PO17A NOVEL IMMUNOHISTOCHEMICAL METHODOLOGY FOR THE INVESTIGATION OF STEM CELL-LIKE SUBPOPULATIONS IN GLIOMA SPHEROIDS AND MIGRATORY CELLS

Abstract

INTRODUCTION: Targeting tumour invasion may represent a complementary treatment when added to standard care in highly infiltrating tumours such as glioblastomas. Invasiveness is believed to be related to stem cell-like qualities of distinct subpopulations of glioma cells. The characterisation of phenotypic differences between invading and non-invading cells will aid the development of specific anti-migratory drugs. A novel immunohistochemical approach was taken to phenotypically characterise invasive migratory glioma cells after drug treatment in a 3D spheroid invasion assay. METHOD: Spheroids generated from U87 and U251 cells were embedded in a collagen matrix and treated over 72 hours with the anti-migratory drugs LiCl and Bio-Indirubin. A protocol was established to prepare the collagen embedded spheroids and migratory cells for immunohistochemistry (IHC). Markers of proliferation, apoptosis and stemness were optimised for IHC. RESULTS: In both U87 and U251, protein expression profiles in cells maintained in the original core were distinct from profiles in migratory cells. The most striking change of expression was observed for SOX-2 in U251, where drug treatment led to a reduction of SOX-2 levels in core-maintained cells whereas migrating cells exhibited higher levels of SOX-2. We could also quantify drug-induced changes in proliferation and apoptotic events in spheroids generated from both cell lines using this method. CONCLUSION: A novel IHC protocol allowed the investigation of the invasive behaviour of cells in a 3D experimental model. Protein expression profiles of markers of stemness indicate that subpopulations of cells exist within glioma spheroids that may respond differently to anti-migratory drugs.