Improving susceptibility of neuroendocrine tumors to radionuclide therapies: personalized approaches towards complementary treatments.

Radionuclide therapies are an important tool for the management of patients with neuroendocrine neoplasms (NENs). Especially [131I]MIBG and [177Lu]Lu-DOTA-TATE are routinely used for the treatment of a subset of NENs, including pheochromocytomas, paragangliomas and gastroenteropancreatic tumors. Some patients suffering from other forms of NENs, such as medullary thyroid carcinoma or neuroblastoma, were shown to respond to radionuclide therapy; however, no general recommendations exist. Although [131I]MIBG and [177Lu]Lu-DOTA-TATE can delay disease progression and improve quality of life, complete remissions are achieved rarely. Hence, better individually tailored combination regimes are required. This review summarizes currently applied radionuclide therapies in the context of NENs and informs about recent advances in the development of theranostic agents that might enable targeting subgroups of NENs that previously did not respond to [131I]MIBG or [177Lu]Lu-DOTA-TATE. Moreover, molecular pathways involved in NEN tumorigenesis and progression that mediate features of radioresistance and are particularly related to the stemness of cancer cells are discussed. Pharmacological inhibition of such pathways might result in radiosensitization or general complementary antitumor effects in patients with certain genetic, transcriptomic, or metabolic characteristics. Finally, we provide an overview of approved targeted agents that might be beneficial in combination with radionuclide therapies in the context of a personalized molecular profiling approach.

Impact of Viscosity on Human Hepatoma Spheroids in Soft Core-Shell Microcapsules.

The extracellular environment regulates the structures and functions of cells, from the molecular to the tissue level. However, the underlying mechanisms influencing the organization and adaptation of cancer in three-dimensional (3D) environments are not yet fully understood. In this study, the influence of the viscosity of the environment is investigated on the mechanical adaptability of human hepatoma cell (HepG2) spheroids in vitro, using 3D microcapsule reactors formed with droplet-based microfluidics. To mimic the environment with different mechanical properties, HepG2 cells are encapsulated in alginate core-shell reservoirs (i.e., microcapsules) with different core viscosities tuned by incorporating carboxymethylcellulose. The significant changes in cell and spheroid distribution, proliferation, and cytoskeleton are observed and quantified. Importantly, changes in the expression and distribution of F-actin and keratin 8 indicate the relation between spheroid stiffness and viscosity of the surrounding medium. The increase of F-actin levels in the viscous medium can indicate an enhanced ability of tumor cells to traverse dense tissue. These results demonstrate the ability of cancer cells to dynamically adapt to the changes in extracellular viscosity, which is an important physical cue regulating tumor development, and thus of relevance in cancer biology.

Anti-Cancer Prodrug Cyclophosphamide Exerts Thrombogenic Effects on Human Venous Endothelial Cells Independent of CYP450 Activation-Relevance to Thrombosis.

Cancer patients are at a very high risk of serious thrombotic events, often fatal. The causes discussed include the detachment of thrombogenic particles from tumor cells or the adverse effects of chemotherapeutic agents. Cytostatic agents can either act directly on their targets or, in the case of a prodrug approach, require metabolization for their action. Cyclophosphamide (CPA) is a widely used cytostatic drug that requires prodrug activation by cytochrome P450 enzymes (CYP) in the liver. We hypothesize that CPA could induce thrombosis in one of the following ways: (1) damage to endothelial cells (EC) after intra-endothelial metabolization; or (2) direct damage to EC without prior metabolization. In order to investigate this hypothesis, endothelial cells (HUVEC) were treated with CPA in clinically relevant concentrations for up to 8 days. HUVECs were chosen as a model representing the first place of action after intravenous CPA administration. No expression of CYP2B6, CYP3A4, CYP2C9 and CYP2C19 was found in HUVEC, but a weak expression of CYP2C18 was observed. CPA treatment of HUVEC induced DNA damage and a reduced formation of an EC monolayer and caused an increased release of prostacyclin (PGI2) and thromboxane (TXA) associated with a shift of the PGI2/TXA balance to a prothrombotic state. In an in vivo scenario, such processes would promote the risk of thrombus formation.

Shell engineering in soft alginate-based capsules for culturing liver spheroids.

Functional interaction between cancer cells and the surrounding microenvironment is still not sufficiently understood, which motivates the tremendous interest for the development of numerous in vitro tumor models. Diverse parameters, for example, transport of nutrients and metabolites, availability of space in the confinement, etc. make an impact on the size, shape, and metabolism of the tumoroids. We demonstrate the fluidics-based low-cost methodology to reproducibly generate the alginate and alginate-chitosan microcapsules and apply it to grow human hepatoma (HepG2) spheroids of different dimensions and geometries. Focusing specifically on the composition and thickness of the hydrogel shell, permeability of the microcapsules was selectively tuned. The diffusion of the selected benchmark molecules through the shell has been systematically investigated using both, experiments and simulations, which is essential to ensure efficient mass transfer and/or filtering of the biochemical species. Metabolic activity of spheroids in microcapsules was confirmed by tracking the turnover of testosterone to androstenedione with chromatography studies in a metabolic assay. Depending on available space, phenotypically different 3D cell assemblies have been observed inside the capsules, varying in the tightness of cell aggregations and their shapes. Conclusively, we believe that our system with the facile tuning of the shell thickness and permeability, represents a promising platform for studying the formation of cancer spheroids and their functional interaction with the surrounding microenvironment.

Investigation of Radiotracer Metabolic Stability In Vitro with CYP-Overexpressing Hepatoma Cell Lines.

The characterization of novel radiotracers toward their metabolic stability is an essential part of their development. While in vitro methods such as liver microsome assays or ex vivo blood or tissue samples provide information on overall stability, little or no information is obtained on cytochrome P450 (CYP) enzyme and isoform-specific contribution to the metabolic fate of individual radiotracers. Herein, we investigated recently established CYP-overexpressing hepatoblastoma cell lines (HepG2) for their suitability to study the metabolic stability of radiotracers in general and to gain insight into CYP isoform specificity. Wildtype HepG2 and CYP1A2-, CYP2C19-, and CYP3A4-overexpressing HepG2 cells were incubated with radiotracers, and metabolic turnover was analyzed. The optimized protocol, covering cell seeding in 96-well plates and analysis of supernatant by radio thin-layer-chromatography for higher throughput, was transferred to the evaluation of three 18F-labeled celecoxib-derived cyclooxygenase-2 inhibitors (coxibs). These investigations revealed time-dependent degradation of the intact radiotracers, as well as CYP isoform- and substrate-specific differences in their metabolic profiles. HepG2 CYP2C19 proved to be the cell line showing the highest metabolic turnover for each radiotracer studied here. Comparison with human and murine liver microsome assays showed good agreement with the human metabolite profile obtained by the HepG2 cell lines. Therefore, CYP-overexpressing HepG2 cells provide a good complement for assessing the metabolic stability of radiotracers and allow the analysis of the CYP isoform-specific contribution to the overall radiotracer metabolism.