Exploration of neuroblastoma xenograft models for tumor extracellular RNA profiling in murine blood plasma

Background: Minimally invasive liquid biopsies are becoming increasingly important in the diagnosis and treatment follow-up of cancer patients, including children with neuroblastoma. Such biopsies contain various biomarker analytes, including extracellular RNA (exRNA) with the potential to reflect dynamic changes in the tumor. However, it is challenging to distinguish tumor-derived exRNA from normal RNA. To overcome this limitation, xenograft models serve as a practical tool. In a mouse engrafted with human tumor cells, human exRNA is by definition originating from the tumor, whereas murine exRNA is host-derived. To study treatment response by monitoring tumor-derived exRNA, xenograft models with a high release of tumor exRNA into the circulation are desirable. Methods: The aim of this study was to evaluate whether and to what extent the cell line, its engraftment site, or the tumor size influence the amount of tumoral exRNA detected in blood plasma. To that end, four different neuroblastoma cell lines were engrafted in nude mice, either subcutaneously in the flank or orthotopically in the adrenal gland. Tumor sizes were monitored by caliper measurements (subcutaneous grafts) or MRI scans (orthotopic grafts) and blood was collected via terminal cardiac puncture to evaluate the tumoral exRNA fraction. Results: We demonstrate that the tumoral exRNA levels are correlated with the size of the subcutaneous tumor grafts. These levels are also highly dependent on the engrafted cell line. Furthermore, orthotopic engraftment potentially results in superior levels of tumoral exRNA, likely because of higher vascularity of the tumor tissue. Conclusions: Factors as cell line, tumor size and injections site should carefully be considered when performing experiments to study circulating RNA biomarkers.