiration, vocalization, and mobility.
Grip strength and rotarod test.
Whole body strength and whole body mobility and coordination were assessed 1 day before end point analyses by means of a grip strength meter (Columbus Instruments, Columbus, OH) and rotarod performance test (Rotamex-5, Columbus Instruments), as described in detail previously (24). Grip strength was assessed five times within 2 min, and the average grip strength was normalized to body mass. Rotarod performance was assessed three times with 15 min between tests, and average latency-to-fall was calculated over the three trials.
On the day of end point analyses, mice were anesthetized with an injection of 0.9% HEPES-buffered pentobarbital sodium (Nembutal; 120 mg/kg; Sigma-Aldrich, Castle Hill, NSW, Australia) via intraperitoneal injection. Supplemental injections of unbuffered Nembutal (60 mg/kg) were administered to maintain unresponsiveness to tactile stimuli. The tibialis anterior (TA), extensor digitorum longus (EDL), soleus, plantaris, gastrocnemius, and quadriceps muscles, as well as the epididymal fat, heart, spleen, and kidneys, were carefully excised, blotted on filter paper, and weighed on an analytical balance. TA muscles were mounted in an embedding medium and frozen in thawing isopentane for later histochemical and biochemical analyses. The TA muscle was chosen because 1) it is of sufficient mass and cross-sectional area (CSA) for conducting these analyses, 2) we have found that it is susceptible to atrophy and functional impairment in the C-26 (24) and LLC tumor-bearing models (23), and 3) we have previously investigated histochemical and biochemical properties of this muscle in the C-26 (24) and LLC tumor-bearing models (23). Although it would be of interest to also examine histochemical and biochemical properties in muscles predominantly composed of type I fibers, such as in the soleus muscle, the small mass of the soleus (5–6 mg) precludes its use in these analyses. Mice were killed as a consequence of heart excision, while still anesthetized deeply.
Skeletal muscle histology.
Serial sections (5 μm) were cut transversely through the TA muscle using a refrigerated (−20°C) cryostat (CTI Cryostat; IEC, Needham Heights, MA). Sections were stained (or reacted) with hematoxylin and eosin (H&E) to determine general muscle architecture, laminin (no. L9393; Sigma-Aldrich) for determination of mean myofiber CSA, succinate dehydrogenase (SDH) to determine activity of oxidative enzymes, and N2.261 (developed by Dr. Helen M. Blau, obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biology, Iowa City, IA) to assess the percentage of myosin IIa isoforms (25). We have shown previously that mouse TA muscle contains a virtual absence of type I fibers (22), and so all non-N2.261 reacting fibers were assumed to represent type IIx/b fibers. Optical density (OD) of SDH was determined after 6 min of reactivity for all samples, and sections were captured in full color using bright-field light microscopy and analyzed, as described previously (25). Digital images were obtained using an upright microscope with camera (Axio Imager D1, Carl Zeiss, Wrek, Göttingen, Germany), controlled and quantified by AxioVision AC software (AxioVision AC Rel. 4.7.1, Carl Zeiss).
Epididymal fat was immersed in Bouin's solution overnight and then transferred to 70% ethanol. Tissues were fixed and embedded in paraffin wax in a random orientation, and 10-μm sections were cut using a cryostat (CTI Cryostat). Sections were stained with H&E, imaged using an upright microscope and camera (Axio Imager F1), and quantified by AxioVision AC software. For each sample, diameter was determined in 251 ± 35 adipocytes (obtained from three independent sections of tissue).
Real-time RT-PCR analyses.
Total RNA was extracted from 10–20 mg of TA muscle using a commercially available kit, according to the manufacturer's instructions (PureLink RNA mini kit; Invitrogen). RNA concentration was determined spectrophotometrically at 260 nm, and the samples were stored at −80°C. RNA was transcribed into cDNA using the Invitrogen SuperScript VILO cDNA synthesis kit, and the resulting cDNA stored at −20°C for subsequent analysis. Real-time RT-PCR was carried out with the Bio-Rad CFX384 Touch real-time PCR detection system (Bio-Rad, Hercules, CA) using the ssoAdvanced SYBR Green Supermix (Bio-Rad). Measurements included a no template control, as well as an RT negative control. Primer sequences for MuRF-1, atrogin-1, IL-6, and TNF-α were as detailed previously (22, 24). The content of single-stranded DNA (ssDNA) in each sample was determined using the Quanti-iT OilGreen ssDNA assay kit (Molecular Probes, Eugene, OR), as described previously (25). Gene expression was quantified by normalizing the logarithmic cycle threshold (CT) value (2−CT) to the cDNA content of each sample to obtain the expression 2−CT/cDNA content (ng/ml).
Western blot analyses.
Western blot analysis was performed as described previously (16, 25). Membranes were incubated overnight at 4°C with the following antibodies (all 1:1,000 in blocking buffer): p-Akt (Ser473; no. 8271; Cell Signaling, Danvers, MA), Akt (no. 9272; Cell Signaling), p-p70S6K (Thr389; no. 9205; Cell Signaling), and p70S6K (no. 9202; Cell Signaling), as detailed elsewhere (16, 25). The signal was imaged using ChemiDoc XRS machine (Bio-Rad), and blots were quantified using Image Lab software (Bio-Rad). A total protein stain (BLOT-FastStain, G-Biosciences, St. Louis, MO) was performed by incubating membranes in Fixer for 3 min at RT, and then in Developer for 1 min at RT followed by 30 min at 4°C. The signal was imaged using ChemiDoc XRS machine, and blots were quantified using Image Lab software.
All values are expressed as means ± SE, unless stated otherwise. Groups were compared using a Student's t-test, a one-way ANOVA, or a two-way ANOVA, where appropriate. Fiber-type proportions are presented as 95% confidence intervals of the mean. Differences were considered significant when no overlap existed between the 95% confidence intervals (31). Bonferroni's post hoc test was used to determine significant differences between individual groups. Correlations were determined by least squares linear regression. The level of significance was set at P < 0.05 for all comparisons.
Body condition score, food intake, body mass, and liver mass in MoCR cohorts.
The body condition score (BCS) assessed the general health and well-being of the mice and was used as a criterion for a humane end point. The BCS of MoCR cohort 1 (mice injected with cells that been passaged once, P1) did not start declining until day 28, and end point analyses were performed on these mice on day 29 (Fig. 1A). In MoCR cohort 2 (mice injected with cells that had been passaged twice, P2), BCS started declining on day 24, and end point analyses were performed on day 26 (Fig. 1A). In MoCR, cohort 3 (mice injected with cells that had been passaged four times, P4), BCS started declining on day 17, and end point analyses were performed on day 20 (Fig. 1A). There was a main effect for the BCS of cohort 3 to be lower than cohort 1 and cohort 2, and for the BCS of cohort 2 to be lower than cohort 1 (P < 0.01, Fig. 1A). The decline in BCS in cohorts 2 and 3 was associated with a plateau in cumulative food intake (Fig. 1B) and relative body mass (Fig. 1C). There was a main effect for food intake of cohort 3 to be lower than cohort 1 and cohort 2, and for food intake of cohort 2 to be lower than cohort 1 (P < 0.01, Fig. 1B). No significant difference between cohorts was found for body mass (Fig. 1C). Despite end point analyses being performed on different days, livers from the three cohorts were ∼95% infiltrated with metastases (Fig. 1D), and there was no significant difference between cohorts in liver mass (P < 0.84, Fig. 1E), indicating that the three MoCR cohorts had a similar extent of metastases. Liver mass in each of the MoCR cohorts was significantly higher than in sham controls (P < 0.01, Fig. 1E). There were also no significant differences among MoCR cohorts in muscle mass (P < 0.98, Fig. 1F). These findings confirm the appropriateness of using BCS in the criteria for a humane end point. Because of the similarity in metastases infiltration, liver mass, and muscle mass, data from the three cohorts were combined for comparison with sham controls, which also comprised three cohorts that had their end point matched to the MoCR cohorts [cohort 1 end point, day 29 (n = 4); cohort 2 end point, day 26 (n = 4); cohort 3 end point (day 20)].
Body mass, body condition score, food and water intake, liver mass, and liver-free body mass in MoCR mice.
Because of the invasive nature of the surgery, relative body mass of sham control mice decreased for the first 5 days after surgery and increased progressively thereafter (Fig. 2A). A similar initial decrease after surgery was seen in the MoCR mice, but despite having an increase in liver mass due to metastases, MoCR mice had lower relative body mass than sham controls from day 10 (Fig. 2A). Sham mice maintained a healthy BCS of 3 over the course of the experiment (data not shown). MoCR mice ate and drank more than sham controls (P < 0.04, Fig. 2, B and C). At end point, MoCR mice had a ∼6.5-fold higher liver mass than controls due to the infiltration of metastases (sham, 1,302 ± 59; n = 12; MoCR, 8,084 ± 852 mg; n = 9; P < 0.001). The increased liver mass was maintained when normalized for initial body mass (P < 0.001, Fig. 2D). Calculation of liver-free body mass revealed that MoCR mice had a 24% reduction in mass compared with sham controls (P < 0.001, Fig. 2E).
Skeletal muscle and fat mass in MoCR mice.
MoCR mice had reduced mass of the soleus (sham, 6.1 ± 0.4; MoCR, 4.8 ± 0.3 mg, P < 0.03), EDL (sham, 12.1 ± 0.5; MoCR, 8.6 ± 0.6 mg, P < 0.001), TA (sham, 44.1 ± 1.1; MoCR, 33.3 ± 1.2 mg, P < 0.001), plantaris (sham, 14.7 ± 0.4; MoCR, 9.1 ± 0.8 mg, P < 0.001), gastrocnemius (sham, 117.7 ± 3.1; MoCR, 91.8 ± 2.6 mg, P < 0.001), and quadriceps muscles (sham, 194.8 ± 5.1; MoCR, 140.8 ± 6.4 mg, P < 0.001) compared with sham controls. When normalized for initial body mass, the lower muscle masses in MoCR mice remained (Fig. 3A). MoCR mice had higher heart mass (sham, 109.9 ± 3.5; MoCR, 136.4 ± 6.6 mg, P < 0.01) and spleen mass (sham, 60.5 ± 3.1; MoCR, 96.2 ± 6.2 mg, P < 0.001) but lower white adipose tissue mass compared with sham controls [white adipose tissue (WAT), sham, 810.1 ± 132.3; MoCR, 92.7 ± 37.3 mg, P < 0.001]. There was no significant difference in kidney mass between groups (sham, 420.8 ± 14.0; MoCR, 429.6 ± 18.9 mg). When normalized for initial body mass, the higher spleen mass and lower WAT mass in the MoCR mice remained (Fig. 3B).
Whole body strength and mobility in MoCR mice.
MoCR mice had a 21% lower average grip strength than sham controls (P < 0.03, Fig. 4A). Latency-to-fall during the rotarod test was 82% lower in MoCR mice compared with sham controls (P < 0.001, Fig. 4B). Impaired mobility has been correlated previously with weight loss in patients with cancer cachexia (13), and so the correlation between rotarod performance and the percentage change in liver-free body mass was investigated. When results for sham and MoCR groups were pooled (n = 17), there was a significant correlation (r = 0.82, P < 0.0001), such that the greater the weight loss, the shorter the latency-to-fall (Fig. 4C).
Muscle fiber size and oxidative enzyme activity in MoCR mice.
TA muscle cross sections were stained with H&E to assess general muscle fiber architecture and reacted for myosin IIa (N2.261, green), laminin (red), and SDH activity (blue) to identify type IIa fibers, visualize all fibers, and indicate oxidative enzyme (SDH) activity, respectively (Fig. 5A). Type I fibers are very rare in mouse TA muscle, so all non-N2.261-reacting fibers were assumed to represent type IIx/b fibers. H&E staining showed that MoCR mice had smaller muscle fibers but similar architecture and extent of nonmuscle tissue as sham controls. There were no differences between groups in the proportion of type IIa and type IIx/b fibers (Fig. 5B) but MoCR mice had a 25% reduction in average fiber CSA (P < 0.001), which was due to atrophy of type IIa (−26%, P < 0.001) and type IIx/b fibers (−25%, P < 0.001, Fig. 5C). Average fiber SDH reaction intensity was also lower in MoCR mice (−19%, P < 0.001), due to lower SDH intensity in type IIa (−18%, P < 0.001) and type IIx/b fibers (−20%, P < 0.001, Fig. 5D).
Remodeling of adipose tissue in MoCR mice.
Examination of cross sections of epididymal fat stained for H&E showed substantial morphological changes in MoCR mice (Fig. 6A). Average adipocyte diameter was 49% smaller in MoCR mice (P < 0.001, Fig. 6B), and a histogram revealed that this was due to a greater proportion of small adipocytes and a lower proportion of large adipocytes (P < 0.05, Fig. 6C). Epididymal fat from MoCR mice also had a 5.6-fold larger area of nonadipocyte tissue (P < 0.01, Fig. 6D).
Pathways involved in protein degradation, inflammation, and protein synthesis in MoCR mice.
To determine the mechanisms involved in the loss of muscle mass in MoCR mice, we assessed the expression of markers involved in muscle protein degradation (MuRF-1 and atrogin-1) and protein synthesis (Akt, p70 S6K) in TA muscles. Because inflammation plays an important role in the pathogenesis of cancer cachexia (2), the expression of inflammatory genes (IL-6 and TNF-α) was also assessed. MuRF-1 and atrogin-1 mRNA expression was onefold and 11-fold higher in MoCR mice compared with sham controls, respectively (P < 0.03, Fig. 7, A and B). IL-6 mRNA expression was twofold higher in MoCR mice compared with controls (P < 0.01, Fig. 7C), but there was no significant difference in TNF-α mRNA expression between groups (Fig. 7D). To examine changes in the protein synthesis pathway, the expression of phosphorylated and total Akt and p70 S6K was assessed (Fig. 8A). There were no significant differences between groups in phosphorylated and total Akt (Fig. 8, B–D), but MoCR mice had a 40% lower expression of total p70 S6K (P < 0.04, Fig. 8F), and a tendency for lower phosphorylated p70 S6K (P < 0.08, Fig. 8E). As a consequence, phosphorylated p70 S6K normalized to total p70 S6K was not different between groups (Fig. 8H).
Cachexia is particularly prevalent and severe in patients with colorectal liver metastases but remains understudied because of the lack of suitable preclinical models. Preclinical models are essential for our understanding of the pathogenesis and relevance of potential therapies. Until now, no preclinical model of cachexia in colorectal liver metastases has been characterized. Because the etiology and response to potential treatments are likely to be very different between models of metastatic and nonmetastatic colorectal cancer, it is essential that a model of cachexia in metastatic colorectal cancer is identified and validated. We report here for the first time a novel preclinical model of cachexia in colorectal liver metastases. In a mouse model of colorectal liver metastases (MoCR), where the intrasplenic injection of murine colorectal cancer cells results in significant liver metastases (17), mice exhibited reductions in body mass, muscle mass, and fat mass, as well as whole body physiological impairments. These changes are consistent with the diagnostic criteria for cachexia (8), and, therefore, confirm the suitability of this model for preclinical studies. Identification of this model represents a significant advance for the translation of preclinical findings and should be used to identify effective treatments for cachexia to improve the quality of life and survival of patients with colorectal liver metastases.
Reproducibility of liver metastases and cachexia in the MoCR model.
Three cohorts of mice were injected with MoCR cells that had been passaged once (P1), twice (P2), or four times (P4). The greater the passage number of injected cells, the faster the deterioration in body condition score and plateau in food intake and body mass. Despite these differences, the infiltration of metastases was similar between cohorts (∼95%). Liver mass and mass of various skeletal muscles were also similar between cohorts, demonstrating the reproducibility of liver metastases and cachexia in the MoCR model. They also show that injection of MoCR cells of differing passages can be used to produce varying speeds of disease progression or when examined at a single time point, can be used to examine different stages of cancer cachexia (i.e., at day 20, P4 cells produce severe cachexia, P2 cells produce cachexia, and P1 cells produce mild cachexia).
Weight loss in the MoCR model of colorectal liver metastases.
The main diagnostic criteria for cachexia in humans is a >5% weight loss (8). Weight loss of >15% is associated with impaired physiological function, and death ensues when weight loss is ∼30% (36). In the present study, liver-free body mass was reduced by 24% in mice with colorectal liver metastases compared with sham controls. In comparison, we have shown previously that severely cachectic mice bearing nonmetastatic subcutaneous colorectal tumors (C-26) lost ∼22% tumor-free body mass (24). Anorexia often but not always accompanies cancer cachexia and can lead to a reduction in nutrient intake of 300–500 kcal/day (32). We have shown previously that severely cachectic mice bearing nonmetastatic C-26 tumors have anorexia (24), but in the current study, when results for the three MoCR cohorts of mice with colorectal liver metastases were combined, anorexia was not observed, and they actually ate and drank more than controls. Previous studies in patients with colorectal liver metastases have shown that despite cachectic patients exhibiting deterioration through depression and physical symptoms, they do not eat less than noncachectic patients (12). These findings reveal that cachexia in mice with colorectal liver metastases is not due to anorexia. Since nutritional supplementation and pharmacological modulation of appetite do not enhance lean muscle mass, these findings are also consistent with the notion that anorexia is not the sole contributor to cancer cachexia (36). The small increase in food intake with the loss of body mass indicates that the MoCR mice may have increased energy expenditure. Although we were unable to directly assess energy expenditure in the present study, a prospective study of patients with colorectal liver metastases found that a 1-kg increase in liver mass (including metastases) resulted in a 343-kcal increase in energy expenditure (19). Assuming a similar relationship exists in mice, the 6.8-g increase in liver mass in the MoCR mice equates to a 2.32 kcal/day elevation in energy expenditure. We have shown previously that 15-wk-old mice have an energy expenditure of ∼14.5 kcal/day (24), and so an elevation of 2.32 kcal/day would equate to a 15% increase in energy expenditure. Together with the reductions in body mass and muscle fiber oxidative enzyme activity (SDH intensity), an increase in energy expenditure in the MoCR mice suggests energy insufficiency, and this has recently been shown in a rat model of cancer cachexia (11). Future studies should confirm this by directly measuring energy expenditure.
Muscle wasting in the MoCR model of colorectal liver metastases.
The magnitude of skeletal muscle loss varies considerably in patients with cancer cachexia. A retrospective study of patients with colorectal liver metastases reported an ∼16% loss of estimated whole body muscle mass (∼4.2 kg) measured at 10.7 to 1.2 mo from death (19). In the present study, the muscle masses of varying fiber types were all significantly (17–37%) smaller in MoCR mice compared with sham controls. This is in contrast to severely cachectic C-26 tumor-bearing mice that demonstrated only (19–21%) reductions in the mass of larger muscles and not in the smaller soleus and plantaris muscles (24). In addition to a loss of muscle mass, patients with cancer cachexia exhibit muscle fiber atrophy with one study reporting a 32% smaller muscle fiber size in cachectic patients with gastrointestinal cancer compared with healthy controls (37). Muscle fiber atrophy was also found in the MoCR mice, with a 25% decrease in fiber area, consistent across the fast oxidative type IIa fibers and fast glycolytic type IIx/b fibers. Real-time RT-PCR and Western blot analyses revealed that the muscle atrophy in MoCR mice involved increased expression of protein degradative pathways (MuRF-1 and atrogin-1), increased inflammatory markers (IL-6), and reduced expression of protein synthesis pathways (p70 S6K). Each of these changes has been reported in animal models of cachexia in nonmetastatic cancer (6, 24, 38) and suggest that the loss of muscle mass in MoCR mice may be due to both decreased signaling of the pathways regulating protein synthesis and increased signaling of the pathways regulating protein degradation.
Severe fatigue affects 70–100% of patients with cancer cachexia and is one of the main factors contributing to reduced quality of life (4). Decreased activity of oxidative enzymes is associated with fatigue and has been reported in patients with cancer cachexia (29). MoCR mice had reduced oxidative enzyme activity, as evidenced by a reduction in SDH activity in type IIa and type IIx/b fibers. MoCR mice, therefore, exhibit similar reductions in muscle mass, fiber size, and oxidative enzyme activity to patients with cancer cachexia.
The MoCR model of colorectal liver metastases has physiological impairments.
One of the most debilitating aspects of cancer cachexia is the impairment in whole body strength and mobility. Grip strength is reduced by more than 25% in patients with cancer cachexia (14), affecting their ability to perform everyday tasks such as rising from a chair or bed, performing home duties, and maintaining personal hygiene. The reduction in grip strength in cachectic patients has also been correlated strongly with postoperative complications (14). We have shown previously that both severely cachectic and mildly cachectic mice bearing nonmetastatic C-26 tumors have a 22% reduction in grip strength compared with controls (24), and in the present study, MoCR mice had a 21% reduction in grip strength. Thus, the magnitude of impairment in grip strength in our nonmetastatic C-26 and metastatic MoCR models is very similar to that in cachectic cancer patients and highlights the suitability of these preclinical models.
The impaired mobility in patients with cancer cachexia results in reduced levels of physical activity (5). MoCR mice exhibited a large 82% impairment in mobility, as assessed by rotarod performance. This is consistent with the reduction in mobility that we have reported previously in severely cachectic C-26 tumor-bearing mice (24), and the consistency between models demonstrates that the impaired mobility in the MoCR mice was not simply due to the large tumor burden in the abdomen. The impaired mobility in patients with cancer cachexia has been correlated significantly with weight loss (13), and we also found a significant correlation between weight loss and impaired mobility, highlighting the clinical relevance of the MoCR model.
Fat loss and remodeling in the MoCR model of colorectal liver metastases.
Cachexia is usually accompanied by a loss of fat mass, which often precedes the loss of muscle mass. Similar to the loss in muscle mass, the most rapid loss of fat in patients with colorectal liver metastases occurs within 2 mo of death (19). In addition to the loss of fat mass, patients with cancer cachexia experience adipose tissue remodeling characterized by the shrinkage of adipocytes and an increase in interstitial spaces (21). Similar to humans, MoCR mice had a loss of fat mass, reduced adipocyte size, and an increased proportion of nonadipocyte tissue. The same characteristics are found in severely cachectic C-26 tumor-bearing mice (34).
An interesting observation in the present study was the increase in spleen and heart mass in the MoCR mice. The increase in spleen mass is consistent with patients with colorectal liver metastases (19) and the nonmetastatic C-26 model of cancer cachexia (35). The cardiac hypertrophy is in contrast to the reduced absolute heart mass reported previously in C-26 tumor-bearing mice (24, 35). However, these same studies found that C-26 tumor-bearing mice had greater heart mass normalized to tumor-free body mass, which was attributed to edema (24, 35). Chronic liver disease is commonly associated with portal hypertension, and the increase in portal vein pressure when the splenic vein drains into the portal system can lead to an increase in splenic venous pressure (15). Portal vein hypertension is a well-documented cause of spleen enlargement (20) and cardiac hypertrophy (28) and is the likely cause of the increased heart and spleen mass in MoCR mice.
Perspectives and Significance
Despite cancer cachexia being a devastating condition affecting more than 80% of cancer patients and causing the death of more than 20–30% of all cancer patients, there is currently no treatment. A contributing reason for the lack of progress has been a lack of suitable preclinical models that closely mimic the human condition necessary for maximizing translational outcomes. Cachexia is particularly severe in patients with colorectal liver metastases, but no preclinical model of cachexia in colorectal liver metastases has been identified. We have identified a novel, reproducible mouse model of colorectal liver metastases with cachexia that represents a suitable preclinical model. Several models of cachexia in nonmetastatic colorectal cancer have been characterized, but this is the first model of cachexia in metastatic colorectal cancer. Since liver metastases affects 70% of patients with colorectal cancer, this model represents a significant advance that will enhance the translation of preclinical findings. This model will increase the chances of identifying an effective treatment for cancer cachexia to improve the quality of life, enhance the response to antineoplastic treatments, and reduce mortality in patients with colorectal liver metastases.
This work was supported by a research grant from the Victorian Cancer Agency (Australia). K.T.M. is supported by an R.D. Wright Biomedical Research Fellowship from the National Health and Medical Research Council (Australia).
No conflicts of interest, financial or otherwise, are declared by the authors.
Author contributions: K.T.M., C.C., and G.S.L. conception and design of research; K.T.M., A.S., and C.M.-W. performed experiments; K.T.M. and A.S. analyzed data; K.T.M., A.S., C.C., and G.S.L. interpreted results of experiments; K.T.M. prepared figures; K.T.M. drafted manuscript; K.T.M., C.C., and G.S.L. edited and revised manuscript; K.T.M., A.S., C.M.-W., C.C., and G.S.L. approved final version of manuscript.
We thank Ms. Annabel Chee (Department of Physiology, The University of Melbourne) for assisting with the preparation of the cells. This manuscript is in honor of Cathy Malcontenti-Wilson, one of the authors of this article, who passed away on February 14, 2013.
- Copyright © 2013 the American Physiological Society