Prostate CancerStem Cell Characteristics in Prostate Cancer Cell Lines
Introduction
The concept that solid cancers are hierarchically organised, with cancer stem or cancer-initiating cells at the top of the organisation, gains acceptance and is based on experimental support. Thus, cancers, like normal organs, consist of cells with different potentials for proliferation and give rise to progeny, consequently establishing a hierarchical organisation of stem cells, transient amplifying cells, and terminally differentiated cells. In fact, cancer can be considered a caricature of normal development. The cancer cells that are able to self-renew give rise to differentiated nonclonogenic progeny and are capable of reconstituting the whole tumour upon transplantation and can thus be called cancer-initiating or cancer stem cells (CSC). The first CSCs were isolated from patients with leukaemia [1], [2]. Since then, CSCs have also been isolated and characterised in solid tumours such as those in the breast [3] and brain [4]. There is also accumulating evidence for CSCs in colon [5], [6], liver [7], pancreatic [8], skin [9], and prostate [10], [11], [12] tumours.
Because CSCs resemble normal stem cells in many of their properties, methods similar to those that have been used to identify normal stem cells can be applied to identify and to enrich CSCs. CSCs have a combination of properties such as renewal ability, colony formation in semisolid media, and dye-exclusion, and they are tumorigenic at low cell numbers. Specific sets of surface markers are an essential part of most stem cell–enrichment procedures.
At low density, nontumorigenic cells generate colonies with different morphologies in vitro that are classified as holoclones, meroclones, and paraclones. These morphologies are believed to derive from stem cells, early progenitor cells, and late progenitor cells, respectively. The holoclones are round colonies with tightly packed, generally small cells, whereas paraclones are highly irregular in shape and contain more flattened and scattered cells. Meroclones are a mixture of the previous two colony types [13]. Only holoclones can be repeatedly passaged, which is in agreement with the existence of self-renewing cells in those colonies [14].
Stem cells express distinct molecular markers that can be used for enrichment of these cells. A surface molecule, CD133 (prominin), has been identified as a haematopoietic [15] and neuronal [16] stem cell marker and has been used to enrich brain tumour–initiating cells [4]. Also, for prostate cancer (PCa) CD133, co-immunophenotyping proved to be of potential utility. In PCa, a CD44+/α2β1-integrin high/CD133+ (0.1–0.3%) subpopulation demonstrated a higher proliferation potential and self-renewal ability in vitro than the negative cells. This population may thus represent prostate CSCs [10]. Another potential marker for CSCs is the breast cancer resistance protein (BCRP), a member of the ATP-binding cassette (ABC) protein family that mediates the resistance to various classes of chemotherapeutic agents [17]. Based on this ability, populations of candidate stem cells have been isolated in various tissues and tumours [18]. Also, the cytoskeletal intermediate filament protein nestin, which was identified as a marker for normal neuroepithelial stem/progenitor cells in the brain [19], is a subject of interest. Since the discovery, expression of nestin has been found in several central nervous system (CNS) tumours [20], [21], and it is strongly expressed in aggressive PCa specimens [22]. Additionally, in prostate epithelial cells, cytokeratin 5 (CK5) presents a marker for basal and, in particular, transient amplifying cells [23]. Several other prostate epithelial basal markers may prove important in identifying the CSC phenotype, such as c-Met and Net [24], [25]. Yet, it is important to note that the characterisation of prostate CSCs is still in the early phase of research.
The use of cancer cell lines that contain stem-like cells would also facilitate the study of the molecular pathways and the behaviour of CSCs in vitro. Recent studies suggest the presence of cells with stem-like behaviour, as dye-exclusion and higher clonogeneity, in several human epithelial cancer cell lines [26], [27], [28], [29], [30], including some PCa cell lines [26], [27]. Despite these findings, it has to be kept in mind that the established cell lines do not completely resemble the original tissue. Changes may have occurred during clonal evolution and culturing in in vitro conditions.
In this study, we examined six PCa cell lines for their expression of the putative stem cell marker CD133 and the presence of self-renewing holoclones. The colonies from the DU145 cell line were further characterised with the differentiation marker CK18; a transient amplifying cell marker, CK5; and stem cell markers CD133, α2-integrin, nestin, and BCRP. We show evidence for cells with stem-like properties within the studied PCa cell lines that can be used in the functional studies and characterisation of prostate CSCs.
Section snippets
Cell culture
All the cell lines were derived from prostate carcinomas. DU145, PC-3, LNCaP, and 22Rv1 cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). DuCaP cells were kindly provided by Dr Ken Pienta (University of Michigan, MI, USA) and LAPC-4 cells by Dr Rob Reiter (University of California, CA, USA). The cell lines were grown in RPMI-1640 supplemented with 10% foetal calf serum (FCS), 2 mM glucose, and 2 mM antibiotics, except for LAPC-4, which was cultured in Iscove’s
CD133 is expressed only in DU145
From six PCa cell lines, DU145 showed expression for CD133 in a small proportion (0.01 ± 0.01%) of the cells (Fig. 1). Because the amount of positive cells was very low, a reanalysis of the flow cytometry–selected cells could not be performed. In DuCaP, LAPC-4, 22Rv1, LNCaP, and PC-3 cell lines, CD133 expression was not detected in FACS analysis. A small population (0.15 ± 0.20%) of BCRP+ DU145 cells was detected cells. Other cell lines were not tested.
Prostate cancer cell lines show different colony morphologies
When plated in vitro at low density, five
Discussion
The aim of this study was to determine whether in the commonly used PCa cell lines cells with stem cell characteristics could be identified. There are previous reports of stem-like cells in cell lines based on the dye-exclusion capacity, revealing a side population in FACS; however, Patrawala et al did not detect a side population in the PCa cell lines DU145, PC-3m and LNCaP [31]. Putative prostate CSCs in cell lines have been found by utilising the cell surface markers CD44 [26] and CD133 [32]
Conclusions
Most of the examined cancer cell lines did not express the potential stem cell marker CD133, implicating that it is not a marker for stem cells in PCa cell lines. Instead, the cell lines DU145, 22Rv1, LAPC-4, LNCaP, and DuCaP did contain cells that can form holoclones. Holoclones contain self-renewing cells and express the putative stem cell markers α2-integrin and BCRP. We suggest that PCa cell lines may contain stem-like cells and should be characterised and studied further.
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