Abstract
Purpose :
Our group previously showed an association of SOX2 tumor expression and invasiveness in eyes with retinoblastoma that overexpressed vascular endothelial growth factor(VEGF). Using tumor spheres we measured the effect of VEGF and anti-VEGF(avastin) and gene expression of angiogenic and stem cell genes.
Methods :
Primary tumor and control Y79(invasive) and WERI(non-invasive) tumor spheres were used. 3-D culture invasion assay: 1-3 tumor spheres(>50uM diameter) were placed in 3-D matrigel wells. Test conditions were done on triplicate and included basal conditions(no treatment), added 50ng/mL of VEGF or 100mg/mL of avastin(anti-VEGF). The effect of each media was estimated by the increase in size of each sphere(volume) at day 0, 1, 3, 6 and 9. Image J software was used to analyze volume difference of day 6 from day 0m that was averaged for each set of samples and conditions. VEGF quantification assay: 1x105 of tumor cells were incubated and ELISA assay for VEGF protein quantification was done at day 1, 5 and 9, from supernatants. Prism 6 software was used for statistical analysis. Gene expression by qPCR for angiogenic genes HIF and VEGF, and stem cell genes PROM1 and SOX2 was analyzed.
Results :
Four invasive(3 primary + Y79 cell line), and 4 non-invasive(3 primary + WERI cell line) tumors were studied. 3-D culture invasion assay: invasive tumors showed significant increase in volume and extension in the matrigel from day 0 to 6 when exposed to VEGF, whereas there was a reduction in volume if treated with Avastin. Non-invasive tumors showed minimal change in volume with VEGF and non-significant decreased size when exposed to Avastin. VEGF Quantification Assay: both invasive and non-invasive tumors had a progressively higher VEGF production. However, concentration increment of VEGF was significantly higher in the invasive tumor cells. PROM1 and SOX2 were upregulated in invasive tumors treated with VEGF(p<0.05) and PROM1 significantly upregulated in invasive tumors treated with anti-VEGF(p<0.0001).
Conclusions :
Invasive retinoblastoma tumor spheres showed enhanced invasiveness and moderate SOX2 upregulation when exposed to VEGF. Both groups showed a modest decrease in growth with anti-VEGF. Invasive tumor had a significant upregulation of PROM1 gene expression under anti-VEGF treatment, therefore, caution is recommended when considering anti-VEGF in retinoblastoma management.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.