Evaluación y selección de componentes

In order to demonstrate the suitability of the chick embryo xenograft model for therapeutic research, the model was assessed using RA.

The dose of 30mg/kg was used throughout chick embryo experiments conducted with RA. This figure was deduced from papers using the drug in xenograft mouse models where the maximum tolerated dose was approximately 60mg/kg and 50-60% of this value was observed in order to observe therapeutic effects (Shalinsky, Bischoff et al. 1995). Dose calculations used the weight of an average egg less the weight of its shell. As tumours in the model exist outside of the embryo itself it seemed logical to include the entire extra-

embryonic weight in drug dose calculations. Although extrapolating drug dosing from

different species is unlikely to provide optimal therapeutic levels this method does provide a bench mark for future experiments. Where new compounds are tested in panels of mouse xenograft models the maximum tolerated dose is administered in order to first establish the presence of a drug effect, before a physiologically acceptable range of doses is later

determined (NCI). The high throughput nature of the chick embryo model would make it amenable to this form of therapeutic research and therefore this approach could also be adapted for future research.

As with cell culture experiments, qPCR was used to detect changes in gene expression signifying differentiation of cells. Retinoic acid administered to tumours in the chick embryo

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model showed a very similar pattern of results to those of cell culture. The general pattern of positive and negative fold change was exactly the same as for culture experiments. Sight differences in the magnitude of these changes, variability and consequent statistical

significance between genes were noted which may reflect the differences in the cells in vitro

and in vivo environment.

The consistency of these results along with their similarity to those seen in cell culture suggests that RA was being delivered effectively to a high proportion of cells. As might be expected a small increase in the variability of results was observed in the in vivo studies perhaps reflecting the more variable drug exposure cells would be expected to receive in this environment. However the changes in gene expression that were observed do suggest that NB cells within the model were differentiating in response to retinoic acid exposure. Furthermore analysis of primary neuroblastoma samples has shown previously that higher levels of Robo2 and STMN4 are commonly identified in lower stage (typically more

differentiated) disease and are associated with better patient survival (Sung, Boulos et al. 2013) adding to the robustness of this finding. Others have attempted to use to analyse gene expression changes in cancer cells implanted on the chick embryo CAM (Hagedorn, Javerzat et al. 2005). During tumour formation on the CAM Hagedorn et al identified up regulation of several genes known to be involved in human glioma tumour progression (Hagedorn, Javerzat et al. 2005). Here we have identified the inverse, that changes in genes associated with cell differentiation, can also be observed.

Many others have suggested the suitability of the chick embryo model for therapeutic research however this is the first time, outside the field of angiogenesis, that the efficacy of a known compound has been reproduced in the chick embryo model. These results are highly promising in confirming our hypothesis that the chick embryo can provide an effective therapeutic model system.

5.1.5.MLN8237

MLN8237 is a small molecule inhibitor of Aurora Kinase A (AURKA) that is currently in early phase clinical testing. AURKA plays a pivotal role in centrosome maturation and spindle formation during mitosis but has also been shown to aid the stabilisation of the N-Myc protein making MLN8237 a potentially powerful tool in the treatment of NB (Brockmann,

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Poon et al. 2013). In order to expand the number of compounds explored in the chick embryo model, as well as to further investigate the role of MYCN in differentiation, we began experimentation with MLN8237.

In order to optimise and characterise the effects of this compound, experiments were conducted in vitro with both BE(2)-C and IMR-32 cell lines using several drug concentrations. At 4µM MLN8237 showed limited toxicity and some morphological changes to cells were visible. Small processes were visible extending from cells, suggesting some influence of the drug on differentiation. However these changes were far less pronounced than those observed with RA, even after 6 days of treatment.

Other works investigating MLN8237 have described evidence of differentiation in vitro. However rather than discussing the effects on cell morphology, the authors state that levels of neurofilament protein L mRNA (NF-L) rose demonstrating differentiation (Brockmann, Poon et al. 2013). Previous studies have found variable levels of NF-L mRNA, but not protein, are expressed in both differentiation induced and control cells. Furthermore NF-L mRNA, and some protein, has been seen to be expressed in both RA-treated and control cells within 6 h of plating, but down-regulated to baseline level thereafter in both

populations (Shea, Sihag et al. 1988, Chiu, Feng et al. 1995). Others have reported the absence of this protein altogether in both cell populations (Andres, Keyser et al.

2013).Therefore the reliability of this marker as an indicator of differentiation is somewhat questionable. Although evidence of differentiation appears less prominent in vitro, in vivo

studies with MLN8237 have been conducted in the Th-MYCN mouse model of NB where partial maturation of tumours was observed. This could suggest other factors such as the tumour-host interactions impact on the response of cells to this compound.

In support of the lack of morphological changes we observed in cell culture, qPCR results failed to show any significant changes in target gene expression. The largest change was observed in STMN4 expression which was 2.51 and 1.89 fold higher in the treated IMR-32 and BE(2)-C cells respectively. However, as stated these changes did not reach statistical significance.

The limited nature of these changes when compared to those observed after 6 days of RA treatment may be explained somewhat by the gene selection process. We chose genes

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based on data published by Sung et al showing significant changes in expression following RA administration (Sung, Boulos et al. 2013). Further experiments involving the siRNA mediated knock down of MYCN and chromosome 1p transfer as means of causing differentiation of NB cells were also conducted by these authors. Each of these methods caused similar patterns of target gene expression, (as seen by up regulation, or down regulation of genes) however the magnitude of these changes was highly variable. It is therefore not surprising that a degree of variation between the two treatments used in our research should be observed. Having selected the genes observed to respond to RA with the greatest fold change, it is also not surprising that these genes show greater change following its administration here.

Our results could be interpreted to suggest that these genes are effective indicators of RA mediated differentiation alone. However, as previously stated although Sung et al saw differing magnitudes of change, they still demonstrated statistically significant differences in expression and observed morphological changes in these cells during each experimental condition. The lack of morphological changes observed after MLN8237 administration, coupled with the lack of statistically significant change observed in gene expression suggests that the differentiation process had not truly begun here. Despite being a negative result these findings are useful in further supporting the reliability of our target gene selection in detecting differentiation in NB cells. The lack of differentiation observed here could support the theory that MYCN levels must rise to initiate the differentiation programme or that other targets of RA are initiating in this process.

MLN8237 did produce a noticeable reduction in cell proliferation which was quantified using Ki67 staining. Overall a 14% decrease in Ki67 staining in the BE(2)-C cells and a 20% decrease in the IMR-32 cells was observed compared to controls. This result was in keeping with published results on the effects of MLN8237 where similar effects were observed over this timescale, although a much greater effect was reported in this paper after 6 days

(Brockmann, Poon et al. 2013). Others have also reported that down regulation of MYCN can reduce proliferation (Shang, Burlingame et al. 2009). It is hypothesised that this effect may be mediated by MYCN induced down regulation of cell-cycle

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Despite consistent results in relation to this compound effects on neuroblastoma

proliferation further work is required to optimise the effects of this molecule before it is utilised in the chick model.