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.

Aquaporin 1 (AQP1) is one of thirteen known mammalian aquaporins. Its main function is the transport of water across cell membranes. Lately, a role of AQP has been attributed to other physiological and pathological functions including cell migration and peripheral pain perception. AQP1 has been found in several parts of the enteric nervous system, e.g., in the rat ileum and in the ovine duodenum. Its function in the intestine appears to be multifaceted and is still not completely understood. The aim of the study was to analyze the distribution and localization of AQP1 in the entire intestinal tract of mice. AQP1 expression was correlated with the hypoxic expression profile of the various intestinal segments, intestinal wall thickness and edema, as well as other aspects of colon function including the ability of mice to concentrate stools and their microbiome composition. AQP1 was found in a specific pattern in the serosa, the mucosa, and the enteric nervous system throughout the gastrointestinal tract. The highest amount of AQP1 in the gastrointestinal tract was found in the small intestine. AQP1 expression correlated with the expression profiles of hypoxia-dependent proteins such as HIF-1α and PGK1. Loss of AQP1 through knockout of AQP1 in these mice led to a reduced amount of bacteroidetes and firmicutes but an increased amount of the rest of the phyla, especially deferribacteres, proteobacteria, and verrucomicrobia. Although AQP-KO mice retained gastrointestinal function, distinct changes regarding the anatomy of the intestinal wall including intestinal wall thickness and edema were observed. Loss of AQP1 might interfere with the ability of the mice to concentrate their stool and it is associated with a significantly different composition of the of the bacterial stool microbiome.

Neuroblastoma, like other cancer types, has an increased need for energy. This results in an increased thermogenic profile of the cells. How tumor cells optimize their energy efficiency has been discussed since Warburg described the fact that tumor cells prefer an anaerobic to an aerobic metabolism in the 1920s. An important question is how far the energy efficiency is influenced by the substrate. The aim of this study was to investigate how the metabolic activity of neuroblastoma cells is stimulated by addition of glucose or fructose to the medium and if this can be measured accurately by using isothermal microcalorimetry. Proliferation of Kelly and SH-EP Tet-21/N cells was determined in normal medium, in fructose-enriched, in glucose-enriched and in a fructose/glucose-enriched environment. Heat development of cells was measured by isothermal microcalorimetry. The addition of fructose, glucose or both to the medium led to increases in the metabolic activity of the cells, resulting in increased proliferation under the influence of fructose. These changes were reflected in an enhanced thermogenic profile, mirroring the results of the proliferation assay. The tested neuroblastoma cells prefer fructose metabolism over glucose metabolism, a quality that provides them with a survival benefit under unfavorable low oxygen and low nutrient supply when fructose is available. This can be quantified by measuring thermogenesis.

Neuroblastoma is a biologically very heterogeneous tumor with its clinical manifestation ranging from spontaneous regression to highly aggressive metastatic disease. Several adverse factors have been linked to oncogenesis, tumor progression and metastases of neuroblastoma including NMYC amplification, the neural adhesion molecule NCAM, as well as CXCR4 as a promoter of metastases. In this study, we investigate to what extent the expression of AQP1 in neuroblastoma correlates with changing cellular factors such as the hypoxic status, differentiation, expression of known adverse factors such as NMYC and NCAM, and CXCR4-related metastatic spread. Our results show that while AQP1 expression leads to an increased migratory behavior of neuroblastoma cells under hypoxic conditions, we find that hypoxia is associated with a reduction of NMYC in the same cells. A similar effect can be observed when using the tetracycline driven mechanism of SH-EP/Tet cells. When NMYC is not expressed, the expression of AQP1 is increased together with an increased expression of HIF-1α and HIF-2α. We furthermore show that when growing cells in different cell densities, they express AQP1, HIF-1α, HIF-2α, NMYC and NCAM to different degrees. AQP1 expression correlates with a hypoxic profile of these cells with increased HIF-1α and HIF-2α expression, as well as with NMYC and NCAM expression in two out of three neuroblastoma cell lines. When investigating cell properties of the cells that actually migrate, we find that the increased APQ1 expression in the migrated cells correlates with an increased NMYC and NCAM expression again in two out of three cell lines. Expression of the tumor cell homing marker CXCR4 varies between different tumor areas and between cell lines. While some migrated tumor cells highly express CXCR4, cells of other origin do not. In the initial phase of migration, we determined a dominant role of AQP1 expression of migrating cells in the scratch assay.

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.

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 pote