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

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

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

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