This study interrogates the effect of transient manipulation of tissue tension via ROCK inhibition on PC progression and response to Gem/Abraxane. In vitro organotypic and CDM experiments were performed in independent biological triplicates, with three technical replicates per repeat and per treatment group. For in vivo experiments, numbers of mice used for each model are outlined in corresponding figures and figure legends. In vivo priming started when tumor volume reached 180 mm3 (average size) or 7 × 105 photons/s (average IVIS signal). Mice for which tumor volume or IVIS signal was 10% lower or higher than the average value before enrollment were excluded from analysis.
FLIM-FRET analysis of CDK1 and SRC activity in vitro was conducted in >30 cells per group in three independent biological repeats. In vivo analysis of CDK1 and SRC was performed in 80 cells per mouse, with measurements in three subcellular areas per cell to generate an average value for CDK1 specifically, whereas measurements of lifetime in the whole cell (one value per cell) were performed for analysis of SRC activity.
IHC, SHG, picrosirius red, and GLCM analyses were conducted on three representative FOVs in organotypic matrices and CDMs and in five representative FOVs in subcutaneous xenograft and intrasplenic experiments. Metastatic burden, extravasation, and metastasis morphology in the liver were analyzed in serial sections (five sections per organ with a 100-μm step). Experimental end points for survival experiments were in compliance with Garvan Ethics Committee guidelines (13/17, 14/06, 14/11, and 16/13 protocols).
FLIM-FRET analysis of CDK1 and SRC activity in vitro was conducted in >30 cells per group in three independent biological repeats. In vivo analysis of CDK1 and SRC was performed in 80 cells per mouse, with measurements in three subcellular areas per cell to generate an average value for CDK1 specifically, whereas measurements of lifetime in the whole cell (one value per cell) were performed for analysis of SRC activity.
IHC, SHG, picrosirius red, and GLCM analyses were conducted on three representative FOVs in organotypic matrices and CDMs and in five representative FOVs in subcutaneous xenograft and intrasplenic experiments. Metastatic burden, extravasation, and metastasis morphology in the liver were analyzed in serial sections (five sections per organ with a 100-μm step). Experimental end points for survival experiments were in compliance with Garvan Ethics Committee guidelines (13/17, 14/06, 14/11, and 16/13 protocols).
