FACS-Free isolation and purification protocol of mouse prostate epithelial cells for organoid primary culture.

The prostate is a gland that contributes to men's fertility. It is highly responsive to androgens and is often the site of carcinogenesis, as prostate cancer is the most frequent cancer in men in over a hundred countries. To study the normal prostate, few in vitro models exist, and most of them do not express the androgen receptor (AR). To overcome this issue, prostate epithelial cells can be grown in primary culture ex vivo in 2- and 3-dimensional culture (organoids). However, methods to purify these cells often require flow cytometry, thus necessitating specialized instruments and expertise. Herein, we present a detailed protocol for the harvest, purification, and primary culture of mouse prostate epithelial cells to grow prostate organoids ex vivo. This protocol does not require flow cytometry approaches, facilitating its implementation in most research laboratories, and organoids grown with this protocol are highly responsive to androgens. In summary, we present a new simple method that can be used to grow prostate organoids that recapitulate the androgen response of this gland in vivo.

Establishment and evaluation of ectopic and orthotopic prostate cancer models using cell sheet technology.

BACKGROUND: The traditional prostate cancer (PCa) model is established by injecting cell suspension and is associated with a low tumor formation rate. Cell sheet technology is one of the advancements in tissue engineering for 3D cell-based therapy. In this study, we established ectopic and orthotopic PCa models by cell sheet technology, and then compared the efficiency of tumor formation with cell suspension injection. METHODS: DU145 cells were seeded on 35 mm temperature-sensitive dishes to form PCa cell sheets, while the cell suspension with the same cell density was prepared. After transplanting into the nude mice, the tumor volumes were measured every 3 days and the tumor growth curves were conducted. At the time points of 2 weeks and 4 weeks after the transplantation, magnetic resonance imaging (MRI) was used to evaluate the transplanting site and distant metastasis. Finally, the mice were sacrificed, and the related tissues were harvested for the further histological evaluation. RESULTS: The orthotopic tumor formation rate of the cell sheet injection group was obviously better than that in cell suspension injection group (100% vs 67%). Compared with cell suspension injection, the tumors of DU145 cell sheet fragments injection had the higher density of micro-vessels, more collagen deposition, and lower apoptosis rate. There was no evidence of metastasis in forelimb, lung and liver was found by MRI and histological tests. CONCLUSION: We successfully cultured the DU145 cell sheet and can be used to establish ectopic and orthotopic PCa tumor-bearing models, which provide an application potential for preclinical drug development, drug-resistance mechanisms and patient individualized therapy.

Effects of the Latex of Synadenium grantii Hook F. (Euphorbiaceae) on a Preclinical Model of Canine Prostate Cancer.

Prostatic cancer (PC) stands out in terms of its occurrence, pathophysiology, and unfavorable prognostics in humans and dogs. Natural drugs bear an integrative potential for conventional antineoplastic treatments. In this context, the bioproducts of Synadenium grantii have been empirically used in different parts of Brazil for the integrative treatment of prostate cancer in humans. However, there is no availability of scientific evidence of the antitumor effects of S. grantii. Therefore, this study aimed to investigate the bioactive compounds in the latex of S. grantii using the high-resolution mass spectrophotometry (HRMS) and to evaluate its cytotoxic effects on primary canine PC cell cultures. Four fragments of phorbol ester were identified as potential bioactive compounds using the HRMS. With the help of an MTT ([3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyltetrazolium bromide]) assay, two canine prostatic carcinoma cell lines (PC 1 and PC2) showed a decrease in the tumor cell count, with an Inhibitory concentration 50 (IC50)of 0.8469 and 0.6068 mg/ml, respectively, for PC1 and PC2. In conclusion, the latex of S. grantii contains phorbol esters in its composition, and its aqueous solution has a cytotoxic effect on canine metastatic PC cells in vitro.

Altered expression of epithelial-to-mesenchymal transition proteins in extraprostatic prostate cancer.

Epithelial to mesenchymal transition (EMT) of cancer cells involves loss of epithelial polarity and adhesiveness, and gain of invasive and migratory mesenchymal behaviours. EMT occurs in prostate cancer (PCa) but it is unknown whether this is in specific areas of primary tumours. We examined whether any of eleven EMT-related proteins have altered expression or subcellular localisation within the extraprostatic extension component of locally advanced PCa compared with other localisations, and whether similar changes may occur in in vitro organotypic PCa cell cultures and in vivo PCa models. Expression profiles of three proteins (E-cadherin, Snail, and α-smooth muscle actin) were significantly different in extraprostatic extension PCa compared with intra-prostatic tumour, and 18/27 cases had an expression change of at least one of these three proteins. Of the three significantly altered EMT proteins in pT3 samples, one showed similar significantly altered expression patterns in in vitro organotypic culture models, and two in in vivo Pten-/- model samples. These results suggest that changes in EMT protein expression can be observed in the extraprostatic extension component of locally invasive PCa. The biology of some of these changes in protein expression may be studied in certain in vitro and in vivo PCa models.