For 3D cul tures, 450 ul PRF Matrigel culture medium was added per well and allowed to polymerise at 37 C with 5% CO2 for 1 hr. thereby Single cell cultures were then seeded at 10000 cells per well while co cultures containing HS5 and PC3 cells were plated at 5000 cells each per well and media was replenished every three days. After 3, 6 and 9 days in culture, 3D bulk cul tures were extracted using Cell Recovery Solution as per the manufacturers instructions. Cell pellets were then lysed and western blotting tech niques were carried out. Integrin 6 and B1 inhibition assays In order to block 6 or B1 integrin subunits, well established functional blocking antibodies were diluted directly into the 3D matrix as follows 1. 6 GoH3, 2. B1 P5B2, 3. 6 and B1 and 4. IgG isotope controls.

Cells were then seeded and grown for 9 days in culture in miniaturised 96 well or a bulk 12 Inhibitors,Modulators,Libraries well plate format. Functional blocking antibodies and IgG isotope controls were replaced during media changes every 3 days at 1. 5 ugmL concentration. Miniaturised 3D cell cultures were then washed and fixed with 4% paraformaldehyde and immunocytochemistry was undertaken. For inhibition assays carried out in a 12 well format, cells were extracted using CRS and western blotting tech niques were undertaken. Western blotting Protein was collected from cells at days 3, 6 and 9 from bulk 3D cultures. Treated cells were lysed in ice cold RIPA buffer containing protease inhibitors, incubated at 4 C for 30 mins prior to centrifugation at 14,100 g for 20 mins to pellet cell debris.

The supernatants were then assayed for pro tein concentration using DC Protein Assay, and equal amounts of protein were loaded onto SDS PAGE gels for electrophoresis. The protein was then transferred to Polyvinylidene Fluoride membranes Inhibitors,Modulators,Libraries in transfer buffer for 30 mins using a Bio Rad Turbo Blot system. PVDF membranes were blocked using 5% non fat milk powder Inhibitors,Modulators,Libraries for 1 hr, washed with TBST and primary antibodies were applied in blocking buffer as follows mouse anti E Cadherin, anti human integrin 6CD49f, anti human integrin B1CD29, goat anti human vimentin, Inhibitors,Modulators,Libraries rabbit anti CXCR7 and anti human N Cadherin applied overnight at 4 C. Membranes were then washed and HRP conjugated secondary antibodies applied for 1 hr at 4 C prior to washing and imaging on a Versa Doc imaging station.

Membranes were stripped and re probed for B actin in the case of vimentin, CXCR7 and CXCR4, whereas E Cadherin, N Cadherin and integrin 6, B1 membranes were directly probed for B actin. Densito metric Inhibitors,Modulators,Libraries analysis was performed using Image Lab software enough and expressed as a fold change in relation to loading controls and normalised against B actin. Immunocytochemistry Miniaturised 3D cultures of PCa cells and co cultures grown in 384 well format were washed and fixed with 4% PFA for 20 mins. For immunofluorescence la belling, cells were washed, permeabilised and blocked ON with 2% BSA, 0. 1% Triton X, 0. 05% TWEEN20 at 4 C.