AQP1 Is Up-Regulated by Hypoxia and Leads to Increased Cell Water Permeability, Motility, and Migration in Neuroblastoma

The water channel aquaporin 1 (AQP1) has been implicated in tumor progression and metastasis. It is hypothesized that AQP1 expression can facilitate the transmembrane water transport leading to changes in cell structure that promote migration. Its impact in neuroblastoma has not been addressed so far. The objectives of this study have been to determine whether AQP1 expression in neuroblastoma is dependent on hypoxia, to demonstrate whether AQP1 is functionally relevant for migration, and to further define AQP1-dependent properties of the migrating cells. This was determined by investigating the reaction of neuroblastoma cell lines, particularly SH-SY5Y, Kelly, SH-EP Tet-21/N and SK-N-BE(2)-M17 to hypoxia, quantitating the AQP1-related water permeability by stopped-flow spectroscopy, and studying the migration-related properties of the cells in a modified transwell assay. We find that AQP1 expression in neuroblastoma cells is up-regulated by hypoxic conditions, and that increased AQP1 expression enabled the cells to form a phenotype which is associated with migratory properties and increased cell agility. This suggests that the hypoxic tumor microenvironment is the trigger for some tumor cells to transition to a migratory phenotype. We demonstrate that migrating tumor cell express elevated AQP1 levels and a hypoxic biochemical phenotype. Our experiments strongly suggest that elevated AQP1 might be a key driver in transitioning stable tumor cells to migrating tumor cells in a hypoxic microenvironment

Metastatic Esophageal Carcinoma Cells Exhibit Reduced Adhesion Strength and Enhanced Thermogenesis

Despite continuous improvements in multimodal therapeutic strategies, esophageal carcinoma maintains a high mortality rate. Metastases are a major life-limiting component; however, very little is known about why some tumors have high metastatic potential and others not. In this study, we investigated thermogenic activity and adhesion strength of primary tumor cells and corresponding metastatic cell lines derived from two patients with metastatic adenocarcinoma of the esophagus. We hypothesized that the increased metastatic potential of the metastatic cell lines correlates with higher thermogenic activity and decreased adhesion strength. Our data show that patient-derived metastatic esophageal tumor cells have a higher thermogenic profile as well as a decreased adhesion strength compared to their corresponding primary tumor cells. Using two paired esophageal carcinoma cell lines of primary tumor and lymph nodes makes the data unique. Both higher specific thermogenesis profile and decreased adhesion strength are associated with a higher metastatic potential. They are in congruence with the clinical patient presentation. Understanding these functional, biophysical properties of patient derived esophageal carcinoma cell lines will enable us to gain further insight into the mechanisms of metastatic potential of primary tumors and metastases. Microcalorimetric evaluation will furthermore allow for rapid assessment of new treatment options for primary tumor and metastases aimed at decreasing the metastatic potential

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

Perspective on Similarities and Possible Overlaps of Congenital Disease Formation—Exemplified on a Case of Congenital Diaphragmatic Hernia and Neuroblastoma in a Neonate

The coincidence of two rare diseases such as congenital diaphragmatic hernia (CDH) and neuroblastoma is exceptional. With an incidence of around 2–3:10,000 and 1:8000 for either disease occurring on its own, the chance of simultaneous presentation of both pathologies at birth is extremely low. Unfortunately, the underlying processes leading to congenital malformation and neonatal tumors are not yet thoroughly understood. There are several hypotheses revolving around the formation of CDH and neuroblastoma. The aim of our study was to put the respective hypotheses of disease formation as well as known factors in this process into perspective regarding their similarities and possible overlaps of congenital disease formation. We present the joint occurrence of these two rare diseases based on a patient presentation and immunochemical prognostic marker evaluation. The aim of this manuscript is to elucidate possible similarities in the pathogeneses of both disease entities. Discussed are the role of toxins, cell differentiation, the influence of retinoic acid and NMYC as well as of hypoxia. The detailed discussion reveals that some of the proposed pathophysiological mechanisms of both malformations have common aspects. Especially disturbances of the retinoic acid pathway and NMYC expression can influence and disrupt cell differentiation in either disease. Due to the rarity of both diseases, interdisciplinary efforts and multi-center studies are needed to investigate the reasons for congenital malformations and their interlinkage with neonatal tumor disease

Perfusion-Based Bioreactor Culture and Isothermal Microcalorimetry for Preclinical Drug Testing with the Carbonic Anhydrase Inhibitor SLC-0111 in Patient-Derived Neuroblastoma

Neuroblastoma is a rare disease. Rare are also the possibilities to test new therapeutic options for neuroblastoma in clinical trials. Despite the constant need to improve therapy and outcomes for patients with advanced neuroblastoma, clinical trials currently only allow for testing few substances in even fewer patients. This increases the need to improve and advance preclinical models for neuroblastoma to preselect favorable candidates for novel therapeutics. Here we propose the use of a new patient-derived 3D slice-culture perfusion-based 3D model in combination with rapid treatment evaluation using isothermal microcalorimetry exemplified with treatment with the novel carbonic anhydrase IX and XII (CAIX/CAXII) inhibitor SLC-0111. Patient samples showed a CAIX expression of 18% and a CAXII expression of 30%. Corresponding with their respective CAIX expression patterns, the viability of SH-EP cells was significantly reduced upon treatment with SLC-0111, while LAN1 cells were not affected. The inhibitory effect on SH-SY5Y cells was dependent on the induction of CAIX expression under hypoxia. These findings corresponded to thermogenesis of the cells. Patient-derived organotypic slice cultures were treated with SLC-0111, which was highly effective despite heterogeneity of CAIX/CAXII expression. Thermogenesis, in congruence with the findings of the histological observations, was significantly reduced in SLC-0111-treated samples. In order to extend the evaluation time, we established a perfusion-based approach for neuroblastoma tissue in a 3D perfusion-based bioreactor system. Using this system, excellent tissue quality with intact tumor cells and stromal structure in neuroblastoma tumors can be maintained for 7 days. The system was successfully used for consecutive drug response monitoring with isothermal microcalorimetry. The described approach for drug testing, relying on an advanced 3D culture system combined with a rapid and highly sensitive metabolic assessment, can facilitate development of personalized treatment strategies for neuroblastoma