Methodological aspects of the molecular and histological study of prostate cancer: focus on PTEN

Abstract

Prostate cancer is among the most frequent cancers in men, and despite its high rate of cure, the high number of cases results in an elevated mortality worldwide. Importantly, prostate cancer incidence is dramatically increasing in western societies in the past decades, suggesting that this type of tumor is exquisitely sensitive to lifestyle changes. Prostate cancer frequently exhibits alterations in the PTEN gene (inactivating mutations or gene deletions) or at the protein level (reduced protein expression or altered sub-cellular compartmentalization). The relevance of PTEN in this type of cancer is further supported by the fact that the sole deletion of PTEN in the murine prostate epithelium recapitulates many of the features of the human disease. In order to study the molecular alterations in prostate cancer, we need to overcome the methodological challenges that this tissue imposes. In this review we present protocols and methods, using PTEN as proof of concept, to study different molecular characteristics of prostate cancer.

Keywords: Fresh tissue; Molecular biology; PTEN; Prostate cancer.

Fig. 1

Preparation of well-diagnosed fresh frozen biopsies. (A–D) Preparation of the punch biopsy (A) and excision with scalpel (B), identification of the harvest point in surgical piece with eosin (C) and longitudinal separation of the punch with scalpel (D). (E) Histological features of punch biopsies with different abundance of tumoral tissue, whole section hematoxylin/eosin staining is shown together with a zoom to show the histological features of the piece.

Fig. 2

Evaluation of the impact on phenol-based lysis and ink/acetic acid contaminants in RNA quality. (A–C) Bioanalyzer analysis of RNA preparations performed in the absence (A) or presence (B) of Trizol lysis (average RIN values for the samples analyzed are presented in C; ^∗∗, significance p < 0.01). (D and E) Representative images of the lysis of samples with increasing amount of ink (the intensity of the dark color reflects the increasing concentration of ink in the sample of origin, which has been separated in three groups as indicated) (D), and RIN values obtained from the RNA preparation (E). (F) Real time quantitative PCR of PTEN (two Taqman probes) and GAPDH shows average Ct amplification values in all samples (left panel) and the lack of correlation between Ct values and the increase in ink (right panel).

Fig. 3

An immunostaining protocol for PTEN in human prostate cancer specimens. (A and B) Representative immunohistochemical images (200×) of PTEN expressing (DU145) and PTEN deficient (PC3) human tumor xenografts. Asterisks indicate stromal cells. (C) Representative micrographs (200×) of PTEN staining in benign hyperplasia tissue (BPH) and prostate cancer (PCa) biopsies with PTEN high and low immunoreactivity, arrows indicate epithelial cells and asterisk depict stromal area.

Fig. 4

A method to harvest RNA from urine EVs. (A) Experimental procedure of the EV isolation from urine samples. (B) Representative image by cryo-Transmission Electron Microscopy (TEM) of the isolated EVs with this approach (scale represents 100 nm). (C) Abundance of PTEN (with two probes) and GAPDH transcript in urine EVs by real time quantitative PCR.