# Detection and Characterisation of Circulating Tumour Cell Clusters in Neuroblastoma

## TL;DR

This study detects and characterizes clusters of circulating tumor cells in neuroblastoma patients, finding that most clusters contain mixed cell types and may play a role in cancer spread.

## Contribution

The study is the first to use imaging flow cytometry to detect and characterize CTC clusters in neuroblastoma.

## Key findings

- CTC clusters were detected in 79% of neuroblastoma patient blood samples.
- Most clusters (93.9%) contained tumor cells grouped with other cell types.
- Clusters contained between 2 and 8 cells, with heterotypic clusters being more common than homotypic ones.

## Abstract

Neuroblastoma is an aggressive childhood cancer. During disease progression, neuroblastoma tumours can release circulating tumour cells (CTCs) into the bloodstream. These cells can group together to form clusters, which may give rise to distant metastases and are linked with worse prognosis in other cancer types. In this study, we used imaging flow cytometry to detect and characterise CTC clusters present in blood samples from neuroblastoma patients. Clusters were detected in 79% of patients. Most clusters (93.9%) contained tumour cells grouped with other cell types, while a smaller proportion (6.1%) consisted of cancer cells only. Each cluster contained between 2 and 8 cells. Our study highlights the importance of studying CTC clusters in neuroblastoma and supports further research into the role of clusters in metastatic spread.

Background: Neuroblastoma (NB) is the most common extracranial paediatric solid cancer, with a 50% survival rate for high-risk patients. Circulating tumour cells (CTCs) are malignant cells shed by the primary tumour and metastatic sites that circulate in the bloodstream. CTCs form clusters with themselves (homotypic) or other cell types (heterotypic). Objectives: To use previously generated ImageStreamX Imaging Flow Cytometer data from blood samples from 24 patients across all NB risk groups, to examine for the presence of CTC clusters. Methods: Immunofluorescence and brightfield morphology were used to identify clusters followed by analysis using IDEAS image analysis software. Results: The mean number of clusters detected per sample was 87 (range, 0–725). Of the clusters detected, 1967/2094 (93.9%) were heterotypic and only 127/2094 (6.1%) were homotypic and found in 6/24 patients. Interestingly, in 3/24 patients, at least one cluster (median, 2 and range, 1–18) was found, but no single CTCs were detected. Clusters mostly comprised two cells (62.8%), with the maximum number of cells in homotypic and heterotypic clusters being four and eight, respectively. Conclusions: These results highlight that imaging flow cytometry can be used to detect and characterise CTC clusters in peripheral blood samples from NB patients, leading to further research exploring the composition and role of CTC clusters in NB metastasis.