5-ALA Fluorescence in Native Pituitary Adenoma Cell Lines: Resection Control and Basis for Photodynamic Therapy (PDT)?

<div><p>Objective: Pituitary adenomas (PA), especially invasive ones, are often not completely resectable. Usage of 5-aminolevulinic acid (5-ALA) for fluorescence guided surgery could improve the rate of total resection and, additionally, open the doors for photodynamic therapy (PDT) in case of unresectable or partially resected PAs. The aim of this study was to investigate the uptake of 5-ALA and the effect of 5-ALA based PDT in cell lines. Methods: GH3 and AtT-20 cell lines were incubated with different concentrations of 5-ALA, protoporphyrin IX (PPIX) fluorescence was measured by flow cytometry and fluorescencespectrometry. WST-1 assays were performed to determine the surviving fraction of cells after PDT. PPIX fluorescence intensities and PDT effect of the pituitary adenoma cells were compared to U373MG, a well-known glioblastoma cell line. Results: Both cell lines showed a 5-ALA dependent intracellular PPIX fluorescence. Significant differences after 24hrs of incubation were observed in AtT-20 cells in comparison to GH3. Regardless of the incubation or metabolism time, there was a proliferation inhibiting effect after PDT, with no statistical significance. Conclusion: Since GH3 cells showed a heterogenous uptake of 5-ALA in the flow cytometry profile, but not constantly high concentrations they might have a 5-ALA efflux mechanism, which still needs to be determined. In the case of AtT-20, the cells might need a longer time for the uptake due to their size or slow metabolism. We showed that the different cell lines have different uptake and metabolism mechanisms, which needs to be further investigated. The general uptake of 5-ALA allows the possibility of resection control and PDT for pituitary adenomas. But, the role of PDT for unresectable pituitary adenomas deserves further investigations.</p></div

Flow cytometry data of PPIX fluorescence in pituitary adenoma cells.

<p>(A) The pituitary adenoma cell lines GH3 and AtT-20 were incubated for 6 and 24hrs with 100 g/ml 5-ALA (black line) or with media alone (gray area). One out of 3–7 independent experiments is shown. (B andC) MFI of all flow cytometry experiments are shown. Error bars represent S.E.M.</p

Spectrofluorometric measurment of GH3, AtT-20 and U373 MG cell lysates after excitation at 405nm.

<p>1x10⁶ cells were seeded and incubated with or without 5-ALA for 6 and 24hrs. The spectra from 450nm to 750nm was analyzed. For each experiment the untreated condition was subtracted from the 5-ALA treated measurement. Peak values at 631nm are compared after 6 and 24hrs of incubation in all three cell lines as arbitrary units (a.u.). Three to four independent experiments were performed and presented as box plot with min.-max. whiskers, one way ANOVA followed by Bonferroni’s post-hoc analysis was used for evaluating the statistical significance, * indicates p ≤ 0.05.</p

Relative cell viability of GH3 cells.

<p>Cells were monitored with Wst-1 assay and compared to the untreated control. The cells were pretreated with different concentrations of 5-ALA for 6 (A) and 24hrs (B). Subsequently, the cells were irradiated with red light (635nm) for 250s and 25 J/cm² (gray bars) or left non irradiated (black bars). Four independent experiments were performed and averaged. Error bars indicates S.E.M., no statistical significance was found as calculated by one way ANOVA followed by Bonferroni’s multiple comparison test.</p

Relative cell viability of U373 cells.

<p>Cells were monitored with Wst-1 assay and compared to the untreated control group without 5-ALA and laser, with 5-ALA and without laser, without 5-ALA and with laser. The cells were treated with 25 μg/ml 5-ALA for 6 hours (n = 12). Subsequently, the cells were irradiated with red light (635nm) for 250s and 25 J/cm². Error bars indicates S.E.M., statistical significance of p<0.05 was found for treated cell group with 5-ALA and with laser.</p

Additional file 2: Figure S1. of Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

A representative image of tumor emboli, a unique feature for tissues of IBC patients showing a positive staining for CD44 and Syndecan-1. (ZIP 2337 kb

Additional file 3: Figure S2. of Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

Flow cytometric analysis of Syndecan-1 expression in control and Syndecan-1 siRNA transfected SUM-149 cells. 500,000 cells were stained for isotype control mouse IgG1-PE and mouse anti-human Syndecan-1 (CD138)-PE and the cells were subjected to flow cytometry. Each plot shows mouse IgG-PE control (dotted line) and CD138-PE-stained cells (solid line). The median fluorescence intensity (MFI) of events is given for each peak. Data are a single experiment representative of three independent experiments. (ZIP 281 kb

Additional file 1: Table S1. of Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

Primers sequences. (DOCX 18 kb

Syndecan-1 (CD138) Modulates Triple-Negative Breast Cancer Stem Cell Properties via Regulation of LRP-6 and IL-6-Mediated STAT3 Signaling

<div><p>Syndecan-1 (CD138), a heparan sulfate proteoglycan, acts as a coreceptor for growth factors and chemokines and is a molecular marker associated with epithelial-mesenchymal transition during development and carcinogenesis. Resistance of Syndecan-1-deficient mice to experimentally-induced tumorigenesis has been linked to altered Wnt-responsive precursor cell pools, suggesting a potential role of Syndecan-1 in breast cancer cell stem function. However, the precise molecular mechanism is still elusive. Here, we decipher the functional impact of Syndecan-1 knockdown using RNA interference on the breast cancer stem cell phenotype of human triple-negative MDA-MB-231 and hormone receptor-positive MCF-7 cells in vitro employing an analytical flow cytometric approach. Successful Syndecan-1 siRNA knockdown was confirmed by flow cytometry. Side population measurement by Hoechst dye exclusion and Aldehyde dehydrogenase-1 activity revealed that Syndecan-1 knockdown in MDA-MB-231 cells significantly reduced putative cancer stem cell pools by 60% and 27%, respectively, compared to controls. In MCF-7 cells, Syndecan-1 depletion reduced the side population by 40% and Aldehyde dehydrogenase-1 by 50%, repectively. In MDA-MB-231 cells, the CD44(+)CD24(-/low) phenotype decreased significantly by 6% upon siRNA-mediated Syndecan-1 depletion. Intriguingly, IL-6, its receptor sIL-6R, and the chemokine CCL20, implicated in regulating stemness-associated pathways, were downregulated by >40% in Syndecan-1-silenced MDA-MB-231 cells, which showed a dysregulated response to IL-6-induced shifts in E-cadherin and vimentin expression. Furthermore, activation of STAT-3 and NFkB transcription factors and expression of a coreceptor for Wnt signaling, LRP-6, were reduced by >45% in Syndecan-1-depleted cells compared to controls. At the functional level, Syndecan-1 siRNA reduced the formation of spheres and cysts in MCF-7 cells grown in suspension culture. Our study demonstrates the viability of flow cytometric approaches in analyzing cancer stem cell function. As Syndecan-1 modulates the cancer stem cell phenotype via regulation of the Wnt and IL-6/STAT3 signaling pathways, it emerges as a promising novel target for therapeutic approaches.</p> </div

siRNA-mediated knockdown of Syndecan-1 downregulates expression of IL-6R, IL-6 and CCL20 and dysregulates epithelial and mesenchymal marker protein expression in MDA-MB-231 breast cancer cells.

<p>A) left panel: RT-PCR analysis of IL-6R expression in MDA-MB-231 cells subjected to Syndecan-1 siRNA knockdown. Following total RNA isolation, mRNA was reverse transcribed and used as a template for PCR amplification of IL-6R. Right panel: PCR band intensities were normalized for actin expression and the data were analyzed using the paired Student's <i>t</i>-test. B) Left panel: Western blot analysis reveals reduction of IL-6R following Syndecan-1 silencing. Lysates of control and Syndecan-1 silenced cells were collected and 30-50µg protein/lane was immunoblotted and probed with sIL-6R antibody. Right panel: Immunoblot band intensities were normalized for tubulin expression and the data were analyzed using the paired Student's <i>t</i>-test. Data shown are triplicates from a single experiment representative of three independent experiments. C) left panel: RT-PCR analysis of IL-6 expression. Right panel: semiquantitative densitometric analysis (see panel A). D) left panel: RT-PCR analysis of CCL20 expression. Right panel: semiquantitative densitometric analysis (see panel A). *=p<0.05, ***=p<0.001, n≥3, error bars=SEM. E,F) The influence of IL-6 treatment on the expression of the epithelial marker E-cadherin (E) and the mesenchymal marker vimentin (F) was studied by Western blotting. Cells were stimulated by 50ng/ml IL-6 24h after transfection with siRNA for 4h and 19h. In control cells, IL-6 treatment for 19h promoted EMT. Syndecan-1 depletion resulted in significant downregulation of E-cadherin expression. IL-6 treatment of Syndecan-1 depleted cells for 4h resulted in marker expression changes suggestive of enhanced mesenchymal-to-epithelial transition. (E,F) Upper panels = representative Western blots, lower panels = quantitative analysis. n≥3,*=P<0.05. G) Confocal immunofluorescence microscopy of phalloidin-labeled actin filaments reveals increased formation of actin stress fibers, filopodia (*) and lamellopodia (#) in Syndecan-1 siRNA-treated compared to control siRNA treated MCF-7 cells. </p