Innovative hypoxia-related factors in neuroblastoma metastasis and therapeutic methods

Pediatric cancer is one of the leading causes of death in children by disease worldwide even in developed countries. Neuroblastoma is derived from the neural crest and is one of the most common solid tumors among children and adolescents, particularly in infants under 1 year of age. It contributes to around 15% of pediatric cancer mortality. It most often occurs in the adrenal glands but can develop anywhere along the sympathetic nerve system (paraspinal ganglia, abdomen, neck and pelvis etc.). It presents in a highly heterogeneous manner, which contributes to difficulties in treatment. Despite the established therapy combining multiple chemoagents, patients who belong to the Children’s Oncology Group (COG) high-risk-group still have a dismal prognosis with a 5-year overall survival of < 50%. Patients in the high-risk group mainly die from metastatic disease. For patients with disease relapse the 5-year overall survival is <10%. Only few new treatment options have been introduced in the last decade, mostly limited to anti-GD2-based therapies.New potential targets for preventing or inhibiting metastatic disease in neuroblastoma are therefore urgently needed. In this study we focus on two hypoxia-induced prognostic factors, one associated with local progression, carbonic anhydrase IX (CAIX), and one associated with cell migration, aquaporin 1 (AQP1). AQP1 is a membrane channel protein that is responsible for both ion and water transport and has been reported as a player in metastasis of some adult tumors and as a promising target to inhibit migration. However, the role and function of AQP1 in neuroblastoma is still unrevealed.
In this study, we showed that the expression of AQP1 is more pronounced in stage IV neuroblastoma patients. As a solid tumor, neuroblastoma is often affected by tumor hypoxia. We could demonstrate that AQP1 was up-regulated in a hypoxia-dependent manner in neuroblastoma cells on both RNA and protein level. In order to get a better understanding of AQP1 function in neuroblastoma, we successfully established AQP1-overexpressing neuroblastoma cell lines by lentiviral transfection. We investigated functional aspects of these modified cells in several assays including wound healing, isothermal microcalorimetry (IMC) and shear stress assay. Compared to wild type cells, AQP1-overexpressing cells migrated more easily, were more thermogenic and less adhesive, suggesting a crucial role of AQP1 in neuroblastoma metastases. We continued by inhibiting the function of AQP1 overexpressing cells using several specific inhibitors of AQP1. In detail, a decrease of migration in the wound healing assay, a reduction of heat production in IMC and an increase of adhesion could be observed when applying the AQP1 inhibitors Aqb011 and Bacopaside II. Following these promising results in 2D cell culture, we modified a perfused bioreactor-based 3D culture system for neuroblastoma. We were able to successfully apply this system to both neuroblastoma cell lines and patient tumor slices. Interestingly, 3D neuroblastoma cell cultures presented with similar morphological features compared to neuroblastoma cells in intact tumor tissue featuring small, round and blue cells. The tissue slices kept their primary structure for at least 7 days in culture. This
perfused 3D model enabled us to culture fresh neuroblastoma tissue for a longer time compared to a static culture. This made it possible to apply different chemotherapy regimens and our small molecular compounds and perform a drug response assessment with IMC.
We furthermore investigated the inhibitory effect of a promising compound SLC-0111, a specific inhibitor of carbonic anhydrase IX (CAIX) and its isoform carbonic anhydrase XII (CAXII), which is currently in clinical trials for adult cancers. Treatment with SLC-0111 was highly effective in the in the neuroblastoma slice culture. It could be considered as a novel therapeutic in CAIX or CAXII positive patients.
Overall, we investigated the function of AQP1 in migrating neuroblastoma cells in the 2D culture and applied 3D culture models to refine neuroblastoma research. We found AQP1 could remarkedly promote neuroblastoma cell migration in vitro while AQP1 specific inhibitors could suppress this process. The newly established perfusion-based 3D culture system is a promising model for further research. SLC-0111, the inhibitor for CAIX and CAXII, could be a potential inhibitor for CAIX/CAXII-positive neuroblastoma patients.