In vivo experiments and tissue collection
To examine the protective effects of the HSP90 inhibitor on cachexia, the cachectic mice received intraperitoneal injection of 17DMAG (HY-12024,MCE,NJ) (15 mg/kg, daily) dissolved in 1% DMSO + 30% PEG300 + 1% Tween 80 or PU-H71 (HY11038,MCE,NJ) (50 mg/kg, daily) dissolved in PBS (as the solvent control) beginning on day 7 when the tumor became palpable; vehicle group were injected with relevant solvent: 17DMAG solvent (1% DMSO + 30% PEG300 + 1% Tween 80) for veh1 and PU-H71 solvent (PBS) for veh2. For the survival experiment, fifty mice were randomly divided into five groups (normal control, C26 + 17DMAG, C26+veh1, C26+PU-H71, C26+veh2), and each group contained 10 mice. The mice were followed until the endpoint criterion was fulfilled or at least 30 days had elapsed after the inoculation of C26 cells to determine the survival rate. The endpoint criterion combined the detected weight loss with the overall condition of the mouse. To evaluate the overall health status of the mice, the following aspects were considered in addition to weight loss: appearance, posture, and natural and provoked behaviour (inactivity, dyskinesia, and reduced response to external stimuli). Mice were euthanized when two researchers confirmed the fulfilment of the endpoint criteria.
To study the potential mechanisms involved, another experiment was conducted with a predetermined endpoint at day 15 after C26 cell inoculation. The numbers of mice and groups were the same as those used in the survival experiment. The following data were collected: body weight, food intake, tumor weight, grip strength, and survival rate. Once the tumors were removed, the gastrocnemius was dissected, harvested, weighed, and then fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) for subsequent histological analysis.