Current developments in modelling the tumour microenvironment in vitro:Incorporation of biochemical and physical gradients

Tumour cell proliferation, metabolism and treatment response depend on the dynamic interaction of the tumour cells with other cellular components and physicochemical gradients present in the tumour microenvironment. Traditional experimental approaches used to investigate the dynamic tumour tissue face a number of limitations, such as lack of biological relevance for the tumour microenvironment and the difficulty to precisely control fluctuating internal conditions, for example in oxygen and nutrients. The arrival of advanced in vitro models represents an alternative approach for modelling the tumour microenvironment using cutting-edge technologies, such as microfabrication. Advanced model systems provide a promising platform for modelling the physiochemical conditions of the tumour microenvironment in a well-controlled manner. Amongst others, advanced in vitro models aim to recreate gradients of oxygen, nutrients and endogenous chemokines, and cell proliferation. Furthermore, the establishment of mechanical cues within such models, e.g., flow and extracellular matrix properties that influence cellular behaviour, are active research areas. These model systems aim to maintain tumour cells in an environment that resembles in vivo conditions. A prominent example of such a system is the microfluidic tumour-on-chip model, which aims to precisely control the local chemical and physical environment that surrounds the tumour cells. In addition, these models also have the potential to recapitulate environmental conditions in isolation or in combination. This enables the analysis of the dynamic interactions between different conditions and their potentially synergistic effects on tumour cells. In this review, we will discuss the various gradients present within the tumour microenvironment and the effects they exert on tumour cells. We will further highlight the challenges and limitations of traditional experimental models in modelling these gradients. We will outline recent achievements in advanced in vitro models with a particular focus on tumour-on-chip systems. We will also discuss the future of these models in cancer research and their contribution to developing more biologically relevant models for cancer research

A Comparison of Cellular Uptake Mechanisms, Delivery Efficacy, and Intracellular Fate between Liposomes and Extracellular Vesicles

A key aspect for successful drug delivery via lipid-based nanoparticles is their internalization in target cells. Two prominent examples of such drug delivery systems are artificial phospholipid-based carriers, such as liposomes, and their biological counterparts, the extracellular vesicles (EVs). Despite a wealth of literature, it remains unclear which mechanisms precisely orchestrate nanoparticle-mediated cargo delivery to recipient cells and the subsequent intracellular fate of therapeutic cargo. In this review, internalization mechanisms involved in the uptake of liposomes and EVs by recipient cells are evaluated, also exploring their intracellular fate after intracellular trafficking. Opportunities are highlighted to tweak these internalization mechanisms and intracellular fates to enhance the therapeutic efficacy of these drug delivery systems. Overall, literature to date shows that both liposomes and EVs are predominantly internalized through classical endocytosis mechanisms, sharing a common fate: accumulation inside lysosomes. Studies tackling the differences between liposomes and EVs, with respect to cellular uptake, intracellular delivery and therapy efficacy, remain scarce, despite its importance for the selection of an appropriate drug delivery system. In addition, further exploration of functionalization strategies of both liposomes and EVs represents an important avenue to pursue in order to control internalization and fate, thereby improving therapeutic efficacy

Liposomes and Extracellular Vesicles as Drug Delivery Systems:A Comparison of Composition, Pharmacokinetics, and Functionalization

Over the past decades, lipid-based nanoparticle drug delivery systems (DDS) have caught the attention of researchers worldwide, encouraging the field to rapidly develop improved ways for effective drug delivery. One of the most prominent examples is liposomes, which are spherical shaped artificial vesicles composed of lipid bilayers and able to encapsulate both hydrophilic and hydrophobic materials. At the same time, biological nanoparticles naturally secreted by cells, called extracellular vesicles (EVs), have emerged as promising more complex biocompatible DDS. In this review paper, the differences and similarities in the composition of both vesicles are evaluated, and critical mediators that affect their pharmacokinetics are elucidate. Different strategies that have been assessed to tweak the pharmacokinetics of both liposomes and EVs are explored, detailing the effects on circulation time, targeting capacity, and cytoplasmic delivery of therapeutic cargo. Finally, whether a hybrid system, consisting of a combination of only the critical constituents of both vesicles, could offer the best of both worlds is discussed. Through these topics, novel leads for further research are provided and, more importantly, gain insight in what the liposome field and the EV field can learn from each other

The gastrointestinal microbiota in colorectal cancer cell migration and invasion

Colorectal carcinoma is the third most common cancer in developed countries and the second leading cause of cancer-related mortality. Interest in the influence of the intestinal microbiota on CRC emerged rapidly in the past few years, and the close presence of microbiota to the tumour mass creates a unique microenvironment in CRC. The gastrointestinal microbiota secrete factors that can contribute to CRC metastasis by influencing, for example, epithelial-to-mesenchymal transition. Although the role of EMT in metastasis is well-studied, mechanisms by which gastrointestinal microbiota contribute to the progression of CRC remain poorly understood. In this review, we will explore bacterial factors that contribute to the migration and invasion of colorectal carcinoma and the mechanisms involved. Bacteria involved in the induction of metastasis in primary CRC include Fusobacterium nucleatum, Enterococcus faecalis, enterotoxigenic Bacteroides fragilis, Escherichia coli and Salmonella enterica. Examples of prominent bacterial factors secreted by these bacteria include Fusobacterium adhesin A and Bacteroides fragilis Toxin. Most of these factors induce EMT-like properties in carcinoma cells and, as such, contribute to disease progression by affecting cell-cell adhesion, breakdown of the extracellular matrix and reorganisation of the cytoskeleton. It is of utmost importance to elucidate how bacterial factors promote CRC recurrence and metastasis to increase patient survival. So far, mainly animal models have been used to demonstrate this interplay between the host and microbiota. More human-based models are needed to study the mechanisms that promote migration and invasion and mimic the progression and recurrence of CRC

Improving Breast Cancer Treatment Specificity Using Aptamers Obtained by 3D Cell-SELEX

Three-dimensional spheroids of non-malignant MCF10A and malignant SKBR3 breast cells were used for subsequent 3D Cell-SELEX to generate aptamers for specific binding and treatment of breast cancer cells. Using 3D Cell-SELEX combined with Next-Generation Sequencing and bioinformatics, ten abundant aptamer families with specific structures were identified that selectively bind to SKBR3, and not to MCF10A cells. Multivalent aptamer polymers were synthesized by co-polymerization and analyzed for binding performance as well as therapeutic efficacy. Binding performance was determined by confocal fluorescence imaging and revealed specific binding and efficient internalization of aptamer polymers into SKBR3 spheroids. For therapeutic purposes, DNA sequences that intercalate the cytotoxic drug doxorubicin were co-polymerized into the aptamer polymers. Viability tests show that the drug-loaded polymers are specific and effective in killing SKBR3 breast cancer cells. Thus, the 3D-selected aptamers enhanced the specificity of doxorubicin against malignant over non-malignant breast cells. The innovative modular DNA aptamer platform based on 3D Cell SELEX and polymer multivalency holds great promise for diagnostics and treatment of breast cancer

Placenta-on-a-Chip as an In Vitro Approach to Evaluate the Physiological and Structural Characteristics of the Human Placental Barrier upon Drug Exposure:A Systematic Review

Quantification of fetal drug exposure remains challenging since sampling from the placenta or fetus during pregnancy is too invasive. Currently existing in vivo (e.g., cord blood sampling) and ex vivo (e.g., placenta perfusion) models have inherent limitations. A placenta-on-a-chip model is a promising alternative. A systematic search was performed in PubMed on 2 February 2023, and Embase on 14 March 2023. Studies were included where placenta-on-a-chip was used to investigate placental physiology, placenta in different obstetric conditions, and/or fetal exposure to maternally administered drugs. Seventeen articles were included that used comparable approaches but different microfluidic devices and/or different cultured maternal and fetal cell lines. Of these studies, four quantified glucose transfer, four studies evaluated drug transport, three studies investigated nanoparticles, one study analyzed bacterial infection and five studies investigated preeclampsia. It was demonstrated that placenta-on-a-chip has the capacity to recapitulate the key characteristics of the human placental barrier. We aimed to identify knowledge gaps and provide the first steps towards an overview of current protocols for developing a placenta-on-a-chip, that facilitates comparison of results from different studies. Although models differ, they offer a promising approach for in vitro human placental and fetal drug studies under healthy and pathological conditions.</p

Spheroid arrays for high-throughput single-cell analysis of spatial patterns and biomarker expression in 3D

We describe and share a device, methodology and image analysis algorithms, which allow up to 66 spheroids to be arranged into a gel-based array directly from a culture plate for downstream processing and analysis. Compared to processing individual samples, the technique uses 11-fold less reagents, saves time and enables automated imaging. To illustrate the power of the technology, we showcase applications of the methodology for investigating 3D spheroid morphology and marker expression and for in vitro safety and efficacy screens. Firstly, spheroid arrays of 11 cell-lines were rapidly assessed for differences in spheroid morphology. Secondly, highly-positive (SOX-2), moderately-positive (Ki-67) and weakly-positive (βIII-tubulin) protein targets were detected and quantified. Third, the arrays enabled screening of ten media compositions for inducing differentiation in human neurospheres. Lastly, the application of spheroid microarrays for spheroid-based drug-screens was demonstrated by quantifying the dose-dependent drop in proliferation and increase in differentiation in etoposide-treated neurospheres

Extracellular vesicles and soluble factors secreted by lung fibroblasts support alveolar organoid formation

Rationale: COPD is characterized by progressive and irreversible airflow limitation as a result of enhanced tissue destruction and defective tissue repair. As current therapeutics do not alter disease progression, new therapies that reactivate lung repair are needed. The secretome of fibroblasts, composed of Extracellular Vesicles (EVs) and other soluble factors (SF), has been linked to alveolar regeneration. We aimed to elucidate the supportive function of lung fibroblast-derived EVs and SF on the regenerative potential of alveolar epithelial progenitor cells in an organoid model.Methods: EVs and SF were purified using ultrafiltration and size exclusion chromatography. Mouse organoids were obtained by co-culturing 10,000 alveolar EpCAM+ cells with 2,500 lung fibroblasts, and then treated with EVs (109 EVs/ml) or SF (30 µg/ml). On day 14, number and size of the organoids was determined, as was the number of differentiated alveolar organoids.Results: Single treatment with EVs or SF increased organoid count, i.e. a 29.50% ± 8.11% increase for EVs and 33.00% ± 20.34% for SF. Neither treatment with EVs nor SF affected organoid size. Immunostaining for prosurfactant protein C revealed that the alveolar organoid count was significantly enhanced upon single treatment with EVs or SF. In addition, consecutive treatment for 14 days with EVs or SF resulted in enhanced organoid count (i.e. a 58.17% ± 39.60% and 91.67% ± 33.28% increase respectively) and size (i.e. a 36.50% ± 10.46% and 37.50% ± 27.02% increase respectively).Conclusions: Lung fibroblast-derived EVs and SF support alveolar epithelial organoid formation, making them an interesting potential treatment to pursue for COPD