Optimization and comprehensive characterization of a faithful tissue culture model of the benign and malignant human prostate

Abstract

Few preclinical models accurately depict normal human prostate tissue or primary prostate cancer (PCa). In vitro systems typically lack complex cellular interactions among structured prostatic epithelia and a stromal microenvironment, and genetic and molecular fidelity are concerns in both in vitro and in vivo models. “Tissue slice cultures” (TSC) provide realistic preclinical models of diverse tissues and organs, but have not been fully developed or widely utilized for prostate studies. Problems encountered include degeneration of differentiated secretory cells, basal cell hyperplasia, and poor survival of PCa. Here, we optimized, characterized, and applied a TSC model of primary human PCa and benign prostate tissue that overcomes many deficiencies of current in vitro models. Tissue cores from fresh prostatectomy specimens were precision-cut at 300-µm and incubated in a rotary culture apparatus. The ability of varied culture conditions to faithfully maintain benign and cancer cell and tissue structure and function over time was evaluated by immunohistological and biochemical assays. After optimization of the culture system, molecular and cellular responses to androgen ablation and to piperlongumine, purported to specifically reduce androgen signaling in PCa, were investigated. Optimized culture conditions successfully maintained the structural and functional fidelity of both benign and PCa TSCs for 5 days. TSCs exhibited androgen-dependence, appropriately undergoing ductal degeneration, reduced proliferation, and decreased prostate-specific antigen expression upon androgen ablation. Furthermore, TSCs revealed cancer-specific reduction of androgen receptor and increased apoptosis upon treatment with piperlongumine, validating data from cell lines. We demonstrate a TSC model that authentically recapitulates the structural, cellular, and genetic characteristics of the benign and malignant human prostate, androgen-dependence of the native tissue, and cancer-specific response to a potential new therapeutic for PCa. The work described herein provides a basis for advancing the experimental utility of the TSC model.

Ex vivo treatment of prostate tumor tissue recapitulates in vivo therapy response

“…Tumor slices from PC295, PC339, and PC310 PCa PDXs were generated using a vibratome and these slices were cultured in prostate growth medium (PGM) for 4 days under the different conditions in which we compared supporting materials (Figure ). Furthermore, stationary condition and 3D orbital movement on a rocking table were compared as smooth 3D orbital movement could increase oxygen and nutrition exchange as reported previously . H&E stained sections of slices of all three tumors showed that slices cultured on cell strainers maintained tissue morphology, while slices cultured without support or on inserts lost tissue integrity and showed an increase in apoptotic nuclei and vacuolated structures over time (Figure A and Figure S1).…”

“…The culture medium composition has vital impact on the viability of tissue slices. To select the optimal medium, we therefore tested four different culture media that have been reported previously for primary prostate cell or tumor culturing (Table ) . Tissue morphology analysis showed that Prostate Growth Medium (PGM) and aDMEM/F12 K outperformed the other media tested (Figure S3).…”

Inhibition of Mouse Breast Tumor-Initiating Cells by Calcitriol and Dietary Vitamin D

“…In initial experiments we used tissue slice cultures that provide realistic preclinical models of diverse tissues and organs. The use of tissue slice cultures to investigate hormone dependence and cancer-specific responses has been validated in studies of benign and malignant human prostate tissue (23). [ 3 H]-labeled ligand binding assays in ex vivo cultures of MMTV- Wnt1 tumor tissue slices demonstrated that these tumors expressed functional ER and VDR proteins (Fig.…”

“…The slices were transferred in a sterile manner to titanium mesh inserts in sterile six-well plates containing culture media mounted on a rotating platform set at a 30° angle in a tissue culture incubator at 37°c with 95% air and 5% CO 2 as described before (22,23). The tumor tissue slices were incubated in phenol-red free DMEM-F12 media containing 5% charcoal-stripped FBS containing vehicle, calcitriol (100 nM), E 2 (10 nM) or a combination of both for 5 h following which RNA was isolated from the tissue slices for the measurement of estrogen receptor α (Erα), progesterone receptor (Pr), 25-hydroxyvitamin D 3 -24 hydroxylase (Cyp24), 25-hydroxyvitamin D 3 -1a hydroxylase (Cyp27B1), Vdr and aromatase (Cyp19) mRNA expression.…”

Patient-Derived Prostate Cancer: from Basic Science to the Clinic

“…Our standard protocols were summarised recently but are constantly updated [20]. For example, we incorporated the approach of Peehl and others to include the use of precision slices for PDXs [21]. There are several advantages to this, including the opportunity to systematically engraft and fix alternate slices, so that pathologies in the engrafted specimens can be accurately recorded and examined.…”