Administration of adiponectin receptor agonist AdipoRon relieves cancer cachexia by mitigating inflammation in tumour‐bearing mice

Abstract Background Cancer cachexia is a life‐threatening, inflammation‐driven wasting syndrome that remains untreatable. Adiponectin, the most abundant adipokine, plays an important role in several metabolic processes as well as in inflammation modulation. Our aim was to test whether administration of AdipoRon (AR), a synthetic agonist of the adiponectin receptors, prevents the development of cancer cachexia and its related muscle atrophy. Methods The effect of AR on cancer cachexia was investigated in two distinct murine models of colorectal cancer. First, 7‐week‐old CD2F1 male mice were subcutaneously injected with colon‐26 carcinoma cells (C26) or vehicle (CT). Six days after injection, mice were treated for 5 days with AdipoRon (50 mg/kg/day; C26 + AR) or the corresponding vehicle (CT and C26). Additionally, a genetic model, the Apc Min/+ mouse, that develops spontaneously numerous intestinal polyps, was used. Eight‐week‐old male Apc Min/+ mice were treated with AdipoRon (50 mg/kg/day; Apc + AR) or the corresponding vehicle (Apc) over a period of 12 weeks, with C57BL/6J wild‐type mice used as controls. In both models, several parameters were assessed in vivo: body weight, grip strength and serum parameters, as well as ex vivo: molecular changes in muscle, fat and liver. Results The protective effect of AR on cachexia development was observed in both cachectic C26 and Apc Min/+ mice. In these mice, AR administration led to a significant alleviation of body weight loss and muscle wasting, together with rescued muscle strength (P < 0.05 for all). In both models, AR had a strong anti‐inflammatory effect, reflected by lower systemic interleukin‐6 levels (−55% vs. C26, P < 0.001 and −80% vs. Apc mice, P < 0.05), reduced muscular inflammation as indicated by lower levels of Socs3, phospho‐STAT3 and Serpina3n, an acute phase reactant (P < 0.05 for all). In addition, AR blunted circulating levels of corticosterone (−46% vs. C26 mice, P < 0.001 and −60% vs. Apc mice, P < 0.05), the predominant murine glucocorticoid known to induce muscle atrophy. Accordingly, key glucocorticoid‐responsive factors implicated in atrophy programmes were—or tended to be—significantly blunted in skeletal muscle by AR. Finally, AR protected against lipid metabolism alterations observed in Apc Min/+ mice, as it mitigated the increase in circulating triglyceride levels (−38%, P < 0.05) by attenuating hepatic triglyceride synthesis and fatty acid uptake by the liver. Conclusions Altogether, these results show that AdipoRon rescued the cachectic phenotype by alleviating body weight loss and muscle atrophy, along with restraining inflammation and hypercorticism in preclinical murine models. Therefore, AdipoRon could represent an innovative therapeutic strategy to counteract cancer cachexia.


C26 kinetic experiment
To study the kinetics of onset of cachexia, C26 animals were necropsied 8, 9, and 10 days after tumor cell injection in a separate experiment.These three time points correspond to different stages in the progression of cachexia in terms of body weight loss and food intake as already reported in a previous experiment [3].Mice showed significant weight loss from day 9 (precachexia) along with a significant depletion of muscle mass.Briefly, after one-week acclimatization, seven-week-old CD2F1 male mice (Charles River Laboratories, MA, USA) were injected subcutaneously in the upper flank with colon carcinoma C26 cells (1 × 10 6 cells/0.1 mL saline; C26 mice) or with saline solution (CT mice).Six groups of mice with equivalent body weight were formed: CT and C26 groups, each euthanized and necropsied 8, 9, and 10 days after tumor cell injection.Body weight was measured daily until euthanasia.

Grip strength test
Whole body grip strength was determined using a grip strength meter (Bioseb, Vitrolles, France).Mice were allowed to use their front and back paws to grab a grid and the tail was slowly pulled back by the same investigator.Tension was recorded at the time the mouse released the paws from the grid.Measurements were repeated three times, and the maximum tension of the three measurements was used in analyses.Each test was repeated twice.

Treadmill test
At the age of 15 weeks, mice were placed on a treadmill with an uphill inclination of 15° to assess their endurance and their resistance to fatigue.The initial speed was set at 5 m/min for 1 min, followed by a progressive increase in speed of 1 m/min up to 18 m/min.After that, the mice were allowed to run at 18 m/min until the end of the protocol (60 min in total).Fatigue was defined as the time at which mice were no longer able or willing to keep up with the treadmill despite gentle electric shock at the back of the treadmill.

Count of intestinal polyps in Apc Min/+ mice
The small intestine was carefully excised and cut into three equal segments (upper, middle, and lower intestine).Large intestines were also dissected.All the segments were flushed with PBS, opened longitudinally, and flattened on paper towel.Total polyps in each section were counted by the same investigator and were categorized according to diameter size (<1 mm, 1-2 mm, 2-3 mm, 3-4mm and >4 mm).

mRNA analysis by RT-qPCR
Total RNA was extracted from frozen tissue samples using the TriPure isolation reagent (Roche Applied Science, Penzberg, Germany) as described by the manufacturer.cDNA was prepared by reverse transcription of 1 µg RNA using RevertAid H Minus Reverse Transcriptase, Random Hexamer, RiboLock RNase Inhibitor and dNTP Mix (all from Thermo Fisher Scientific, Waltham, MA, USA).Sybr Green® Real-time quantitative PCR (RT-qPCR) was performed as previously described [4].Relative mRNA levels were calculated using the comparative CT method and normalized by the expression of housekeeping gene Cyclophilin.Primer sequences used for amplification during real-time qPCR are listed in Table S2 (Supplementary Materials).
Primers were tested to avoid primer dimers, self-priming formation, or any unspecific amplification.

Mitochondrial DNA content analysis
Quadriceps tissue (20 to 30 mg) was digested with 0.1 mg/mL proteinase K (100 μg/mL) in lysis buffer (100mM Tris-HCL pH8, 1mM EDTA pH8, 100mM NaCl, 1% (w/v) SDS) at 50 °C overnight.Then, total DNA was isolated with phenol/chloroform/isoamyl alcohol method, pelleted with 100% ethanol and re-suspended in DEPC H2O.To estimate mtDNA copy number, sybr Green® RT-qPCR was performed as previously described [4] using specific mitochondrial gene primers: Cytochrome B (Cyt-b) and NADH-ubiquinone oxidoreductase chain 1 (Nd1) and nuclear gene primers: H19 imprinted maternally expressed transcript (H19) listed in Table S2 (Supplementary Materials).Relative mitochondrial DNA content, corresponding to the ratio between mtDNA and nuclear DNA gene levels, was calculated using the comparative CT method and normalizing by the expression of H19 gene.

Blood analyses
Blood samples were collected at sacrifice, allowed to clot for 30 min at room temperature and then centrifuged at 2000 g for 20 min at 4°C to obtain serum.Serum was aliquoted and stored at −80°C until analysis.Full-length adiponectin and IL-6 levels were measured with specific enzyme-linked immunosorbent assay (ELISA) kits (both from R&D Systems) according to the recommendations of the manufacturer.Total corticosterone was measured by competitive ELISA (MyBioSource, San Diego, USA) following the manufacturers' instructions.
Concentrations of glycerol (Abcam, Cambridge, UK) and non-esterified fatty acids (NEFA; Randox, Crumlin, UK) were quantified by commercial kits based on colorimetric methods according to the manufacturer's instructions.Triglycerides levels were determined by colorimetric method FUJI DRI-CHEM NX500 biochemical analyzer (Fujifilm, Tokyo, Japan).
For Apc mice, during tissue collection, a small part of blood was collected with heparinized capillary tubes, placed on ice, and centrifuged at 4000 rpm for 10 min using a centrifuge Haematokrit 210 (Hettich, Kirchlengern, Germany) to determine the haematocrit.

Muscle fiber cross-sectional area
Gastrocnemius muscle samples were fixed in 10% formalin for 48 h and embedded in paraffin.
Five µm thick formalin fixed paraffin embedded (FFPE) tissue sections were mounted on SuperFrost Plus slides (Menzel-Gläser, Braunschweig, Germany), and deparaffinized with xylene and rehydrated in a graded series of ethanol baths.Gastrocnemius tissue sections were then stained with rhodamine-labeled Wheat Germ Agglutinin (WGA; Vector Laboratories Newark, CA, USA) and Hoechst (Sigma, Darmstadt, Germany).Entire muscle sections were then scanned using the Axio Scan.Z1 slide scanner (Zeiss, Oberkochen, Germany) at 20X magnification.Five fields per section of each mouse (n=6 per group) were randomly pictured at magnification 20X using ZEISS ZEN 3.4 (blue edition) software (Zeiss).Cross-sectional area was determined for each fiber using ImageJ 1.47v software (NIH, Bethesda, MD, USA).

Adipocyte cross-sectional area
Epipidymal white adipose tissue samples were fixed in 10% formalin for 48 h and then embedded in paraffin.Five µm thick FFPE tissue sections were mounted on SuperFrost Plus slides (Menzel-Gläser), and deparaffinized with xylene and rehydrated in a graded series of ethanol baths.Adipose tissue sections were then stained with mayer haematoxylin (Sigma) and Erythrosine B (Merck, Darmstadt, Germany) and after scanned under Pannoramic Scan II slide scanner (3DHISTECH, Budapest, Hungary) at 20X magnification.Five fields per section were randomly pictured using slideviewer software (3DHISTECH).Adipocyte diameter was measured automatically using Adiposoft 1.16 software (under ImageJ 1.47win software) followed by manual correction.

Clinical study in cancer patients
Patients with colorectal or lung cancer, confirmed by pathology, were enrolled in a crosssectional prospective study (NCT01604642) at the time of diagnosis or at relapse and before any therapeutic intervention, between January 2012 and March 2014 at the Cliniques universitaires Saint-Luc, Brussels, Belgium.The study protocol was approved by the local ethics committee of the UCLouvain (B403201111269) and written consent was given by patients prior to entry into the study.Exclusion criteria were: non-caucasian subjects, obvious malabsorption, major depression, artificial nutrition, high doses of steroids (>1 mg/kg hydrocortisone equivalent), hyperthyroidism, other causes of malnutrition, major walking handicap, ECOG performance status ≥4 and psychological, familial, social or geographic conditions that would preclude participation in the full protocol.The diagnosis of cachexia was established according to the definition proposed by Fearon et al. [5], as at least one of these criteria: an involuntary weight loss > 5% over the past 6 months; or weight loss > 2% and body mass index (BMI) < 20 kg/m 2 ; or weight loss > 2% and low muscularity.Patient distribution is reported in supplementary materials (Table S3).Blood samples were collected from cancer patients at the time of recruitment, in standardized conditions.Plasma was aliquoted and stored at −80°C until analysis.Plasma sample from age-matched healthy subjects was used as control group (CT).Plasma human total adiponectin levels was measured with ELISA kit (R&D Systems) according to the recommendations of the manufacturer.

Table S3 . Characteristics of healthy subjects and cancer patients Healthy subjects Colorectal cancer patients Lung cancer patients
Comparison of AdipoR gene (Adipor1 and Adipor2) cycle thresholds (Ct) from RT-qPCR analysis in skeletal muscle, liver and fat (eWAT) of C57BL/6J wild-type mice (WT), Apc Min/+ mice (Apc), and Apc Min/+ mice receiving AdipoRon in their water (Apc+AR) at 20 weeks (n = 9-12/group).(C) Relative gene expression levels of Adipor1 and Adipor2 in skeletal muscle,