Figure 3. Identification of CCL5/RANTES as an OCy secreted mediator of enhanced PCa invasion.

A) CM prepared from MLO-Y4 cells pressurized at 0 and 40 mmHg for 24 hours was compared using a cytokine screening antibody array and subjected to densitometry. Results are shown as change in optical density (OD) of the cells subjected to 40 mmHg relative to the 0 mmHg. B) Real time PCR of CCL5 from pressurized MLO-Y4 cells was performed and normalized to GAPDH. C) CCL5 secreted from OCy, normalized by protein concentration, increases as the level of hydrostatic pressure increases as measured by ELISA. D) Recombinant murine CCL5 (rCCL5; 10 μg/ml) was used as a chemoattractant for PCa migration and invasion. Neutralization of rCCL5 was accomplished by incubating with anti-CCL5 neutralizing antibody (1 μg/ml) but not with isotype control antibody (1 μg/ml). rCCL5 neutralization inhibited migration across all cell lines. However, invasion of LNCaP and PC3 was not altered by the neutralization of rCCL5. E) CM derived from MLO-Y4 cells pressurized at 0, 20, and 40 mmHg was incubated with CCL5 neutralizing antibody before being used as a chemoattractant in migration and invasion assays. Neutralization of CM derived CCL5 led to significant inhibition of PCa migration and invasion despite increasing pressure. Secretion of CCL5 by MLO-Y4 is induced by tumor generated pressure leading to subsequent increases in tumor cell invasiveness. One way ANOVA with Bonferroni post-test (C/D) or two-way ANOVA (E) was performed; bars represent significant differences where p<0.05.