Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

Summary

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Abstract

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

Introduction

Intratumoral heterogeneity is considered to be a hallmark of cancer¹. Indeed, several mechanisms of intratumoral heterogeneity have been described, including genetic mutation and interactions with the microenvironment. In addition, some cancers may contain a cellular hierarchy with a subpopulation of cancer stem cells (CSCs) that exhibits the properties of tumor-initiating capacity and self-renewal in serial transplantation assays^2-5. Initially described in hematological⁶, breast⁷, and brain⁸ malignancies, CSCs have also been studied in prostate cancer^9-12 as well as other tumor types^2-5.

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Protocol

1. Harvesting and Processing of Human Prostate Cancer Tissue from Surgical Specimens

1. Prepare two 50 ml polystyrene conical tubes containing 15 ml of Roswell Park Memorial Institute (RPMI) 1640 culture medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
2. Pathology service personnel harvest primary and metastatic prostate cancer samples from prostatectomy and palliative surgery specimens, respectively, under an Institutional Review Board approved protocol. Metastatic samples may be obtained from lymph node dissections during primary surgery, the vertebrae during palliative surgical procedures, such as....

Results

Figure 1. Human prostate cancer tumor harvesting and flow cytometry plot illustrating specific populations from a human prostate cancer. (A) Tumor nodules are har.......

Discussion

This protocol describes the isolation of CSCs from fresh human prostate cancer tissues. Several important considerations influence the successful outcome of this protocol.

The recovery of high numbers of viable prostate cancer cells is dependent on careful gross assessment of surgical samples. In our experience, the successful isolation of tumorigenic prostate cells is best assured when macroscopic tumor nodules are observed and processed¹⁰.

However, nowa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI	Gibco Life Technologies	11875-093
Fetal bovine serum	Gibco Life Technologies	10437-028
Penicillin Streptomycin	Gibco Life Technologies	15140-122
PBS	Corning Cell Gro	21-031-CM
60 mm, 15 mm Cell culture dish	Corning	3295
35 µm Cell Strainer	BD Falcon	352340
50 ml conical tube	Crystalgen	23-2263
15 ml conical tube	Crystalgen	23-2265
Red blood cell lysing buffer	Sigma	R7757
HLA class I (W6/32) PE antibody	Abcam	ab43545
CD31 FITC antibody	eBioscience	11-0319-42
CD45 FITC antibody	Abcam	ab27287
IgG2aκ PE antibody	BD Pharminogen	555574
IgG1κ FITC antibody	BD Pharminogen	551954
DAPI	Invitrogen	d3571
12 mm x 75 mm Polystyrene tubes with cell strainer cap	BD Falcon	352235
Vortex Mixer	Crystalgen	CG-BV1000
10% Neutral Buffer Formalin	Fisher	RBBP-0480