Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Summary

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Abstract

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Introduction

The scientific community has been attempting to elucidate the complex mechanism of human cancer development for decades. Whereas identification of potential key players and drug targets begins with patient cells and tissue studies, the translational application of such findings often requires the use of pre-clinical animal models. The use of genetically engineered mice models (GEMMs) to model human cancers has steadily risen since the establishment of the Mouse Models of Human Cancers Consortium (NCI-MMHCC), a committee which sought to describe and unify characteristics of mouse cancer models for scientists worldwide^1,

Protocol

All mouse experiments described here were performed according to the guidelines outlined in the institutional IACUC-approved protocols at SUNY Upstate Medical University.

1. The Urogenital System (UGS) Dissection

Note: The schematic is presented in Figure 1.

1. Euthanize a 3-month-old male C57BL/6 mouse using the CO₂ inhalational euthanasia method or another approved technique.
   Note: Mice between the ages of 3.......

Discussion

This paper outlines the methods for dissection of the mouse prostate and identification of individual lobes. Also described is the protocol for culturing mouse prostate cells in a 3D culture for in vitro analysis.

A critical step in the dissection protocol is (1) harvesting the entire UGS out of the mouse and separating the individual organs under a dissection microscope. The prostate tissue is very small and surrounded by the rest of the UGS; thus, it is practically impossible to har.......

Materials

Name	Company	Catalog Number	Comments
Mouse surgical instruments (Mouse Dissecting kit)	World Precision Instruments	MOUSEKIT
Dissection microscope
RPMI medium	Thermofisher Scientific	11875093
Dissection medium (DMEM + 10%FBS)	Thermofisher Scientific	11965-084
Fetal Bovine Serum	Thermofisher Scientific	10438018
PBS (Phosphate buffered saline)	Thermofisher Scientific	10010031
Collagenase	Thermofisher Scientific	17018029	Make 10x stock (10mg/ml) in RPMI, filter sterilize, aliquot and store at -20 °C
Trypsin-EDTA (0.05%)	Thermofisher Scientific	25300054
DNase I	Sigma-Aldrich	10104159001 ROCHE
Syringes and Needles	Fisher Scientific
Fisherbran Sterile Cell Strainers, 40μm	Fisher Scientific	22-363-547
PrEGM BulletKit	Lonza	CC-3166	Add all componenets, aliquot and store at -20 °C.
Matrigel membrane matrix	Thermofisher Scientific	CB-40234
Dispase II powder	Thermofisher Scientific	17105041	Make 10x stock (10mg/ml) in PrEGM, filter sterilize, aliquot and store at -20 °C