Comparison of markers for selection of monolayer- and spheroid-CFCs.

<p>[<b>A</b>] Following MACS, total monolayer-colonies arising from each (+)ve and (−)ve fraction were counted to determine the monolayer CFC-recovery, i.e. proportion of input CFCs which are fractionated to the (+)ve fraction (n = 3). The CD49f+ fraction contained 97.9±0.3% of monolayer-CFCs, in contrast to CD44+ and CD133+ cell fractions which contained 13.9±17.9% and 3.1±1.9%, respectively. [<b>B</b>] A typical monolayer CFC-assay is shown. (+)ve and (−)ve fractions were derived from immunomagnetic sorting of 50,000 cells, each plated onto 10 cm culture dishes. [<b>C</b>] Following MACS, total spheroid-colonies arising from each (+)ve and (−)ve cell fractions were counted to determine the spheroid CFC-recovery (n = 3). The CD49f+ fraction contained 98.9±1.1% of spheroid CFCs, in contrast to CD44+ and CD133+ cell fractions which contained 5.7±2.1% and 0.7±0.6%, respectively (p<0.001). [<b>D</b>] CD49f+ were 10.6 fold more enriched in CFCs compared to unsorted cells, and significantly more enriched in CFCs compared to CD49f− cells (p<0.05) (n = 6). No significant CFC enrichment was detected upon comparison of CD44+ and CD44− cells (<i>n</i> = 5), or between CD133+ and CD133− cells (n = 3), respectively. ** p<0.05, * p>0.05.</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

Clinico-pathological characteristics and CFC-recovery following immunomagnetic cell separation.

<p>All patients had Gleason score 7 or above, clinical stage T3, and one patient had metastatic disease at presentation. CFC-recovery (defined as the proportion of total CFCs within the positive fraction) was measured for each marker, with CD49f demonstrating the highest recovery.</p><p>AB = androgen ablation, SD = standard deviation.</p>*<p>sample infected.</p

Flow cytometric live-cell analysis of freshly-isolated prostate cells for the identification of CD49f+, CD133+ and CD44+ subpopulations.

<p>[<b>A</b>] Sequential gating is used to exclude debris by positive selection of scatter gate R1, select single cells by positive selection of pulse width gate R2, and exclude dead cells by negative selection of propidium iodide-positive gate R3. [<b>B</b>] Representative dot plots of prostate cells labelled with each antibody. Scatter profiles of prostate cells expressing CD49f, CD44 or CD133 are also shown on the right. FS = Forward scatter, SS = Side scatter.</p

CD133 expression in frozen prostate tissue sections.

<p>Validation of CD133 (clones AC133 and C24B9) antibody specificity and expression in the human prostate. [<b>A</b>] Punctate expression was shown on the cell surface of Caco-2 cells for clones AC133 and C24B9, as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046979#pone.0046979-Corbeil1" target="_blank">[21]</a>. [<b>B</b>] Orthogonal sectioning following three-dimensional reconstruction of 150 slices (red lines marked by hollow and solid arrowheads indicate the x-z planes shown below, or to the right of the confocal image, respectively) indicate CD133 expression only along the apical border of the plasma cell membrane <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046979#pone.0046979-Corbeil1" target="_blank">[21]</a>. [<b>C</b>] Immunohistochemical expression of AC133 and C24B9 in prostate tissue. Each frozen tissue section measured 10×10 mm in cross-sectional area. Following examination of 20 slides each from 5 patients, we found no cell with definitive membrane expression. [<b>D</b>] Flow cytometric co-expression analysis of CD133 and CD49f. A representative flow cytometric analysis of 3 patients shows that CD133+ cells and CD49f+ cells are mutually exclusive populations, with no significant increase in the percentage of cells within the CD49f+/CD133+ cell gate compared to control. Scale bar = 20 µm.</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

Additional file 1: Appendix 1. of Metformin and longevity (METAL): a window of opportunity study investigating the biological effects of metformin in localised prostate cancer

Informed Consent Form. (DOC 213 kb

Additional file 2: Appendix 2. of Metformin and longevity (METAL): a window of opportunity study investigating the biological effects of metformin in localised prostate cancer

SOP serum and whole blood preperation. (DOCX 23.8 kb