Fig. 4.

Prostate-derived ECM invasion by PCa cells. Invasion of prostate tumor microenvironment by PCa cells was studied by using a 3D ex-vivo/in-vitro model consisting of prostate-derived extracellular matrix in co-culture with prostate cell lines. Cells were seeded on ECMs, placed into a 25 cm² sterile flask, at a density of 800,000/ml in the appropriate growth medium. After 1–3 days in the incubator, ECMs were transferred into a new flask with fresh medium which was replaced every 3–4 days for a total of 12 days. Thereafter, ECMs from co-cultures were fixed in 10% buffered formalin for 24–48 h at room temperature, embedded in paraffin and processed for haematoxylin and eosin (H&E) staining. ECM local invasion by prostate cell lines was quantified by counting the number of infiltrated cells on at least 3 FOVs (Fields of View)/slice of ECM. At least 3 slices/ECM were considered for cell quantification. In order to avoid misinterpretations due to either a different cell doubling-time or the different size of the ECM slices, the number of invading cells was normalized to the number of total cells (i.e., invading + non-invading cells). (A) Representatives FOVs of H&E-stained non tumoral-prostate derived ECM, co-cultured with RWPE-1, RWPE-2 or PC3 cell lines. Cells within the ECM are considered as invading cells, whereas the cells along the outer border and along the lumen as well as cells located in the cavities of the ECM are considered as non-invading. (B) Quantification of invading cells from ECM-cells co-cultures. Plot showing number of invading cells, expressed as a percentage of the total number of cells; (C) Representatives 10-fold-magnified FOVs (left panels) and 20-fold-magnified FOVs (right panels) of H&E-stained non tumoral-prostate derived ECM, co-cultured with the PSA- PC3 cells or the PSA+ MDaPCa2b cells. A totally different pattern of invasion is evident as the PC3 completely and uniformly invaded the matrix, whereas the MDaPCa2b were not able to infiltrate the matrix but got stuck on the outer border and along the lumen of the matrix; (D) Plot showing the effect of PSA treatment on invasion ability of prostate cells. For each cell line the number of invading cells, normalized to the number of total cells, is expressed as a percentage of untreated cells. At least 3 FOVs/slice/sample have been counted. * p < 0.05; ** p < 0.01. Mann-Whitney U test.