Poster Presentation Advances in Neuroblastoma Research Congress 2016

Characterisation of neuroblastoma cells isolated from bone marrow aspirates of children with stage 4 disease at diagnosis: an NCRI CCL CSG Neuroblastoma Group Study. (#356)

Sue A Burchill 1 , Charlotte Haunch-Smith 1 , Andrea M Berry 1 , David R Westhead 2 , Walter M Gregory 3 , Martin Elliott 4 , Kate Wheeler 5 , Deborah A Tweddle 6 , Ruth Ladenstein 7
  1. Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom
  2. Astbury Centre for Structural Molecular Biology , University of Leeds, Leeds, United Kingdom
  3. Leeds Institute of Clinical Trials Research, Leeds, United Kingdom
  4. Paediatric Oncology and Haematology, , Leeds Teaching Hospital Trust , Leeds, United Kingdom
  5. University of Oxford, Oxford, United Kingdom
  6. Northern Institute for Cancer Research, Newcastle upon Tyne , United Kingdom
  7. St Anna Children's Hospital, Vienna, Austria

Purpose

Neuroblastoma (NB) cells in the bone marrow are a hallmark of high-risk disease, selecting children for more intensive treatment. These cells can contribute to disease progression and relapse, their elimination being one of the greatest challenges for cure of some children. We have therefore isolated and characterised these cells, to reveal pathways that might be exploited to develop more effective treatments targeting bone marrow disease.

Methods

NB cells were isolated from bone marrow aspirates (BM; n=52) from children with stage 4 disease using immune-magnetic bead selection for the cell surface disialoganglioside GD2. Self-renewal was assessed by plating single cells in low and substrate adherent plates, and in soft agar. Migratory capacity was analysed using a 3D gelatin-based assay. Self-renewing cells were characterised using TaqMan® MicroRNA arrays, reverse transcriptase quantitative polymerase chain reaction (RTqPCR), immunocytology and microscopy.

Results     

Infiltration of BM with GD2 positive NB cells was 10% (range 0.02 – 43%). Spheroid forming efficiency from a single cell was 6% (range 0.5-27%) and colony formation efficiency in soft agar 3% (range 0-9%). Migratory cells were identified in all NB cultures; the migration index was highly heterogeneous (126; range 10-428). Cells were isolated that expressed tyrosine hydroxylase and nestin, but not NB84, CD57 or MRP4. Interestingly the ABC transporter protein MRP-1 was expressed in cells from 96% of cultures. Cell cultures could not be established from the GD2 negative cells. Importantly clonal populations of self-renewing cells have been isolated, and propagated. HIF-1, PI3K-AKT and ErbB signalling pathways are highly expressed in self-renewing NB cells (n=38) isolated from the bone marrow, compared to published pathway profiles in primary tumour.

Conclusion

NB cells with heterogeneous self-renewing capacity and a migratory phenotype have been successfully isolated and maintained in culture from BM aspirates taken at diagnosis from children with stage 4 disease. Signalling pathways have been identified which might in the future be exploited for the development of treatment targeting bone marrow disease.