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

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

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

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

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

Tumourigenesis of ABCG2^hiSP and ABCG2^lowNSP cells in nude mice at 30 days.

<p>Tumourigenesis of ABCG2^hiSP and ABCG2^lowNSP cells in nude mice at 30 days.</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

Identification of SP phenotype in bladder cancer cell lines.

<p>RT112, RT4, J82 and 253JB-V cells were stained with Hoechst 33342 dye alone or in the presence of reserpine and analysed by flow cytometry measuring Hoechst blue vs Hoechst red fluorescence. The SP was gated and represented as a percentage of the whole viable cell population following PI exclusion (mean ±SE).</p

Patient demographics and clinicopathological data.

<p>Abbreviations: CIS, carcinoma in situ; IQR, interquartile range; SD, standard deviation.</p>a<p>n = 221.</p>b<p>Percentage of patients excluding those with stage T0 and Tx.</p>c<p>Percentage of recorded cases only.</p

Receiver operating characteristics curves for Mcm5 and NMP22 tests for the detection of bladder cancer in all studied patients with valid test results.

<p>Receiver operating characteristics curves for Mcm5 and NMP22 tests for the detection of bladder cancer in all studied patients with valid test results.</p