Morphological, immunocytochemical and flow cytometric in vitro characterisation of a surface-adherent medulloblastoma

Well-characterised cell lines derived from paediatric intrinsic brain tumours are rare. The different repertoire of cell adhesion molecules expressed by primitive neuro-ectodermal tumours, when compared with gliomas, results in a general lack of propensity for surface adherence. In this study, a highly cellular, medulloblastoma biopsy with a Ki-67 index of 20%, obtained by posterior fossa craniotomy of a two-year-old boy, was maintained in surface-adherent culture for twelve sequential in vitro passages. The culture (VC312R) was characterised by immunocytochemistry and flow cytometry using antibodies against cluster of differentiation 44 (CD44), glial fibrillary acidic protein (GFAP), intermediate filament proteins (Nestin and Vimentin), neural cell adhesion molecules (NCAMs) (ERIC and UJ13A), ganglioside (GD3) and neuron-glial 2 (NG2). GD3, GFAP, ERIC-1, UJ13A and NG2 were detected by neither immunocytochemistry nor flow cytometry. It is of particular interest that we have previously reported that the progenitor cell-associated NG2 heparan sulphate proteoglycan was not expressed in a series of medulloblastoma biopsy sections in our laboratories, while NG2 positivity was seen in supratentorial primitive neuro-ectodermal tumours (PNETs). Strong CD44 positivity was detected on most cells (mean=93.5% of cells on flow cytometry). In one previous case of medulloblastoma, maintained in our laboratories (IPNN-8) as a substrate-adherent culture, no CD44 staining was detected. Twenty-five percent of cells were strongly Vimentin-positive while 54.5% of cells showed Nestin positivity. The expression of Nestin, Vimentin and CD44 is consistent with primitive neural cell evolution. Non-expression of NCAMs may be consistent with the lack of cell-cell adhesion in this culture, which results in surface adherence. The high expression of CD44 may also indicate a distinct phenotype within primitive neuroectodermal tumours, which determines cell-cell and cell-extra cellular matrix adhesive properties.