AR expression and activity within the prostate epithelial hierarchy of differentiation.

<p>A) Representative images of expression of CD133 and AR counterstained with DRAQ5™ within prostate EpCAM^+VE α₂β₁^HI CD133^+VE (Left panel), α₂β₁^HI CD133^–VE (central panel) and α₂β₁^LOW CD133^–VE cells (right panel). <b>B)</b> Expression of the AR regulated genes PSA, KLK2 and TMPRSS2 normalised to GAPDH (n = 10) (p<0.001 comparing α₂β₁^HI and α₂β₁^LOW). Error bars represent standard error of the mean.</p

Expression of the AR within the prostate epithelial hierarchy of differentiation.

<p>Error bars represent standard error of the mean. <b>A)</b> Expression of AR transcript relative to GAPDH. Error bars represent standard error of the mean for n = 10. <b>B)</b> Upper dotplot representative of CD133 staining for progenitor α₂β₁^HI cells. Lower left dotplot representative of AR expression in CD133^–VE gated α₂β₁^HI cells. Lower right dotplot representative of AR expression in CD133^+VE gated α₂β₁^HI cells. Gates were set according to appropriate isotype controls. <b>C)</b> Mean percentage of cells expressing the AR in CD133^+VE and CD133^–VE α₂β₁^HI cells (n = 6). <b>D)</b> Representative histograms for fluorescence of α₂β₁^HI and α₂β₁^LOW isotype controls and AR detection. <b>E)</b> Mean fold change in median staining relative to isotype control for AR stained α₂β₁^HI cells and α₂β₁^LOW cells (n = 6).</p

Side Population in Human Non-Muscle Invasive Bladder Cancer Enriches for Cancer Stem Cells That Are Maintained by MAPK Signalling

<div><p>Side population (SP) and ABC transporter expression enrich for stem cells in numerous tissues. We explored if this phenotype characterised human bladder cancer stem cells (CSCs) and attempted to identify regulatory mechanisms. Focusing on non-muscle invasive bladder cancer (NMIBC), multiple human cell lines were used to characterise SP and ABC transporter expression. <em>In vitro</em> and <em>in vivo</em> phenotypic and functional assessments of CSC behaviour were undertaken. Expression of putative CSC marker ABCG2 was assessed in clinical NMIBC samples (n = 148), and a role for MAPK signalling, a central mechanism of bladder tumourigenesis, was investigated. Results showed that the ABCG2 transporter was predominantly expressed and was up-regulated in the SP fraction by 3-fold (ABCG2^hi) relative to the non-SP (NSP) fraction (ABCG2^low). ABCG2^hi SP cells displayed enrichment of stem cell markers (Nanog, Notch1 and SOX2) and a three-fold increase in colony forming efficiency (CFE) in comparison to ABCG2^low NSP cells. <em>In vivo</em>, ABCG2^hi SP cells enriched for tumour growth compared with ABCG2^low NSP cells, consistent with CSCs. pERK was constitutively active in ABCG2^hi SP cells and MEK inhibition also inhibited the ABCG2^hi SP phenotype and significantly suppressed CFE. Furthermore, on examining clinical NMIBC samples, ABCG2 expression correlated with increased recurrence and decreased progression free survival. Additionally, pERK expression also correlated with decreased progression free survival, whilst a positive correlation was further demonstrated between ABCG2 and pERK expression. In conclusion, we confirm ABCG2^hi SP enriches for CSCs in human NMIBC and MAPK/ERK pathway is a suitable therapeutic target.</p> </div

Validation of AR detection with flow cytometry.

<p><b>A)</b> Percentage of cells staining above a no antibody control for either isotype antibody (hollow points) or PG-21 AR antibody (solid points) in LNCaP (circles) or PC3 (triangles) across a dilution series. <b>B)</b> Representative staining patterns for PC3 (upper dotplots) and LNCaP (lower dotplots) for either 1∶200 isotype antibody (left dotplots) or 1∶200 PG-21 AR antibody (right dotplots) of equivalent concentrations. Gates set according to isotype control. <b>C)</b> Left dotplot representative of staining of LNCaP with isotype control, right dotplots representative of AR staining in non-transfected LNCaP (upper), LNCaP transfected with scrambled siRNA (middle dotplot) and LNCaP transfected with AR siRNA (lower). Gates were set according to an appropriate isotype control. <b>D)</b> Percentage of cells staining positive for AR relative to an isotype control in non-transfected LNCaP, LNCaP transfected with scrambled siRNA and LNCaP transfected with AR siRNA. Error bars represent standard error of the mean for n = 3. <b>E)</b> Western blots of AR expression for non-transfected LNCaP, LNCaP transfected with scrambled siRNA and LNCaP transfected with AR siRNA are shown using a different AR antibody (G122-434, BD Pharmingen).</p

Strategy of enrichment for required cell types.

<p><b>A)</b> Schematic work flow for enrichment of epithelial cells for assessment of AR expression. <b>B)</b> Purity of selection by expression of the lineage specific markers CD24 (epithelial), CD146 (endothelial) and CD45 (haematopoietic) normalised to GAPDH following real-time PCR for unsorted prostate epithelia and EpCAM/HEA sorted epithelia, error bars represent standard error of the mean for n = 3. <b>C)</b> CD133/1 Sorted samples were assessed for purity by co-expression of the CD133/2 epitope, confirming that these two epitopes are co-expressed in the prostate and that our CD133 selection efficiently enriches for CD133^+VE cells: Upper dotplot representative of CD133/2 staining for unsorted α₂β₁^HI epithelial cells; lower left dotplot representative of CD133/2 staining for α₂β₁^HI CD133/1^–VE cells; lower right dotplot representative of CD133/2 staining for α₂β₁^HI CD133/1^+VE cells. Gates are set according to appropriate isotype controls. <b>D)</b> Confirmation of CD133 enrichment with real-time PCR. CD133 expression is shown normalised to GAPDH, error bars represent standard error of the mean n = 10.</p

Immunohistochemical expression of ABCG2 in clinical bladder cancer.

<p>(A) ABCG2 expression in normal urothelium, low grade (LG) and high grade (HG) NMIBC is typically non-specific and can be seen nuclear and cytoplasmic. (B) Correlation of ABCG2 immunostaining intensity with grade and stage in NMIBC. (C) Kaplan-Meier curves illustrating outcomes of ABCG2 intensity (0, 1, 2 and 3) with recurrence and progression free survival (p<0.001, Log Rank analyses). (D) Kaplan-Meier curves illustrating outcomes of pERK intensity (0, 1, 2 and 3) with progression free survival (p<0.001, Log Rank analyses). (E) Correlation of ABCG2 and pERK expression (p = 0.005, Pearson’s r = 0.99).</p

SP cells are more tumourigenic.

<p>(A) 1×10³ ABCG2^hi SP and ABCG2^low NSP sorted RT112 cells were subcutaneously injected into nude mice and monitored for tumour growth. (B) Immunohistochemical staining of serial sections, of tumours derived from mice injected with 1×10³ ABCG2^hi SP and residual cell pellets from mice injected with 1×10³ ABCG2^low NSP sorted RT112 cells, with H&E and ABCG2 and at higher magnification (20x). Markedly increased immunoreactivity for ABCG2 is seen in the ABCG2^hiSP derived tumours. (C) Relative mRNA expression of ABCG2 in tumours formed following injection of 1×10³ ABCG2^hi SP and ABCG2^low NSP sorted RT112 cells was determined using real time PCR. (D–F) Relative mRNA expression of Nanog, Notch1 and SOX2 in tumours formed following injection of 1×10³ ABCG2^hi SP and ABCG2^low NSP sorted RT112 cells was determined using real time PCR. (G) Immunohistochemical staining of serial sections, of tumours formed following injection of 1×10³ ABCG2^hi SP and ABCG2^low NSP sorted RT112 cells, with Nanog, Notch1 and SOX2.</p

NMIBC SP cells show elevated ABCG2 transporter expression.

<p>Relative mRNA expression of ABCA2, ABCG2, MRP1 and P-glycoprotein was determined in SP and NSP fractions of RT112 (A) and RT4 (B) cells using real time PCR. Data shown are the mean of three independent experiments each performed in triplicate (mean ±SE). *P<0.05 (Student’s two-tailed <i>t</i>-test). Inhibition studies were performed on RT112 (C) and RT4 (D) cells using ABCG2-specific inhibitor fumitremorgin C (FTC) and P-glycoprotein inhibitor verapamil.</p

ABCG2^hi SP cells have a higher clonogenic ability in comparison to ABCG2^low NSP cells.

<p>(A) FACS sorted cells were seeded in 6-well plates at a density of 1×10² cells/well and colonies were counted after 2 weeks. Colony forming efficiency (CFE) was calculated as described in Materials and Methods. Data shown are the mean (±SE) of three independent experiments each done in triplicate. *P<0.05 (Student’s two-tailed <i>t</i>-test). (B) Cell cycle profiles of RT112 ABCG2^hi SP and ABCG2^low NSP cells showing an increase in S-phase in the ABCG2^hi SP fraction. (C) Serial selection, sub-culture and cytometric fractioning between RT112 SP and NSP cells. SP and NSP cells sorted from RT112 were cultured for two weeks, restained with Hoechst 33342 dye and reanalysed by flow cytometry. This process was repeated 4 times.</p

Inhibition of ERK signalling attenuates the fraction of ABCG2^hi SP RT112 cells.

<p>(A) Western blot analysis was performed with phospho-ERK1/2 and ERK1/2 antibodies. (B) RT112 cells were placed in FM alone or in the presence of U0126 (10 µM) for 30 min prior to staining with Hoechst 33342. The effect of U0126 on the ‘tail’ and ‘top’ end compartments of ABCG2^hiSP⁺ was investigated. (C) CFE of ABCG2^hi SP cells with increasing concentrations of UO126 and different sized colonies (≥1 mm, ≥2 mm and ≥3 mm in diameter).</p