The proliferative fraction of DU145 colonies types determined by Ki67 staining.

<p>The percentage of Ki67 positive cells was determined by counting the number of green (FITC) cells as a proportion of blue DAPI positive nuclei. Representative colonies shown.</p

Tumourigenicity of DU145 colonies.

<p>DU145 colonies of each type were injected subcutaneously into the flanks of nude mice. Holoclones (A) and Meroclones (B) both formed tumours which were excised and stained with H&E (C&D). (E) The tumours derived from 10000 cells were measured by digital callipers across 2 diameters at 180° weekly and tumor volume calculated (mean ± S.E.). (F) Clonogenicity of tumors. Following dissection, tumors were digested in collagenase to produce a single cell suspension. 200 cells were seeded into petri dishes to determine colony forming efficiency (%) and the types of colonies formed by tumors of parent colonies.</p

Expression of epithelial and stem cell markers by DU145 colonies.

<p>Expression of luminal (K18) and basal (K5) epithelial and stem cell markers (CD44, α2β1 integrin, Oct4 and BMI1) in DU145 colonies was determined by immunocytochemistry. (A) Holo, mero and paraclone DU145 colonies were stained by immunocytochemistry with monoclonal antibodies against the target, detected with a FITC conjugated secondary antibody (Green) and counter stained with DAPI (blue). (B) The number of positive cells for each maker were determined as a percentage of the total number of cells counted. Holoclone and meroclones contained more CD44 positive cells than paraclones. Hololcones colonies contained more α2β1 positive cells than meroclones. p<0.05 (MANOVA).</p

Number of Cell Divisions during Serial Cloning.

<p>Each colony type was serially cloned and the proliferative capacity of each clone determined. Colony size was used to estimate the number of cell divisions at each generation displayed as mean cell number and minimum number of cell divisions in brackets. The sum of divisions at each generation provides an estimate of how many total cell divisions the original cell which formed the original colony of each type can undergo.</p

Clonogenicity: Holoclones and Meroclones Contain Stem Cells

<div><p>When primary cultures of normal cells are cloned, three types of colony grow, called holoclones, meroclones and paraclones. These colonies are believed to be derived from stem cells, transit-amplifying cells and differentiated cells respectively. More recently, this approach has been extended to cancer cell lines. However, we observed that meroclones from the prostate cancer cell line DU145 produce holoclones, a paradoxical observation as meroclones are thought to be derived from transit-amplifying cells. The purpose of this study was to confirm this observation and determine if both holoclones and meroclones from cancer cell lines contain stem cells. We demonstrated that both holoclones and meroclones can be serially passaged indefinitely, are highly proliferative, can self-renew to form spheres, are serially tumorigenic and express stem cell markers. This study demonstrates that the major difference between holoclones and meroclones derived from a cancer cell line is the proportion of stem cells within each colony, not the presence or absence of stem cells. These findings may reflect the properties of cancer as opposed to normal cells, perhaps indicating that the hierarchy of stem cells is more extensive in cancer.</p></div

Tumorigenicity of DU145 colonies.

<p>DU145 colonies were pooled and were injected s.c into the flanks of Nude mice in a mixture of 1∶1 Matrigel: RPMI. Vehicle control (VC) animals received and injection of Matrigel: RPMI alone. Tumor latency was determined as the first day tumours were palpable and animals sacrificed after 12 weeks and tumors weighed. Mean latency and tumor weights display, standard error in brackets.</p

Single cell origin of DU145 colonies.

<p>(A) Formation of DU145 colonies from single cells was tracked by time lapse photography (Incucyte) every 4 hours from initial adherence for two weeks. (B) The number of colonies originating from 1 or more cells was determined. Colonies which were derived from a single cell upon initial adherence, but merged with other colonies were also determined. Results are displayed as mean ± S.E. from 5 experiments tracking 40 cells per experiment.</p

CFC-recovery following CD49f+, CD44+, and CD133+ selection, in advanced prostate cancer.

<p><b>[A]</b> A representative monolayer colony-formation assay arising from positive and negative fractions of putative markers is shown. Amongst the positively selected fractions, the greatest numbers of colonies is found in the CD49+ fraction. <b>[B]</b> CD49f+ selection recovers the largest number of monolayer-CFCs.</p

Characterization of monolayer- and spheroid-CFCs.

<p>[<b>A</b>] (<b>i</b>) A representative monolayer colony formation assay on Day 12 is shown. (<b>ii</b>) The frequency of monolayer-CFCs within unsorted cells was 0.42±0.07% (n = 5). (<b>iii</b>) Cells within monolayer colonies expressed cytokeratin 5 (an epithelial cell marker <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046979#pone.0046979-Uzgare1" target="_blank">[20]</a>), but no smooth muscle actin (a stromal cell marker). [<b>B</b>] (<b>i</b>) A representative image of prostate spheroids on day 12. (<b>ii</b>) The frequency of spheroid-CFCs within unsorted cells was 0.45±0.08% (n = 4). (<b>iii</b>) Spheroids when kept in culture developed further branching buds (indicated by white arrowheads on day 21 and 28), suggestive of branching ductal structures. [<b>C</b>] To show that monolayer- and spheroid-CFCs could represent the same population of cells, cells from monolayer-colonies were used to develop spheroid colonies (<b>i</b>), and cells isolated from spheroids were used to form monolayer colonies (<b>ii</b>). [<b>D</b>] Spheroids expressed markers of both basal (CK5) and luminal (CK18) epithelial cells. CFC = colony-forming cell, CK5 = cytokeratin 5, SMA = smooth muscle actin.</p

Characterization of CD49+ cells in the benign prostate.

<p><b>A.</b> CD49f expression was assessed in frozen sections of prostate tissue at low (i), medium (ii) and high magnification (iii, iv) by confocal microscopy. Expression was polarised towards the outer surface of the basal cell layer as reported previously (iii, iv) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046979#pone.0046979-Knox1" target="_blank">[22]</a>. <b>B.</b> Co-expression of CD49f with CK5, a basal cell specific marker. <b>C.</b> CD49f expression was also found in endothelial cells as demonstrated by co-expression with CD31, a pan-endothelial cell marker. Endothelial cells formed either a luminal (rows (i) & (ii)), or a linear structure (rows (iii) & (iv)) within the stroma. Co-localization of CD31 with CD49f (indicated by yellow color) was only observed in the stromal compartment but not in the basal layer. <b>D.</b> Human prostate cells labeled with CD31 and CD49f (representative of 3 samples). CD31+ cells alone represented in 3.0±1.5% of human prostate cells. CD31+ cells formed a distinct subpopulation within CD49f+ cells, and all CD31+ cells were CD49f+. <b>E.</b> Colony-forming cell assays conducted using CD31+ and CD31− populations conducted by sorting 50,000 cells by MACS (n = 3) showed, in all assays, almost no colonies in the CD31+ fraction. Scale bar = µm. <b>F.</b> A representative flow cytometric co-expression analysis of CD49f with androgen receptor (AR) or PSA is shown.</p