Introduction

Cachexia and skeletal muscle wasting are highly prevalent in patients with ovarian cancer and are associated with poor disease outcome.1 The mechanisms underlying the development of cachexia in ovarian cancer remain elusive, although several lines of evidence have implicated tumour-derived compounds and their direct and indirect effects on skeletal muscle and adipose tissue.2-5 In particular, skeletal muscle protein metabolism alterations induced by changes in muscle RING-finger protein-1 (MuRF1), Atrogin-1, REDD1 (regulated in development and DNA damage responses 1),6, 7 and/or NF-κB activity8 have been the focus of research.

About one-third of all newly diagnosed ovarian cancer patients and almost all patients with recurrent disease accumulate ascites.9 In the intraperitoneal cavity, small amounts of free fluids are produced by capillary membranes to allow for lubrication of serosal surfaces. Under physiological conditions, the vast majority of these fluids are reabsorbed by the lymphatic system. However, under influence of tumour-derived growth factors such as vascular endothelial growth factor, the peritumoural microvasculature becomes increasingly leaky. Furthermore, disseminated disease can cause obstruction of the lymphatic vessels.10 This combination of increased build-up and decreased reabsorption causes rapid accumulation of ascites fluid in the peritoneal cavity under malignant conditions.11 Importantly, excessive accumulation of ascites has been associated with detrimental nutritional status in ovarian cancer patients.12

Ovarian cancer ascites is a complex reservoir of soluble factors and cell components, which collectively provide a pro-inflammatory and tumour promoting micro-environment.13 Interestingly, cytokine expression profiling of ascites from ovarian cancer patients revealed high levels of IL-6, IL-8, and Mcp-1,14 factors that have been suggested to promote tissue wasting in individuals with cachexia.15 Furthermore, the concentration of cachexia-related inflammatory cytokines in ovarian cancer ascites has been shown to be significantly higher in comparison with the serum concentrations of the same patient.16 Because ovarian cancer ascites is relatively easily accessible, generally present in large quantities, and contains a high concentration of tumour-derived compounds, it represents an attractive experimental tool to study the impact of ovarian cancer-derived factors on skeletal muscle physiology.

We hypothesized that factors present in ovarian cancer ascites from sarcopenic patients would induce protein metabolism disturbances characteristic of cachexia-associated sarcopenia in skeletal muscle cells. C2C12 skeletal muscle cells were exposed to ascites from well-phenotyped sarcopenic vs. non-sarcopenic patients with malignant or benign ovarian tumours, followed by analysis of protein synthesis and breakdown. We found that ascites from sarcopenic cancer patients decreased C2C12 protein synthesis in correspondence with their degree of sarcopenia.

Methods Patients and cachexia screening

Between March 2017 and March 2018, 15 consecutive patients with a suspected malignancy of the ovary, as indicated by computed tomography scan analysis and the presence of abdominal ascites, were prospectively enrolled at the Maastricht University Medical Centre+. Patients were eligible for either primary cytoreductive surgery or neoadjuvant chemotherapy. Before start of the treatment, patients received a physical screening including assessment of handgrip strength, triceps skinfold assessment, upper arm circumference, and wrist circumference. The physical screening was complemented with the Patient-Generated Subjective Global Assessment, Mini Nutritional Assessment, and subjective assessment of fat, muscle, and fluid status. Patient-reported weight loss was assessed over the past 6 months. Venous blood was drawn, and concentrations of haemoglobin, leucocytes (and differentiation), kidney function markers, liver function indicators, lipids, insulin, glucose, and acute phase proteins were assessed to characterize cancer cachexia and to identify possible promoters of sarcopenia.

For body composition analysis, one single axial slice of the abdominal computed tomography scan at the third lumbar level was used. Standard Hounsfield unit ranges of −30 to +150 Hounsfield units (HU) for skeletal muscle, −190 to −30 HU for intramuscular adipose tissue and subcutaneous adipose tissue, and −150 to −50 HU for visceral adipose tissue were used to demarcate tissue using SliceOmatic software (v5.0, TomoVision, Montreal, Canada). Following demarcation, surface areas were standardized by height to compute the skeletal muscle index (SMI) in cm2/m2. Skeletal muscle radiation attenuation was calculated using the mean HU values of skeletal muscle. Patients with a malignancy (n = 12) were divided into a sarcopenic and a non-sarcopenic group based on their L3-SMI. The cut-off for sarcopenia was determined by 1 SD below the mean SMI (SMI 39.1 cm2/m2).1 Three patients with a benign ovarian condition served as non-sarcopenic controls. This study was approved by the Medical Ethics Committee of Maastricht University Medical Centre+ and has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and all its revisions. All patients gave their informed consent prior to their inclusion in the study.

Collection and analysis of ascites

Ascites was collected either during an abdominal paracentesis or during primary cytoreductive surgery, but in all instances before any systemic treatment was started. Although more ascites was present, between 30 mL and 200 mL of ascites were collected for further analysis (see Table 1). After aspiration, the ascites was kept on ice before centrifugation for 10 min at 200× g. The supernatant was centrifuged again for 15 min at 350× g. Cell-free supernatant was aliquoted and stored at −80°C. The ascites was processed under sterile conditions in a flow cabinet.

Table 1. Baseline characteristics, body composition parameters, and biochemical serum analysis of the patients included Patient and tumour characteristics Unit All patients (n = 15) Sarcopenia (n = 6) No sarcopenia (n = 6) Non-sarcopenic benign controls (n = 3) P-value Reference values Age Years Median (range) 65 (22–81) 68 (65–75) 59.5 (22–63)* 67 (66–81) 0.008 Body mass index kg/m2 Mean ± SD 25.8 ± 3.7 22.4 ± 1.4* 29.0 ± 2.3 26.0 ± 3.6 0.011 Weight loss % (range) 1.6 (0–4.4) 3.3 (0–4.4) 1.5 (0–3.6) 0.8 (0–1.3) 0.164 FIGO stage II n (%) 2 (13.3) 2 (33.3) 0 (0) n.a III n (%) 8 (53.3) 3 (50) 5 (83.3) n.a IV n (%) 2 (13.3) 1 (16.7) 1 (16.7) n.a. Tumour grade Low n (%) 2 (13.3) 1 (16.7) 1 (16.7) n.a High n (%) 10 (66.7) 5 (83.3) 5 (83.3) n.a Ascites volume mL Median (range) 1200 (200–8000) 1200 (500–8000) 2750 (500–7000) 350 (250–500) 0.264 Measurements Unit All patients (n = 15) Sarcopenia (n = 6) No sarcopenia (n = 6) Control (n = 3) P-value Reference values Wrist circumference (WC) cm Mean ± SD 16.8 ± 1.3 15.8 ± 1.2 17.4 ± 0.9 17.5 ± 0.7 0.211 Triceps skinfold assessment (TSA) cm Mean ± SD 1.7 ± 0.4 1.3 ± 0.2* 2.0 ± 0.4 1.9 ± 0.1 0.005 Upper arm circumference (UAC) cm Mean ± SD 27.7 ± 3.1 24.9 ± 2.7 30.0 ± 1.1 29.0 ± 1.4 0.069 Handgrip strengtha kg Mean ± SD 24 ± 4.2 22.5 ± 4.9 26.2 ± 2.4 22 ± 5.7 0.069 L3-SMI cm2/m2 Mean ± SD 41.1 ± 4.8 36.6 ± 2.9* 45.2 ± 2.4 41.7 ± 3.6 0.002 L3-SAT cm2/m2 Mean ± SD 198.7 ± 90.3 125.4 ± 66.1 282.7 ± 45.6 177.4 ± 52.3 0.06 L3-VAT cm2/m2 Mean ± SD 95.5 ± 70.1 48.9 ± 40.1 105.2 ± 66.6 169.4 ± 65.4 0.06 L3-IMAT cm2/m2 Mean ± SD 14.6 ± 9.3 12.4 ± 10.1 17.9 ± 10.5 12.3 ± 4.0 0.448 L3-MRA HU Mean ± SD 37.6 ± 10.7 40.1 ± 9.0 38.4 ± 13.8 30.9 ± 6.2 0.164 Electrolytes and kidney function Unit All patients (n = 15) Sarcopenia (n = 6) No sarcopenia (n = 6) Control (n = 3) P-value Reference values Sodium mM Median (range) 139 (135–144) 138 (135–140) 139.5 (137–141) 142 (140–144) 0.06 135–145 Potassium mM Median (range) 4.6 (4.0–5.2) 4.5 (4.0–5.2) 4.7 (4.1–5.0) 4.4 (4.4–4.6) 0.875 3.6–5.0 Urea mM Median (range) 4.4 (2.2–10.8) 4.1 (2.2–10.8) 4.6 (3.5–7.0) 4.4 (3.8–5.4) 0.875 3.0–8.0 Creatinine μM Median (range) 68 (52–88) 63.5 (52–74) 73 (56–88) 68 (65–73) 0.448 50–100 MDRD mL/min Median (range) 80 (60.5–90) 86.8 (73.5–90) 77.70 (60.5–90) 76.8 (74.2–80.3) 0.448 >90 Liver function and parameters Unit All patients (n = 15) Sarcopenia (n = 6) No sarcopenia (n = 6) Control (n = 3) P-value Reference values Alkaline phosphatase U/L Median (range) 93.5 (47–751) 92 (61–751) 100 (47–403) 89 (83–95) 0.819 <98 γ-GT U/L Median (range) 25 (8–657) 25 (11–407) 88 (8–657) 25 (25–25) 0.517 <38 ASAT U/L Median (range) 27 (14–197) 23 (14–122) 28 (17–197) 18 (20–38) 0.627 <31 ALAT U/L Median (range) 17 (5–221) 11 (5–99) 51 (16–221) 24 (14–34) 0.11 <34 Lactate dehydrogenase U/L Median (range) 185 (125–375) 171 (126–375) 186 (125–218) 185 (185–185) 0.517 47–247 Total bilirubin μM Median (range) 4.6 (2.1–6.5) 5.0 (4.0–6.5) 2.2 (2.1–5.2) 5.4 (4.6–6.1) 0.557 <20 Direct bilirubin μM Median (range) 2.3 (2.0–4.4) 2.7 (2.3–4.2) 2 (2.0–4.4) 2.0 (2.0–2.0) 0.136 <5 Lipids Unit All patients (n = 15) Sarcopenia (n = 6) No sarcopenia (n = 6) Control (n = 3) P-value Reference values Cholesterol mM Median (range) 3.9 (2.4–8.8) 3.1 (2.4–4.7) 4.2 (5.5–8.8) 5.5 (5.1–5.8) 0.264 5.0–6.4 HDL mM Median (range) 1.0 (0.6–1.7) 1.0 (0.6–1.7) 1.1 (0.8–1.6) 1.2 (0.9–1.4) 0.842 >0.9 LDL mM Median (range) 1.8 (1.0–6.1) 1.5 (1.0–3.0) 1.7 (1.4–6.1) 3.6 (3.1–4.0) 0.264 3.5–4.4 Triglycerides mM Median (range) 1.4 (0.8–3.1) 1.2 (0.8–1.5) 2.8 (2.7–3.1) 1.7 (1.3–2.1) 0.036 0.8–1.94 Free fatty acids mM Median (range) 0.7 (0.2–2.0) 0.6 (0.5–0.8) 1.4 (0.2–1.9) 0.4 (0.2–0.7) 0.325 0.1–0.6 Proteins and inflammation Unit All patients (n = 15) Sarcopenia (n = 6) No sarcopenia (n = 6) Control (n = 3) P-value Reference values CRP mg/L Median (range) 37 (1–212) 77 (2–212) 67 (16–87) 2 (1–3) 0.145 <10 Total protein g/L Median (range) 61.1 (57.0–78.6) 60.7 (57.3–66.1) 61 (57.0–78.6) 65 (62.5–66.9) 0.246 60–80 Albumin g/L Median (range) 30.3 (15.9–36.9) 24.6 (15.9–32.1) 31 (20.6–36.9) 32.7 (30.3–36.2) 0.226 32–47 Leucocytes 109/L Median (range) 7.6 (6.2–12.9) 10.9 (7.0–12.9) 8.1 (6.7–9.7) 6.4 (6.2–6.5) 0.345 3.5–11 Haemoglobin mM Median (range) 7.7 (5.2–9.1) 6.8 (5.2–7.7)* 8.2 (6.0–9.1) 8.2 (8.1–8.4) 0.008 7.3–9.7 Glucose mM Median (range) 5.8 (4.8–9.0) 5.4 (4.8–6.8) 5.9 (5.6–6.7) 7.2 (5.4–9.0) 0.842 3.1–7.8 Insulin pM Median (range) 46.3 (12.0–245.0) 28.7 (12.0–135.0) 150.4 (78.7–222.0) 145.7 (46.3–245.0) 0.155 12–150 Questionnaires All patients (n = 15) Sarcopenia (n = 6) No sarcopenia (n = 6) Control (n = 3) P-value Reference values PG-SGA Median (range) 12 (2–23) 11 (2–23) 13 (3–17) 8 (7–12) 0.325 MNA Median (range) 12 (6–14) 11 (6–14) 12 (12–12) 12 (12–13) 0.773 ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; BMI, body mass index; CRP, C-reactive protein; FIGO, International Federation of Gynaecology and Obstetrics; HDL, high-density lipoprotein; IMAT, intramuscular adipose tissue; LDL, low-density lipoprotein; MDRD, modification of diet in renal disease; MNA, Mini Nutritional Assessment; MRA, muscle radiation attenuation; PG-SGA, Patient-Generated Subjective Global Assessment; SAT, subcutaneous adipose tissue; SD, standard deviation; SMI, skeletal muscle index; VAT, visceral adipose tissue; γ-GT, γ-glutamyltransferase.

The concentrations of IL-6, IL-8, GDF-8, GDF-15, tumour necrosis factor (TNF)-α, IL-1β, leukaemia inhibitory factor, and Mcp-1 in ascites were quantified with enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN, USA) as per the manufacturer's protocol.

C2C12 cell culture

C2C12 murine myoblasts (American Type Culture Collection No. CRL1772, Manassas, VA, USA) were cultured in growth medium (GM), composed of low-glucose (1 g/L) Dulbecco's modified Eagle's medium (DMEM) (Gibco, Dublin, Ireland) supplemented with 10% (v/v) foetal bovine serum and 1% (v/v) antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, Gibco). Cells were maintained at 37°C and 5% CO2 until 70–80% confluency was reached at which point the cells were passaged or used for experiments.

Depending on the experiment, cells were seeded at a density of 1.5 × 104 cells/cm2 on BD Matrigel-coated (Matrigel® Matrix Basement Membrane—Growth Factor Reduced, Corning) (1:50 in low-glucose DMEM) cell culture plates (Eppendorf). Myoblasts were cultured for 24 h in GM. After 24 h, differentiation was initiated after washing the cells with Dulbecco's phosphate-buffered saline (PBS) (Gibco) and switching the GM to differentiation medium (DM),17 which consisted of high-glucose (4.5 g/L) DMEM supplemented with 1% (v/v) heat-inactivated foetal bovine serum (30 min at 56°C), 1% (v/v) sodium pyruvate, and 0.5% (v/v) antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin). DM was refreshed every 48 h for 5–6 days at which point the cells were used for experiments (see Supporting Information, Video S1, for an example of a fully differentiated myotube culture used for experimentation). Myotubes were monitored during experiments using an IncuCyte® S3 Live-Cell Analysis System (Sartorius). Phase-contrast images were captured every 2 h using the 10× objective.

Analysis of protein synthesis

Fully differentiated myotubes were treated with ascites or indicated control compounds for 24 h in humidified conditions at 37°C and 5% CO2. Ascites was diluted in DM (25% v/v). Controls consisted of Hank's balanced salt solution (HBSS) in DM (25% v/v), 100 nM insulin (Eli Lilly, Indianapolis, IN, USA) in DM (positive control), or 10 μM dexamethasone (Sigma, St. Louis, MO, USA) solubilized in absolute ethanol and diluted in DM (negative control). All compounds and media were pre-warmed to 37°C before they were added to the cells. Before treatment, the C2C12 cells were washed twice with warm PBS. After treatment, cells