The role of heparan sulfate in neuroblastoma and potential for intervention with engineered heparins

Heparan sulfate (HS) denotes sugar-type chemicals that adorn most cells and their surrounding matrix. Binding to proteins called growth factors, HS regulates cell behaviour and growth. Whilst DNA is a renowned focus in cancer research, understanding HS is also essential: HS can carry vastly more information than DNA and HS abnormalities feature in several human tumours. Neuroblastoma (NB) is a common childhood tumour whose sufferers often have incurable disease; HS is abnormal in NB cells and patients. Engineered heparins are synthetic agents that modulate effects of HS: studying HS will allow us to develop this new potential therapy. However analysing cancer HS had been limited by technology. Now, we have developed tools to elucidate roles of HS in NB. We have shown NB subtypes have distinct HS profiles that may underpin their behaviour. Moreover we can also perform HS profiling in stored tumours. We now propose to study the effects of HS modulation on NB cells? behaviour. We will determine which observed changes in HS profile are important to NB growth and which of those we can target with engineered heparins

Binding key growth factors, heparan sulfates (HS) are integral to cell signalling and normal development. HS is dysregulated in several human cancers. Neuroblastoma (NB) is a common, often incurable, childhood tumour in which HS abnormalities were identified in the 1980s. Moreover elevated serum heparanase correlates with poor prognosis NB. These discoveries have not been pursued largely due to lack of technology: whilst DNA?s template for protein synthesis facilitates analysis, HS synthesis is non-template driven. This hampers HS analysis whilst also meaning that HS is vastly more information-rich than DNA. Understanding HS in cancer therefore remains both challenging and essential. We developed techniques to show that (i) different NB cell lines vary characteristically in HS elaboration, (ii) HS is overexpressed in archival NB relative to surrounding non-tumour tissue, (iii) phage-display antibodies help overcome HS?s limited immunogenicity to study HS immunohistochemically and (iv) phage-display antibodies? HS binding provides a functional rather than a purely structural read-out. We also developed engineered heparins as potential novel therapies, which signal selectively via growth factor without anticoagulation side effects. Realising this promise requires us to determine how HS regulates NB.
Hypothesis: NB phenotype is regulated by HS and can be altered by engineered heparins.
Aim 1: To determine how specific HS alterations regulate NB cell fate & migration in vitro.
Aim 2: To determine how specific HS alterations regulate NB growth and spread in vivo.
NB cell lines will be used to examine effects of HS modulation on cell fate and migration. The latter will be achieved using pharmacological agents, genetic knockdown of HS synthetic enzymes and deployment of engineered heparins. Untreated NB cells will serve as controls. To determine how HS-modified NB cells behave in vivo, NB cell lines +/- the HS modulations described will be introduced into a murine xenotransplant NB model. We will compare resulting NB tumours for size, growth, metastatic spread and responsiveness to engineered heparins (based on the in vitro work). Xenotransplanted NB tumours will be compared between experimental groups by (i) HS immunohistochemistry (generic and phage-display antibodies), (ii) HS extraction and sequencing, (iii) NB cell fate assays (proliferation, differentiation, migration, apoptosis) and (iv) expression and phosphorylation of HS-binding growth factors (themselves targets for tyrosine kinase inhibitor strategies in human cancer studies). We will determine how differential HS elaboration by NB lines regulates phenotype and HS-binding growth factor activity and, for the first time, whether engineered heparins can restrain NB growth.