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. 2009;26(5):433-46.
doi: 10.1007/s10585-009-9242-2. Epub 2009 Feb 17.

Invasive prostate cancer cells are tumor initiating cells that have a stem cell-like genomic signature

George J Klarmann  1 , Elaine M Hurt, Lesley A Mathews, Xiaohu Zhang, Maria A Duhagon, Tashan Mistree, Suneetha B Thomas, William L Farrar
Affiliations

Invasive prostate cancer cells are tumor initiating cells that have a stem cell-like genomic signature

George J Klarmann et al. Clin Exp Metastasis. 2009.

Abstract

Development of metastasis is a leading cause of cancer-induced death. Acquisition of an invasive tumor cell phenotype suggests loss of cell adhesion and basement membrane breakdown during a process termed epithelial-to-mesenchymal transition (EMT). Recently, cancer stem cells (CSC) were discovered to mediate solid tumor initiation and progression. Prostate CSCs are a subpopulation of CD44(+) cells within the tumor that give rise to differentiated tumor cells and also self-renew. Using both primary and established prostate cancer cell lines, we tested the assumption that CSCs are more invasive. The ability of unsorted cells and CD44-positive and -negative subpopulations to undergo Matrigel invasion and EMT was evaluated, and the gene expression profiles of these cells were analyzed by microarray and a subset confirmed using QRT-PCR. Our data reveal that a subpopulation of CD44(+) CSC-like cells invade Matrigel through an EMT, while in contrast, CD44(-) cells are non-invasive. Furthermore, the genomic profile of the invasive cells closely resembles that of CD44(+)CD24(-) prostate CSCs and shows evidence for increased Hedgehog signaling. Finally, invasive cells from DU145 and primary prostate cancer cells are more tumorigenic in NOD/SCID mice compared with non-invasive cells. Our data strongly suggest that basement membrane invasion, an early and necessary step in metastasis development, is mediated by these potential cancer stem cells.

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Figures

Fig. 1
Fig. 1
Prostate Cancer Cell Invasiveness. Representative photos of prostate cancer cell migration across an uncoated, porous membrane, “control” or invasion across a Matrigel-coated membrane, “Matrigel”. A. LNCaP cells (25,000-50,000) were plated in 500 μl RPMI-1640 in the upper chamber, and 750μl of RPMI/10% heat inactivated fetal bovine serum (Gemini Bioproducts; West Sacramento, CA) was used as the attractant in the lower reservoir. After 22-48 hr at 37°C, 5% CO2, 90% humidity, non-invasive cells were removed from the upper surface of the membrane with cotton swabs, and invasive cells were stained with the Richard-Allen Scientific 3 Step Stain Kit (Kalamazoo, MI). B, DU145 cells (25,000) were plated and treated as in “A”, C. PCSC3 cells (25,000) were plated as described in “A”, D. Percent invasion relative to control migration± standard deviation, calculated as described in “Materials and Methods.”
Fig. 2
Fig. 2
Invasive Prostate Cancer Cells go Through Epithelial-to-Mesenchymal Transition. LNCaP, DU145 and PCSC3 staining for E-cadherin. Duplicate Matrigel invasion chambers were used for each antibody; one each for staining invasive cells non-invasive cells. Cells not being stained were removed from each insert, and cells of interest were fixed to the membrane in ice-cold methanol for 5 min followed by 3 PBS washes. Non-specific antibody binding sites were blocked for 30 min with PBS/0.3% fish skin gelatin (Sigma, St. Louis, MO) and briefly washed. Cells were incubated with a mouse antibody (25°C, 1.5-2 hrs) to E-Cadherin (Abcam; 1:60 dilution). Following 3 PBS washes, rabbit anti-mouse antibody (Texas Red conjugated; Abcam) was added for 30 min at room temp and membranes received 3 PBS washes and then air-dried. Membranes were mounted on glass slides with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA). Cells were visualized with an Olympus IX70 fluorescent microscope, separate images were obtained for Texas Red and DAPI fluorescence, and images were overlaid with Adobe Photoshop (Adobe Systems, Inc; San Jose, CA). “Non-invasive” cells were stained on the topside of the membrane, while “Invasive cells” were stained on the underside of the membrane. Controls using the Texas Red-coupled secondary antibody and no primary antibody indicated that little, if any, fluorescence was contributed by non-specific binding of this antibody (data not shown).
Fig. 3
Fig. 3
Gene Expression Analysis of Invasive Prostate Cancer Cells. RNA from non-invasive “top” and invasive “bottom” cells was isolated and analyzed using microarrays as described in “Materials and Methods.” Changes in gene expression are relative to the “top” data sets for each cell line. A. Invasive DU145 and LNCaP cells go through EMT as judged by change in expression of the EMT-related genes, where green indicates a decrease and red indicates an increase, black indicates no change, and gray indicates missing data due to array imperfections or data that fell below cut-offs. B. ‘Stemness’ genes are preferentially expressed in the invasive subpopulation of PCSC1, DU145 and LNCaP cells. “Depleted” are LNCaP cells depleted of the CD44+CD24- cells. “Stem” are LNCaP CD44+CD24- cells.
Fig. 4
Fig. 4
Purified Prostate Cancer Stem Cells are Invasive. Magnetic bead purified CD44+ and CD44- cells were evaluated for migration across control membranes and Matrigel-coated membranes. A. LNCaP B. DU145 C. Invasion percentage relative to control migration (average of 3 experiments ±standard deviation). “Bulk” refers to the unsorted cell population.
Fig. 5
Fig. 5
Top 20 functional categories of genes as determined by Ingenuity Pathway analysis showing an equal to or greater than 1.8-fold change in Matrigel-invasive cells compared with non-invasive cells. Y-axis shows functional category while X axis are the number of genes in each category.
Fig. 6
Fig. 6
Cell Signaling Pathways Active in the Invasive Subpopulation. Data extracted from the microarrays to examine key genes in the A. Wnt signaling pathway; B. Hedgehog pathway; C. Notch pathway; and D. Nanog/Sox2/Oct3 network. Fold changes in expression are depicted for the invasive cells relative to the non-invasive cells. The asterisk indicates data averaged from multiple oligonucleotides that bind different regions of the same gene.
Fig. 7
Fig. 7
Depiction of Cellular Changes Upon Matrigel Invasion
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