Aldehyde Dehydrogenase 2 (ALDH2): A novel sorafenib target in hepatocellular carcinoma unraveled by the proteome-wide cellular thermal shift assay.

Sorafenib is a multikinase inhibitor indicated for first-line treatment of unresectable hepatocellular carcinoma. Despite its widespread use in the clinic, the existing knowledge of sorafenib mode-of-action remains incomplete. To build upon the current understanding, we used the Cellular Thermal Shift Assay (CETSA) coupled to Mass Spectrometry (CETSA-MS) to monitor compound binding to its target proteins in the cellular context on a proteome-wide scale. Among the potential sorafenib targets, we identified aldehyde dehydrogenase 2 (ALDH2), an enzyme that plays a major role in alcohol metabolism. We validated the interaction of sorafenib with ALDH2 by orthogonal methods using pure recombinant protein, proving that this interaction is not mediated by other cellular components. Moreover, we showed that sorafenib inhibits ALDH2 activity, supporting a functional role for this interaction. Finally, we were able to demonstrate that both ALDH2 protein expression and activity were reduced in sorafenib-resistant cells compared to the parental cell line. Overall, our study allowed the identification of ALDH2 as a novel sorafenib target and sheds light on its potential role in both hepatocellular carcinoma and sorafenib resistance condition.

Flexible 3D Cell-Based Platforms for the Discovery and Profiling of Novel Drugs Targeting Plasmodium Hepatic Infection.

The restricted pipeline of drugs targeting the liver stage of Plasmodium infection reflects the scarcity of cell models that mimic the human hepatic phenotype and drug metabolism, as well as Plasmodium hepatic infection. Using stirred-tank culture systems, spheroids of human hepatic cell lines were generated, sustaining a stable hepatic phenotype over 4 weeks of culture. Spheroids were employed in the establishment of 3D Plasmodium berghei infection platforms that relied on static or dynamic culture conditions. P. berghei invasion and development were recapitulated in the hepatic spheroids, yielding blood-infective merozoites. The translational potential of the 3D platforms was demonstrated by comparing the in vitro minimum inhibitory concentration of M5717, a compound under clinical development, with in vivo plasma concentrations that clear liver stage P. berghei in mice. Our results show that the 3D platforms are flexible and scalable and can predict the efficacy of antiplasmodial therapies, constituting a powerful tool for integration in drug discovery programs.

3D-3-culture: A tool to unveil macrophage plasticity in the tumour microenvironment.

The tumour microenvironment (TME) shapes disease progression and influences therapeutic response. Most aggressive solid tumours have high levels of myeloid cell infiltration, namely tumour associated macrophages (TAM). Recapitulation of the interaction between the different cellular players of the TME, along with the extracellular matrix (ECM), is critical for understanding the mechanisms underlying disease progression. This particularly holds true for prediction of therapeutic response(s) to standard therapies and interrogation of efficacy of TME-targeting agents. In this work, we explored a culture platform based on alginate microencapsulation and stirred culture systems to develop the 3D-3-culture, which entails the co-culture of tumour cell spheroids of non-small cell lung carcinoma (NSCLC), cancer associated fibroblasts (CAF) and monocytes. We demonstrate that the 3D-3-culture recreates an invasive and immunosuppressive TME, with accumulation of cytokines/chemokines (IL4, IL10, IL13, CCL22, CCL24, CXCL1), ECM elements (collagen type I, IV and fibronectin) and matrix metalloproteinases (MMP1/9), supporting cell migration and promoting cell-cell interactions within the alginate microcapsules. Importantly, we show that both the monocytic cell line THP-1 and peripheral blood-derived monocytes infiltrate the tumour tissue and transpolarize into an M2-like macrophage phenotype expressing CD68, CD163 and CD206, resembling the TAM phenotype in NSCLC. The 3D-3-culture was challenged with chemo- and immunotherapeutic agents and the response to therapy was assessed in each cellular component. Specifically, the macrophage phenotype was modulated upon treatment with the CSF1R inhibitor BLZ945, resulting in a decrease of the M2-like macrophages. In conclusion, the crosstalk between the ECM and tumour, stromal and immune cells in microencapsulated 3D-3-culture promotes the activation of monocytes into TAM, mimicking aggressive tumour stages. The 3D-3-culture constitutes a novel tool to study tumour-immune interaction and macrophage plasticity in response to external stimuli, such as chemotherapeutic and immunomodulatory drugs.

Three-dimensional co-culture of human hepatocytes and mesenchymal stem cells: improved functionality in long-term bioreactor cultures.

The development of human cell models that can efficiently restore hepatic functionality and cope with the reproducibility and scalability required for preclinical development poses a significant effort in tissue engineering and biotechnology. Primary cultures of human hepatocytes (HHs), the preferred model for in vitro toxicity testing, dedifferentiate and have short-term viability in two-dimensional (2D) cultures. In this study, hepatocytes isolated from human liver tissue were co-cultured with human bone marrow mesenchymal stem cells (BM-MSCs) as spheroids in automated, computer-controlled, stirred-tank bioreactors with perfusion operation mode. A dual-step inoculation strategy was used, resulting in an inner core of parenchymal liver tissue with an outer layer of stromal cells. Hepatocyte polarization and morphology as well as the mesenchymal phenotype of BM-MSCs were maintained throughout the culture period and the crosstalk between the two cell types was depicted. The viability, compact morphology and phenotypic stability of hepatocytes were enhanced in co-cultures in comparison to monocultures. Gene expression of phase I and II enzymes was higher and CYP3A4 and CYP1A2 activity was inducible until week 2 of culture, being applicable for repeated-dose toxicity testing. Moreover, the excretory activity was maintained in co-cultures and the biosynthetic hepatocellular functions (albumin and urea secretion) were not affected by the presence of BM-MSCs. This strategy might be extended to other hepatic cell sources and the characterization performed brings knowledge on the interplay between the two cell types, which may be relevant for therapeutic applications. Copyright © 2015 John Wiley & Sons, Ltd.

Drug screening in 3D in vitro tumor models: overcoming current pitfalls of efficacy read-outs.

There is cumulating evidence that in vitro 3D tumor models with increased physiological relevance can improve the predictive value of pre-clinical research and ultimately contribute to achieve decisions earlier during the development of cancer-targeted therapies. Due to the role of tumor microenvironment in the response of tumor cells to therapeutics, the incorporation of different elements of the tumor niche on cell model design is expected to contribute to the establishment of more predictive in vitro tumor models. This review is focused on the several challenges and adjustments that the field of oncology research is facing to translate these advanced tumor cells models to drug discovery, taking advantage of the progress on culture technologies, imaging platforms, high throughput and automated systems. The choice of 3D cell model, the experimental design, choice of read-outs and interpretation of data obtained from 3D cell models are critical aspects when considering their implementation in drug discovery. In this review, we foresee some of these aspects and depict the potential directions of pre-clinical oncology drug discovery towards improved prediction of drug efficacy.

Validation of Bioreactor and Human-on-a-Chip Devices for Chemical Safety Assessment.

Equipment and device qualification and test assay validation in the field of tissue engineered human organs for substance assessment remain formidable tasks with only a few successful examples so far. The hurdles seem to increase with the growing complexity of the biological systems, emulated by the respective models. Controlled single tissue or organ culture in bioreactors improves the organ-specific functions and maintains their phenotypic stability for longer periods of time. The reproducibility attained with bioreactor operations is, per se, an advantage for the validation of safety assessment. Regulatory agencies have gradually altered the validation concept from exhaustive "product" to rigorous and detailed process characterization, valuing reproducibility as a standard for validation. "Human-on-a-chip" technologies applying micro-physiological systems to the in vitro combination of miniaturized human organ equivalents into functional human micro-organisms are nowadays thought to be the most elaborate solution created to date. They target the replacement of the current most complex models-laboratory animals. Therefore, we provide here a road map towards the validation of such "human-on-a-chip" models and qualification of their respective bioreactor and microchip equipment along a path currently used for the respective animal models.

Adaptable stirred-tank culture strategies for large scale production of multicellular spheroid-based tumor cell models.

Currently there is an effort toward the development of in vitro cancer models more predictive of clinical efficacy. The onset of advanced analytical tools and imaging technologies has increased the utilization of spheroids in the implementation of high throughput approaches in drug discovery. Agitation-based culture systems are commonly proposed as an alternative method for the production of tumor spheroids, despite the scarce experimental evidence found in the literature. In this study, we demonstrate the robustness and reliability of stirred-tank cultures for the scalable generation of 3D cancer models. We developed standardized protocols to a panel of tumor cell lines from different pathologies and attained efficient tumor cell aggregation by tuning hydrodynamic parameters. Large numbers of spheroids were obtained (typically 1000-1500 spheroids/mL) presenting features of native tumors, namely morphology, proliferation and hypoxia gradients, in a cell line-dependent mode. Heterotypic 3D cancer models, based on co-cultures of tumor cells and fibroblasts, were also established in the absence or presence of additional physical support from an alginate matrix, with maintenance of high cell viability. Altogether, we demonstrate that 3D tumor cell model production in stirred-tank culture systems is a robust and versatile approach, providing reproducible tools for drug screening and target verification in pre-clinical oncology research.

Modelling the tumour microenvironment in long-term microencapsulated 3D co-cultures recapitulates phenotypic features of disease progression.

3D cell tumour models are generated mainly in non-scalable culture systems, using bioactive scaffolds. Many of these models fail to reflect the complex tumour microenvironment and do not allow long-term monitoring of tumour progression. To overcome these limitations, we have combined alginate microencapsulation with agitation-based culture systems, to recapitulate and monitor key aspects of the tumour microenvironment and disease progression. Aggregates of MCF-7 breast cancer cells were microencapsulated in alginate, either alone or in combination with human fibroblasts, then cultured for 15 days. In co-cultures, the fibroblasts arranged themselves around the tumour aggregates creating distinct epithelial and stromal compartments. The presence of fibroblasts resulted in secretion of pro-inflammatory cytokines and deposition of collagen in the stromal compartment. Tumour cells established cell-cell contacts and polarised around small lumina in the interior of the aggregates. Over the culture period, there was a reduction in oestrogen receptor and membranous E-cadherin alongside loss of cell polarity, increased collective cell migration and enhanced angiogenic potential in co-cultures. These phenotypic alterations, typical of advanced stages of cancer, were not observed in the mono-cultures of MCF-7 cells. The proposed model system constitutes a new tool to study tumour-stroma crosstalk, disease progression and drug resistance mechanisms.

Establishing Liver Bioreactors for In Vitro Research.

In vitro systems that can effectively model liver function for long periods of time are fundamental tools for preclinical research. Nevertheless, the adoption of in vitro research tools at the earliest stages of drug development has been hampered by the lack of culture systems that offer the robustness, scalability, and flexibility necessary to meet industry's demands. Bioreactor-based technologies, such as stirred tank bioreactors, constitute a feasible approach to aggregate hepatic cells and maintain long-term three-dimensional cultures. These three-dimensional cultures sustain the polarity, differentiated phenotype, and metabolic performance of human hepatocytes. Culture in computer-controlled stirred tank bioreactors allows the maintenance of physiological conditions, such as pH, dissolved oxygen, and temperature, with minimal fluctuations. Moreover, by operating in perfusion mode, gradients of soluble factors and metabolic by-products can be established, aiming at resembling the in vivo microenvironment. This chapter provides a protocol for the aggregation and culture of hepatocyte spheroids in stirred tank bioreactors by applying perfusion mode for the long-term culture of human hepatocytes. This in vitro culture system is compatible with feeding high-throughput screening platforms for the assessment of drug elimination pathways, being a useful tool for toxicology research and drug development in the preclinical phase.

HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism.

The need for models that recapitulate liver physiology is perceived for drug development, study of liver disease and bioartificial liver support. The bipotent cell line HepaRG constitutes an efficient surrogate of liver function, yet its differentiated status relies on high concentrations of DMSO, which may compromise the study of drug metabolism and limit the applicability of this hepatic model. Herein, we present a three-dimensional (3D) strategy for the differentiation of HepaRG based on alginate microencapsulation of cell spheroids and culture in dimethyl sulfoxide (DMSO)-free conditions. A ratio of 2.9:1 hepatocyte-like to biliary-like cells was obtained in the 3D culture, with an improvement of 35.9 % in the hepatocyte differentiation when compared with two-dimensional (2D) cultures. The expression of the hepatic identity genes HNF4α and PXR in 3D cultures was comparable to 2D differentiated cultures, while the expression of homeostatic-associated genes albumin and carbamoyl phosphate synthase 1 was higher in 3D. Moreover, the spheroids presented a polarized organization, exhibiting an interconnected bile canalicular network and excretory functionality, assessed by specific activity of MRP2. Importantly, despite variability in basal gene expression levels, the activity of the phase I enzymes cytochrome P450 family 3, subfamily A, polypeptide 4 and cytochrome P450 family 1, subfamily A, polypeptide 2 upon induction was comparable to differentiated 2D cultures and albumin production and ammonia detoxification were enhanced in 3D. The presented model is suitable for toxicological applications, as it allows high throughput analysis of multiple compounds in a DMSO-free setting. Due to the high xenobiotic metabolism and maintenance of biosynthetic functions, the applicability of this model might be broadened to understand liver physiology and for bioartificial liver applications.