Growth of ovarian cancer xenografts causes loss of muscle and bone mass: a new model for the study of cancer cachexia

Abstract

Background: Cachexia frequently occurs in women with advanced ovarian cancer (OC), along with enhanced inflammation. Despite being responsible for one third of all cancer deaths, cachexia is generally under-studied in OC due to a limited number of pre-clinical animal models. We aimed to address this gap by characterizing the cachectic phenotype in a mouse model of OC.

Methods: Nod SCID gamma mice (n = 6-10) were injected intraperitoneally with 1 × 10⁷ ES-2 human OC cells to mimic disseminated abdominal disease. Muscle size and strength, as well as bone morphometry, were assessed. Tumour-derived effects on muscle fibres were investigated in C2C12 myotube cultures. IL-6 levels were detected in serum and ascites from tumour hosts, as well as in tumour sections.

Results: In about 2 weeks, ES-2 cells developed abdominal tumours infiltrating omentum, mesentery, and adjacent organs. The ES-2 tumours caused severe cachexia with marked loss of body weight (-12%, P < 0.01) and ascites accumulation in the peritoneal cavity (4.7 ± 1.5 mL). Skeletal muscles appeared markedly smaller in the tumour-bearing mice (approximately -35%, P < 0.001). Muscle loss was accompanied by fibre atrophy, consistent with reduced muscle cross-sectional area (-34%, P < 0.01) and muscle weakness (-50%, P < 0.001). Body composition assessment by dual-energy X-ray absorptiometry revealed decreased bone mineral density (-8%, P < 0.01) and bone mineral content (-19%, P < 0.01), also consistent with reduced trabecular bone in both femurs and vertebrae, as suggested by micro-CT imaging of bone morphometry. In the ES-2 mouse model, cachexia was also associated with high tumour-derived IL-6 levels in plasma and ascites (26.3 and 279.6 pg/mL, respectively) and with elevated phospho-STAT3 (+274%, P < 0.001), reduced phospho-AKT (-44%, P < 0.001) and decreased mitochondrial proteins, as well as with increased protein ubiquitination (+42%, P < 0.001) and expression of ubiquitin ligases in the skeletal muscle of tumour hosts. Similarly, ES-2 conditioned medium directly induced fibre atrophy in C2C12 mouse myotubes (-16%, P < 0.001), consistent with elevated phospho-STAT3 (+1.4-fold, P < 0.001) and altered mitochondrial homoeostasis and metabolism, while inhibition of the IL-6/STAT3 signalling by means of INCB018424 was sufficient to restore the myotubes size.

Conclusions: Our results suggest that the development of ES-2 OC promotes muscle atrophy in both in vivo and in vitro conditions, accompanied by loss of bone mass, enhanced muscle protein catabolism, abnormal mitochondrial homoeostasis, and elevated IL-6 levels. Therefore, this represents an appropriate model for the study of OC cachexia. Our model will aid in identifying molecular mediators that could be effectively targeted in order to improve muscle wasting associated with OC.

Keywords: Animal model; Cancer cachexia; ES-2; IL-6; Ovarian cancer; Skeletal muscle.

Figure 1

The ES‐2 tumours cause significant body weight (BW) loss. Body weight curves (A) and body weight change (i.e. body weight at time of sacrifice vs. initial body weight) (B) in mice bearing the ES‐2 ovarian cancer (n = 6–10). Data expressed as means ± standard deviation. Significance of the differences: ^*** P < 0.001 vs. Control.

Figure 2

Growth of the ES‐2 ovarian cancer causes marked muscle depletion. Muscle weights in ES‐2 tumour hosts (n = 6–10). Weights were normalized to the initial body weight (IBW) and expressed as weight/100 mg IBW. Data expressed as means ± standard deviation. Significance of the differences: ^*** P < 0.001 vs. Control.

Figure 3

Organ size is affected by the occurrence of ES‐2 ovarian cancer. Liver, spleen, and gonadal fat weights in mice bearing the ES‐2 ovarian cancer (n = 6–10). Weights were normalized to the initial body weight (IBW) and expressed as weight/100 mg IBW. Data expressed as means ± standard deviation. Significance of the differences: ^** P < 0.01, ^*** P < 0.001 vs. Control.

Figure 4

Development of ovarian cancer is accompanied by muscle atrophy and progressive muscle weakness. Muscle morphology (haematoxylin and eosin staining) and quantification of the cross‐sectional area (CSA) in the tibialis anterior muscle of ES‐2 hosts. Scale bar: 100 μm (A). Whole body grip strength in animals bearing the ES‐2 tumours, reported as peak force, was measured weekly by taking advantage of a grip strength metre and expressed as the average of the three top pulls from each animal (n = 6–10) (B). Data expressed as means ± standard deviation. Significance of the differences: ^** P < 0.01, ^*** P < 0.001 vs. Control.

Figure 5

Body composition is affected by the growth of ES‐2 tumours. Body composition was assessed by means of dual‐energy X‐ray absorptiometry scanning of carcasses from ovarian cancer hosts (n = 6–10). Fat and lean tissue, total bone mineral density (BMD), and bone mineral content (BMC) measurements of the whole body are shown. Data (means ± standard deviation) are expressed as g (for fat, lean, and BMC) or g/cm² (for BMD). Significance of the differences: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. Control.

Figure 6

Growth of the ES‐2 tumour affects bone quality. Representative three‐dimensional rendering of microcomputed tomography scan images and quantification of bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), and trabecular connectivity density (Conn.Dn) in the femurs (A) and vertebrae (B) of mice bearing the ES‐2 tumour (n = 6–10). Data are expressed as means ± standard deviation. Significance of the differences: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. Control.

Figure 7

Development of ovarian cancer is accompanied by changes in the expression of key regulators of muscle size. Representative western blotting and quantification for pSTAT3, pAKT, pP38, and pERK1/2 (A) and OPA1, Mitofusin‐2, PGC1α, and Cytochrome C (Cyt C) (B) in whole muscle protein extracts from mice bearing ovarian cancers (n = 6–10). Levels of phosphorylated proteins were normalized to the respective total protein expression. Tubulin was used as loading control. Data are expressed as fold change vs. control and reported as means ± standard deviation. Succinate dehydrogenase (SDH) enzymatic activity (expressed as mU/μL) in quadriceps muscle from control mice and ES‐2 hosts (n = 6–10) (C). Succinate dehydrogenase staining performed on 8 μm thick sections from tibialis anterior muscles excised from controls and ES‐2 (n = 6–10) (D). The number of oxidative (dark purple) and glycolytic (light purple) fibres was assessed and reported as % of total fibres per field (E). Scale bar: 100 μm. Data are expressed as means ± standard deviation. Significance of the differences: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. Control.

Figure 8

Elevated muscle protein catabolism is a hallmark of ovarian cancer cachexia. Representative western blotting and quantification for total ubiquitin (A) in whole muscle protein extracts from mice bearing ovarian cancers (n = 6–10). Tubulin was used as loading control. Gene expression levels for Atrogin‐1, MuRF‐1, Fbxo21, and Fbxo31 ubiquitin ligases (B) were performed by quantitative real‐time PCR. Gene expression was normalized to TBP levels. Data (fold change vs. control) were expressed as means ± standard deviation.

Figure 9

ES‐2‐derived factors directly affect fibre size in C2C12 myotube cultures. Assessment of myofibre size in C2C12 murine myotube cultures exposed for up to 48 h to 25% and 50% ES‐2 unconditioned medium (UCM) or conditioned medium (CM) in 2% horse serum‐containing differentiation medium (n = 250–350). Green staining: myosin heavy chain. Scale bar: 100 μm (A). Representative western blotting for pSTAT3 and STAT3 in whole protein extracts from C2C12 myotubes exposed to different concentrations of ES‐2 CM (n = 3) (B). Representative western blotting and quantification for pSTAT3 (C) and myotubes size (D) in C2C12 myotubes exposed to 50% ES‐2 CM, with or without INCB018424 (400 nM). pSTAT3 levels were normalized to STAT3 expression. Tubulin was used as loading control. ND, not detected. Data (means ± standard deviation) are expressed as fold change vs. UCM. Significance of the differences: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. UCM; ^# P < 0.05 vs. CM.

Figure 10

IL‐6 is elevated in ES‐2 hosts. IL‐6 levels assessed in plasma and ascites from control and ES‐2 bearing mice (n = 6–10) by means of ELISA (A). Ascites was collected at time of euthanasia and subjected to centrifugation to remove cells and cellular debris. Data (pg/mL) are expressed as means ± standard deviation. Immunohistochemistry for IL‐6 in two representative sections from ES‐2 solid tumours infiltrating murine pancreas (n = 10) (B). Normal rabbit IgG was used as negative control. Scale bar: 50 μm.

Figure 11

Mitochondrial homoeostasis and respiration are impaired in C2C12 myotubes exposed to ES‐2 conditioned medium (CM). Representative western blotting and quantification for PGC1α, Cytochrome C (Cyt C), OPA1, and DRP1 in protein extracts from C2C12 myotubes exposed to different concentrations of ES‐2 CM (n = 3). Tubulin was used as loading control (A). Mitochondrial polarization assessed by means of MitoTracker red CMXRos staining in C2C12 exposed to 50% ES‐2 CM (n = 3). Red signal intensity was quantified by using the ImageJ software (five fields per sample). Scale bar: 100 μm (B). Basal respiration (BR), mitochondrial respiration (MR), spare respiratory capacity (SRC), proton leak (PL), and ATP production (data expressed in pmol/min) determined by extracellular flux analysis in C2C12 myotubes exposed to 50% ES‐2 CM (n = 3) (C). Data are reported as means ± standard deviation. Significance of the differences: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. unconditioned medium (UCM).