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.

Chondrosarcoma is the most common type of primary bone sarcoma in adults but with a high risk of local recurrence and metastasis. Most chondrosarcomas are resistant to chemotherapy and radiotherapy, which means that surgery is the only effective treatment option for the majority of patients. Therefore, new therapeutic targets are required. CD44 is a transmembrane protein that has roles in cell proliferation, adhesion and migration and it has already been shown to be overexpressed in several cancer types. In this study, chondrosarcoma patient tissue sections were characterised for CD44 expression using immunohistochemistry. The relationship between CD44 expression in the patient tissue and overall and event-free survival was then investigated. CRISPR/Cas9 gene editing was also used to knockout CD44 from chondrosarcoma cells which were used in a spheroid invasion assay to understand the role of CD44 in chondrosarcoma cell invasion. Cox multivariate analysis of CD44 expression by chondrosarcoma patient tissue revealed that high CD44 expression was linked to decreased overall and event-free survival. Furthermore, the invasion of the CD44 knockout cells in the spheroid invasion assay was less than the invasion of the wild-type cells. Increased expression of CD44 for intermediate and high grades of chondrosarcoma as well as reduced invasion of CD44 knockout cells suggests that CD44 plays an important role in chondrosarcoma metastasis. CD44 is therefore worthy of further investigation as an imaging and therapeutic target.