Abstract
Medulloblastomas (MBs) are the most common solid malignant childhood brain tumour. Recent genomic research advances have led to the classification of this tumour type into four distinct subroups: Wnt, Shh, Group 3 and Group 4. Group 3 and 4 have the worst prognosis due to the nature of the tumour to disemminate away from the cerebellum via the leptomeninges. Leptomeningeal dissemination is complex and most likely involves bi-directional communication between tumour cells, host cells and extracellular matrix molecules (ECM). We conducted a study to identify differentially expressed ECM molecules using a human ‘leptomeningeal’ cell-MB model. BMEN1 is a low-grade meningioma that has been genetically modified and has been used by others as a model for MB dispersal. Two human Group 4 MB cell lines, CHLA-01-Med and CHLA-01R-Med were derived from the same patient (the CHLA-01R being from a recurrent metastatic lesion). BMEN1 cells were treated with conditioned media from either CHLA-01 or CHLA-01R for 48 hrs. RNA was isolated and an ECM focused PCR array was conducted to identify potential differentially expressed molecules. Following the initial screening, bioinformatic analyses on identified differential expressed genes were conducted using available MB subgroup and metastatic versus non-metastatic (M0-M3) databases. In parallel, functional experiments examining MB cell motility were performed using fluorescent live cell image analysis. Validation of one of the identified ECM molecules discovered in the focus RT-PCR arrays using Western blot and flow cytometric analysis has been conducted. Twenty of 84 genes were found to be upregulated in leptomeningeal cells treated with conditioned media from the recurrent metastatic Group 4 cell line. Of these, 6 ECM related genes were upregulated greater than 3-fold. Initial data analyses examining the 20 upregulated genes revealed several interesting findings. For example, MMP-9, MMP-15, COL15A1, VCAN and THBS1 were found to be over expressed in Group 3 or Group 4. MMP-15 was highest in M(2) and M(3). To gain further understanding of cell motility, BMEN1 cells were plated and following attachment, the MB cell lines CHLA-01 or CHLA-01R were fluorescent labelled and added. The MB cell line CHLA-01R was significantly more motile (P value < 0.0001) and travelled further on the BMEN1 cells than the CHLA-01 MB cells, reflecting their origin of biopsy from the same patient. These results suggest there may be specific ECM molecules that may play a role in medulloblastoma dispersal. In addition to identifying differential expressed ECM molecules, we have begun to develop an all human in vitro model with which to explore the role of these molecules.