TUMOUR SECRETED EXTRACELLULAR MATRIX PREDICTS SURVIVAL AND INFLUENCES MIGRATION AND CELL DEATH IN SHH MEDULLOBLASTOMA 3D MODELS

Abstract Medulloblastoma is the most common malignant paediatric brain tumour. Four molecular sub-groups exist (WNT, Sonic hedgehog (SHH), Group 3 and Group 4), each associated with different patterns of metastasis and chemoresistance. We have shown that within these sub-groups further clinically relevant sub-types exist, characterised by differential expression of extracellular matrix (ECM) proteins. For example, good overall survival in two-thirds of SHH sub-group patients is associated with the expression of specific ECM proteins. Our aim here is to further characterise these ECM components in SHH medulloblastoma and to determine how they confer better overall survival in this sub-group. Using a combination of 3D OrbiSIMs and immunohistochemical staining we have identified, that when grown in 3D hyaluronic acid hydrogels or as spheroids, SHH medulloblastoma cell lines form an ECM shell-like structure composed of laminin, collagen and lumican. In addition, scRNAseq of SHH hydrogel nodules revealed sub-group specific clusters of cells with high levels of ECM interaction and adhesion. We therefore hypothesise that ECM interaction restricts SHH tumour invasion and metastasis through this shell-like structure. To understand how this ECM shell-like structure potentiates better survival in SHH medulloblastoma we have created a CRISPRcas9 laminin knockout SHH medulloblastoma cell line. 3D culture of the laminin knockout cell line demonstrated that laminin is essential for the formation of 3D cell structures as well as migration in SHH medulloblastoma. Furthermore, we have identified that apoptosis is increased in the laminin knockout SHH cell line, suggesting that apoptosis-targeted therapeutics may represent a beneficial treatment option for SHH patients whose tumours exhibit this ECM shell. In summary, tumour-secreted ECM plays a major role in SHH medulloblastoma progression. Expression of laminin, collagen or lumican can be used to classify SHH patients into low and high-risk groups with different therapeutic outcomes and treatment options.