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

Patient and tumor characteristics from 23 consecutive surgically resected chordomas.

<p>Abbreviations: LOH, Loss of heterozygosity.</p><p>Patient and tumor characteristics from 23 consecutive surgically resected chordomas.</p

PTEN reconstitution enhances Chordoma cell sensitivity PDGFR inhibition

<p>. (A and B) Representative apoptosis assays for (A) C18 cell line (<i>PTEN</i> +/-) and (B) UCH1 cell line (<i>PTEN</i> +/-), respectively, assessed by Annexin-V and 7-AAD florescence demonstrating significantly higher rates of apoptosis with restoration of <i>PTEN</i> and administration of PDGFR inhibitor compared with control. *P<0.05, Two sample independent student’s t test. This is representative of three independent experiments. (C and D) Representative matrigel invasion assays with the C18 cell line (<i>PTEN</i> +/-) and UCH1 cell line (<i>PTEN</i> +/-), respectively, demonstrating significant decrease in invasion in response to PDGF inhibition with restoration of <i>PTEN</i> expression. These images are representative of 3 independent experiments. *P<0.05, Two sample independent student’s t test.</p

Hypoxic expansion of Chordoma cells is mediated by stabilization of HIF-1α.

<p>(A) A representative cell proliferation assay assessed by the MTS method for the UCH1 cell line (<i>PTEN</i> +/-) demonstrating increased proliferation with exposure to hypoxia (2% Oxygen) compared to normoxia (21% Oxygen). This is representative of 3 independent experiments. *P<0.05, Two sample independent student’s t test. (B) A representative VEGF expression assay as assessed by ELISA from cell culture supernatants for the UCH1 cell line (<i>PTEN</i> +/-) demonstrating increased expression of VEGF with exposure to hypoxia compared with normoxic controls. This is representative of 3 independent experiments. *P<0.05, Two sample independent student’s t test. (C) A representative immunoblot for HIF-1α demonstrating increased expression of HIF-1α with hypoxia exposure compared with normoxia and abrogation of this increase with administration of HDAC inhibitor.</p

Restoration of PTEN expression attenuates <i>in vitro</i> proliferation of Chordoma cells.

<p>(A) A representative immunoblot demonstrating reconstitution of PTEN expression via adenoviral delivery. (B) A representative immunoblot for phospho-PDGFRb demonstrating decreased expression with administration of PDGFR inhibitor compared with vehicle controls. (C and D) Cell proliferation assays assessed by the MTS method for C18 cell line (<i>PTEN</i> +/-) and UCH1 cell line (<i>PTEN</i> +/-), respectively, demonstrating decreased proliferation with restoration of <i>PTEN</i> and administration of PDGFR inhibitor. *P<0.05, Two sample independent student’s t test compared with untreated control. **P<0.05, Two sample independent student’s t test compared with PDGFR inhibitor group.</p

Combined PDGFR and HDAC inhibition strikingly reduces proliferation and <i>in vitro</i> invasion of Chordoma cells.

<p>(A) Cell proliferation assay assessed by MTS method using C18 (<i>PTEN</i> +/-) cells demonstrating significantly decreased proliferation in response to HDAC inhibition and combined treatment with a PDGFR inhibitor compared to controls. This is representative of 3 independent of experiments. *P<0.05, Two sample independent student’s t test. (B) Cell proliferation assays assessed by MTS method using UCH1 (PTEN +/-) and UCH2 (PTEN +/-) cells demonstrating significantly decreased proliferation in response to HDAC inhibition and combined treatment with a PDGFR inhibitor compared to controls. This is representative of 3 independent of experiments. *P<0.05, Two sample independent student’s t test. (C) Representative matrigel invasion assays with the C18 cell line (<i>PTEN</i> +/-) and UCH1 cell line (<i>PTEN</i> +/-), respectively, demonstrating significant decrease in invasion in response to PDGFR inhibition, HDAC inhibition and PDGFR and HDAC inhibition together. These images are representative of 3 independent experiments. *P<0.05, Two sample independent student’s t test compared to untreated control. **P<0.05, Two sample independent student’s t test compared to PDGFR inhibitor alone.</p