Characterization of cachexia in the human fibrosarcoma HT-1080 mouse tumour model

Abstract

Background: Cancer cachexia is a complex metabolic disease with unmet medical need. Although many rodent models are available, none are identical to the human disease. Therefore, the development of new preclinical models that simulate some of the physiological, biochemical, and clinical characteristics of the human disease is valuable. The HT-1080 human fibrosarcoma tumour cell line was reported to induce cachexia in mice. Therefore, the purpose of this work was to determine how well the HT-1080 tumour model could recapitulate human cachexia and to examine its technical performance. Furthermore, the efficacy of ghrelin receptor activation via anamorelin treatment was evaluated, because it is one of few clinically validated mechanisms.

Methods: Female severe combined immunodeficient mice were implanted subcutaneously or heterotopically (renal capsule) with HT-1080 tumour cells. The cachectic phenotype was evaluated during tumour development, including body weight, body composition, food intake, muscle function (force and fatigue), grip strength, and physical activity measurements. Heterotopic and subcutaneous tumour histology was also compared. Energy balance was evaluated at standard and thermoneutral housing temperatures in the subcutaneous model. The effect of anamorelin (ghrelin analogue) treatment was also examined.

Results: The HT-1080 tumour model had excellent technical performance and was reproducible across multiple experimental conditions. Heterotopic and subcutaneous tumour cell implantation resulted in similar cachexia phenotypes independent of housing temperature. Tumour weight and histology was comparable between both routes of administration with minimal inflammation. Subcutaneous HT-1080 tumour-bearing mice presented with weight loss (decreased fat mass and skeletal muscle mass/fibre cross-sectional area), reduced food intake, impaired muscle function (reduced force and grip strength), and decreased spontaneous activity and voluntary wheel running. Key circulating inflammatory biomarkers were produced by the tumour, including growth differentiation factor 15, Activin A, interleukin 6, and TNF alpha. Anamorelin prevented but did not reverse anorexia and weight loss in the subcutaneous model.

Conclusions: The subcutaneous HT-1080 tumour model displays many of the perturbations of energy balance and physical performance described in human cachexia, consistent with the production of key inflammatory factors. Anamorelin was most effective when administered early in disease progression. The HT-1080 tumour model is valuable for studying potential therapeutic targets for the treatment of cachexia.

Keywords: Anamorelin; Cachexia; Fibrosarcoma; Food intake; HT-1080 tumour model; Muscle force.

Figure 1

Heterotopic and subcutaneous HT‐1080 tumour implantation result in similar changes in body weight, body composition, and food intake independent of housing temperature. (A–D) Heterotopic (renal capsule) HT‐1080 tumour implantation at standard housing temperature decreased (A) body weight, (B) fat mass, (C) tumour‐free lean mass, and (D) cumulative food intake (Days 4–15). n = 9–15 animals/group. (E–H) Subcutaneous HT‐1080 tumour implantation at standard housing temperature decreased (E) tumour‐free body weight, (F) fat mass, (G) tumour‐free lean mass, and (H) cumulative food intake (Days 7–21). n = 9–10 animals/group. (I–L) Subcutaneous HT‐1080 tumour implantation at thermoneutral housing temperature decreased (I) tumour‐free body weight, (J) fat mass, (K) tumour‐free lean mass, and (L) cumulative food intake (Days 5–15). n = 13–16 animals/group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; comparison of body weight changes with mixed‐effects models; comparison of body composition and food intake with Student's t‐test. Data represented as mean ± SEM. NTB = non‐tumor bearing, SC = subcutaneous.

Figure 2

Heterotopic and subcutaneous HT‐1080 tumour implantation result in comparable tumour growth and histological characteristics independent of housing temperature. (A and B) Heterotopic (renal capsule) HT‐1080 tumour implantation at standard housing temperature; (A) approximate tumour weight on Day 8 (n = 3) and 15 (n = 13) post‐implantation (right kidney weight subtracted from the weight of tumour with primary left kidney (Figure S1C) for each animal on Day 8 and Day 15) and (B) histology showing well‐encapsulated tumour nodules with geographic necrosis. (C and D) Subcutaneous HT‐1080 tumour implantation at standard housing temperature; tumour weight up to 21 days post‐implantation (n = 10) and histology showing well‐encapsulated tumour nodules with geographic necrosis. (E and F) Subcutaneous HT‐1080 tumour implantation at thermoneutral housing temperature; tumour weight up to 15 days post‐implantation (n = 16) and histology showing well‐encapsulated tumour nodules with geographic necrosis. Sections were stained with haematoxylin and eosin. Scale bar (right panel): 4 mm. Yellow asterisks, tumour cells; red asterisks, necrosis. **P < 0.01 by Student's t‐test or mixed‐effects models were applied for tumour weight as appropriate. Data are expressed as mean ± SEM. SC = subcutaneous.

Figure 3

Whole‐body metabolic changes in cancer cachexia. (A and B) The daytime and night‐time energy expenditure, respiratory exchange ratio, and oxygen consumption (VO₂) (non‐normalized) in tumour‐bearing mice during early (Days 8–10) and late (Days 15–18) was lower than in the non‐tumour‐bearing (NTB) group. Shadows represented SEM. n = 8 animals/group; *P < 0.001 by mixed‐effects models. Data represented as mean ± SEM. RER = respiratory exchange ratio, SC = subcutaneous.

Figure 4

Several key circulating catabolic and pro‐inflammatory cytokines are produced by the HT‐1080 tumour (subcutaneous, thermoneutral). Catabolic factors: (A) growth differentiation factor 15 (GDF‐15), (B) GDF‐11, (C) GDF‐8, and (D) Activin A. Pro‐inflammatory cytokines: (E) interleukin (IL)‐1β, (F) IL‐2, (G) IL‐4, (H) IL‐6, (I) IL‐8, (J) IL‐10, (K) tumor necrosis factor alpha (TNFα), and (L) interferon gamma (INFγ). n = 15 animals/group; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by false discovery rate (FDR)‐adjusted Student's t‐test. These data represented as mean ± SEM. LLOD = lower limit of detection, NTB = non‐tumor‐bearing, SC = subcutaneous.

Figure 5

The HT‐1080 tumour model (subcutaneous, thermoneutral) causes skeletal muscle wasting and reduced muscle function (force and fatigue). (A and B) Tibialis anterior (TA) fibre cross‐sectional area reduced in HT‐1080 tumour‐bearing mice. TA muscle fiber average cross‐sectional area distribution demonstrates that the overall fibre sizes are smaller in the HT‐1080 tumour group. n = 11–14 animals/group. (C) Isometric twitch and maximum contraction of the gastrocnemius/soleus complex in vivo was reduced HT‐1080 tumour‐bearing mice. n = 13–14 animals/group. (D) Maximum rate of contraction of the gastrocnemius/soleus complex at 150 Hz during isometric contraction in vivo was decreased in HT‐1080 tumour‐bearing mice. n = 13–14 animals/group. **P < 0.01; ***P < 0.001; ****P < 0.0001 by Student t‐test, Kolmogorov–Smirnov test, and mixed‐effects model. Data represented as mean ± SEM. CSA = cross‐sectional area, NTB = non‐tumor‐bearing, SC = subcutaneous.

Figure 6

Robust decline in voluntary activity but not grip strength in HT‐1080 tumour‐bearing mice. (A) Muscle strength was assessed via grip strength measurement. Grip strength was reduced in HT‐1080 tumour‐bearing mice. n = 16 animals/group. (B) Ambulatory activity during early (Days 8–10) and late (Days 15–18) by comprehensive laboratory animal monitoring system home cage (CLAMS) system. The night‐time ambulatory activity in SC HT‐1080 tumour‐bearing was lower than in non‐tumour‐bearing (NTB) control group. n = 8 animals/group; *P < 0.05 by mixed‐effects models. (C–F) Total daily wheel running distance (Columbus magnetic wheels) was decreased in HT‐1080 tumour‐bearing mice from Day 17 to Day 28. Total daily spontaneous home cage motion (mm/s) measured in the VIUM Smart Cage system was decreased in HT‐1080 tumour‐bearing mice. n = 10–15 animals/group; *P < 0.05; **P < 0.01; grip strength were tested by Student's t‐test. Pairwise comparison with false discovery rate (FDR) adjustment was applied for wheel running activation and motion, and comparison of body weight changes with mixed‐effects models. Data represented as mean ± SEM. NTB = non‐tumor‐bearing, SC = subcutaneous.

Figure 7

Anamorelin prevents but does not reverse cachexia in the HT‐1080 tumour model (subcutaneous, thermoneutral). (A–F) Anamorelin treatment was initiated on Day 8 post‐implantation prior to weight loss (prevention). In both non‐tumour‐bearing (NTB) and HT‐1080 tumour‐bearing mice, anamorelin increased (A) tumour‐free body weight and (B) fat mass but not (C) tumour‐free lean mass. (D) Anamorelin also increased cumulative food intake in both NTB and HT‐1080 tumour‐bearing mice (Days 8–18). (E) Tumour weight was not affected by anamorelin treatment. n = 7–11 animals/group. (F and G) Anamorelin treatment was initiated on Day 13 post‐implantation at ~10% weight loss (intervention). In HT‐1080 tumour‐bearing mice, (F) anamorelin did not increase tumour‐free body weight. (G) Tumour weight was decreased by anamorelin treatment at the end of the study. n = 10 animals/group, *P < 0.05; comparison of body weight changes with mixed‐effects models. Comparison of body composition (fat and lean mass) and food intake changes with one‐way analysis of variance. Comparison of tumour weight by mixed‐effect models. Data represented as mean ± SEM. SC = subcutaneous.