TGF-β Blockade Reduces Mortality and Metabolic Changes in a Validated Murine Model of Pancreatic Cancer Cachexia

Abstract

Cancer cachexia is a debilitating condition characterized by a combination of anorexia, muscle wasting, weight loss, and malnutrition. This condition affects an overwhelming majority of patients with pancreatic cancer and is a primary cause of cancer-related death. However, few, if any, effective therapies exist for both treatment and prevention of this syndrome. In order to develop novel therapeutic strategies for pancreatic cancer cachexia, appropriate animal models are necessary. In this study, we developed and validated a syngeneic, metastatic, murine model of pancreatic cancer cachexia. Using our model, we investigated the ability of transforming growth factor beta (TGF-β) blockade to mitigate the metabolic changes associated with cachexia. We found that TGF-β inhibition using the anti-TGF-β antibody 1D11.16.8 significantly improved overall mortality, weight loss, fat mass, lean body mass, bone mineral density, and skeletal muscle proteolysis in mice harboring advanced pancreatic cancer. Other immunotherapeutic strategies we employed were not effective. Collectively, we validated a simplified but useful model of pancreatic cancer cachexia to investigate immunologic treatment strategies. In addition, we showed that TGF-β inhibition can decrease the metabolic changes associated with cancer cachexia and improve overall survival.

Fig 1. Mice challenged with Pan02 developed lethal tumor burden associated with progressive weight loss.

(A) Mice administered Pan02 exhibited gross and (B) microscopic evidence of peritoneal carcinomatosis as well as microscopic liver implants. Representative images and summary data are shown. (C) Kaplan-Meier analysis of survival was performed in PBS and Pan02 treated animals (n = 20/group, p<0.05). (D) The average body weights of mouse cohorts treated with PBS or Pan02 were calculated at various time points as was the total weight change over the course of the study (n = 20; *p<0.05, ***p<0.001).

Fig 2. Mice challenged with Pan02 developed evidence of cachexia based on body composition.

(A) Average fat mass B) and average percent body fat of mouse cohorts treated with PBS or Pan02 was calculated at various time points as well as total change over the course of the study. (C) Changes in lean mass, (D) bone mineral content, (E) bone mineral density, (F) arm circumference, and (G) skinfold thickness were also calculated (n = 20; *p<0.05, **p<0.01, ***p<0.001).

Fig 3. Evidence of muscle wasting and systemic inflammation.

(A) mRNA levels of Atrogin-1 and (B) MuRF1 in quadriceps muscles of mice treated with PBS or Pan02 were calculated by qPCR. (C) Similarly, levels of Atrogin-1, Myostatin, MuRF1, and β-actin in quadriceps muscle were calculated by Western blotting. (D) ZAG expression was tested in visceral adipose tissue from cohorts of mice treated with either PBS or Pan02. (E) Weight of quadriceps muscles from mouse cohorts treated with PBS or Pan02 was calculated. (F) Serum levels of MCP-1 and IL-6 were compared in PBS- and Pan02-treated mice (n = 5; *p<0.05, **p<0.01, ***p<0.001).

Fig 4. Combined TLR7/9 blockade mitigates inflammation but does not substantially improve the cachectic phenotype.

(A) Mice were treated with Pan02 or Pan02 + IRS 954 and tested for serum cytokines (n = 20; *p<0.05) and (B) survival using the Kaplan-Meier method (n = 20; p = ns). (C) Similarly, mice were treated with PBS, IRS 954, Pan02, or Pan02 + IRS 954 and tested for overall weight change, and changes in (D) lean mass, (E) fat mass, (F) arm circumference and skinfold thickness, (G) bone mineral density, (H) and bone mineral content (n = 20/group; *p<0.05).

Fig 5. Effects of TGF-β inhibition on pancreatic cancer growth.

(A) Serum levels of TGF-β were measured in mice treated with Pan02, Pan02 + 1D11.16.8, or PBS + ID11.16.8 (n = 10/group; *p<0.05). (B) Intramuscular levels of Smad2, p-Smad2/3, TGF-β, and Ezrin (a loading control) were calculated by Western blotting. Density analysis was done based on triplicates (*p<0.05, **p<0.01, ***p<0.001). (C) Pan02 cell proliferation was measured after treatment with increasing doses of 1D11.16.8. (D) 10⁷ Pan02 cells were implanted subcutaneously into the right flank of mice treated with either PBS or 1D11.16.8. Tumors were measured twice weekly by caliper and explanted tumors were compared for tumor size (n = 5/group).

Fig 6. TGF-β inhibition improves overall survival and body composition in pancreatic cancer cachexia.

(A) Mice were treated with Pan02 or Pan02 + 11D1.16.8 and tested for survival using Kaplan-Meier analysis. (B) Cohorts of mice were also tested for weight change, and changes in (C) fat mass, (D) skinfold thickness, or (E) bone mineral density (n = 10/group; *p<0.05, **p<0.01). (F) Mice were treated with Pan02 or Pan02 + 11D1.16.8 and were evaluated for gross loss of lean body mass. (G) Cohorts of mice were also tested for change in lean body mass (n = 10/group; *p<0.05).