Selective Targeting of Tumor Cells in a Microfluidic Tumor Model with Multiple Cell Types.

Organ-on-a-chip technology allows researchers to precisely monitor drug efficacy in 3D tissue culture systems that are physiologically more relevant to humans compared to 2D cultures and that allow better control over experimental conditions as compared to animal models. Specifically, the high control over microenvironmental conditions combined with the broad range of direct measurements that can be performed in these systems makes organ-on-a-chip devices a versatile tool to investigate tumor targeting and drug delivery. Here, we describe a detailed protocol for studying the cell-selective targeting of protein drugs to tumor cells on an organ-on-a-chip system using a co-culture consisting of BT-474 cancer cells and C5120 human fibroblasts as an example.

Potent and selective eradication of tumor cells by an EpCAM-targeted Ras-degrading enzyme.

Despite decades of efforts, an urgent need remains to develop tumor cell-selective rat sarcoma (Ras)-targeting therapies that can treat patients with Ras-driven tumors. Here we report modular engineered proteins that degrade Ras selectively in tumor cells that overexpress the tumor cell marker epithelial cell adhesion molecule (EpCAM) by fusing the Ras degrader Ras-Rap1-specific endopeptidase with the translocation domain of the Pseudomonas aeruginosa exotoxin A (ETA) or diphtheria toxin (DT). Redirection to EpCAM is achieved by a designed ankyrin repeat protein. In two-dimensional tumor cell cultures, complete degradation of Ras proteins after 24 h was observed with EpCAM-targeted Ras degraders fused to ETA or DT in EpCAM-overexpressing MCF7 and HCT116 cells, with median inhibition concentration values at sub-nanomolar levels. The viability of EpCAM-low non-cancerous fibroblasts remained unaffected. In a three-dimensional (3D) tumor-on-a-chip system that mimics the natural tumor microenvironment, effective Ras degradation and selective toxicity toward tumor cells, particularly with the ETA-fused constructs, was determined on-chip. To conclude, we demonstrate the potential of modular engineered proteins to kill tumor cells highly selectively by simultaneously exploiting EpCAM as a tumor-specific cell surface molecule as well as Ras as an intracellular oncotarget in a 3D system mimicking the natural tumor microenvironment.

Co-Culture of Glomerular Endothelial Cells and Podocytes in a Custom-Designed Glomerulus-on-a-Chip Model Improves the Filtration Barrier Integrity and Affects the Glomerular Cell Phenotype.

Crosstalk between glomerular endothelial cells and glomerular epithelial cells (podocytes) is increasingly becoming apparent as a crucial mechanism to maintain the integrity of the glomerular filtration barrier. However, in vitro studies directly investigating the effect of this crosstalk on the glomerular filtration barrier are scarce because of the lack of suitable experimental models. Therefore, we developed a custom-made glomerulus-on-a-chip model recapitulating the glomerular filtration barrier, in which we investigated the effects of co-culture of glomerular endothelial cells and podocytes on filtration barrier function and the phenotype of these respective cell types. The custom-made glomerulus-on-a-chip model was designed using soft lithography. The chip consisted of two parallel microfluidic channels separated by a semi-permeable polycarbonate membrane. The glycocalyx was visualized by wheat germ agglutinin staining and the barrier integrity of the glomerulus-on-a-chip model was determined by measuring the transport rate of fluorescently labelled dextran from the top to the bottom channel. The effect of crosstalk on the transcriptome of glomerular endothelial cells and podocytes was investigated via RNA-sequencing. Glomerular endothelial cells and podocytes were successfully cultured on opposite sides of the membrane in our glomerulus-on-a-chip model using a polydopamine and collagen A double coating. Barrier integrity of the chip model was significantly improved when glomerular endothelial cells were co-cultured with podocytes compared to monocultures of either glomerular endothelial cells or podocytes. Co-culture enlarged the surface area of podocyte foot processes and increased the thickness of the glycocalyx. RNA-sequencing analysis revealed the regulation of cellular pathways involved in cellular differentiation and cellular adhesion as a result of the interaction between glomerular endothelial cells and podocytes. We present a novel custom-made glomerulus-on-a-chip co-culture model and demonstrated for the first time using a glomerulus-on-a-chip model that co-culture affects the morphology and transcriptional phenotype of glomerular endothelial cells and podocytes. Moreover, we showed that co-culture improves barrier function as a relevant functional readout for clinical translation. This model can be used in future studies to investigate specific glomerular paracrine pathways and unravel the role of glomerular crosstalk in glomerular (patho) physiology.

Generation of Protein-Phosphorodiamidate Morpholino Oligomer Conjugates for Efficient Cellular Delivery via Anthrax Protective Antigen.

Phosphorodiamidate morpholino oligomers (PMOs) offer great promise as therapeutic agents for translation blocking or splice modulation due to their high stability and affinity for target sequences. However, in spite of their neutral charge as compared to natural oligonucleotides or phosphorothioate analogs, they still show little permeability for cellular membranes, highlighting the need for effective cytosolic delivery strategies. In addition, the implementation of strategies for efficient cellular targeting is highly desirable to minimize side effects and maximize the drug dose at its site of action. Anthrax toxin is a three-protein toxin of which the pore-forming protein anthrax protective antigen (PA) can be redirected to a receptor of choice and lethal factor (LF), one of the two substrate proteins, can be coupled to various cargoes for efficient cytosolic cargo delivery. In this protocol, we describe the steps to produce the proteins and protein conjugates required for cytosolic delivery of PMOs through the cation-selective pore generated by anthrax protective antigen. The method relies on the introduction of a unique cysteine at the C-terminal end of a truncated LF (aa 1-254), high-yield expression of the (truncated) toxin proteins in E. coli, functionalization of a PMO with a maleimide group and coupling of the maleimide-functionalized PMO to the unique cysteine on LF by maleimide-thiol conjugation chemistry. Through co-administration of PA with LF-PMO conjugates, an efficient cytosolic delivery of PMOs can be obtained.

Oxygen control: the often overlooked but essential piece to create better in vitro systems.

Variations in oxygen levels play key roles in numerous physiological and pathological processes, but are often not properly controlled in in vitro models, introducing a significant bias in experimental outcomes. Recent developments in microfluidic technology have introduced a paradigm shift by providing new opportunities to better mimic physiological and pathological conditions, which is achieved by both regulating and monitoring oxygen levels at the micrometre scale in miniaturized devices. In this review, we first introduce the nature and relevance of oxygen-dependent pathways in both physiological and pathological contexts. Subsequently, we discuss strategies to control oxygen in microfluidic devices, distinguishing between engineering approaches that operate at the device level during its fabrication and chemical approaches that involve the active perfusion of fluids oxygenated at a precise level or supplemented with oxygen-producing or oxygen-scavenging materials. In addition, we discuss readout approaches for monitoring oxygen levels at the cellular and tissue levels, focusing on electrochemical and optical detection schemes for high-resolution measurements directly on-chip. An overview of different applications in which microfluidic devices have been utilized to answer biological research questions is then provided. In the final section, we provide our vision for further technological refinements of oxygen-controlling devices and discuss how these devices can be employed to generate new fundamental insights regarding key scientific problems that call for emulating oxygen levels as encountered in vivo. We conclude by making the case that ultimately emulating physiological or pathological oxygen levels should become a standard feature in all in vitro cell, tissue, and organ models.

Mimicking the Biology of Engineered Protein and mRNA Nanoparticle Delivery Using a Versatile Microfluidic Platform.

To investigate the delivery of next-generation macromolecular drugs, such as engineered proteins and mRNA-containing nanoparticles, there is an increasing push towards the use of physiologically relevant disease models that incorporate human cells and do not face ethical dilemmas associated with animal use. Here, we illustrate the versatility and ease of use of a microfluidic platform for studying drug delivery using high-resolution microscopy in 3D. Using this microfluidic platform, we successfully demonstrate the specific targeting of carbonic anhydrase IX (CAIX) on cells overexpressing the protein in a tumor-mimicking chip system using affibodies, with CAIX-negative cells and non-binding affibodies as controls. Furthermore, we demonstrate this system's feasibility for testing mRNA-containing biomaterials designed to regenerate bone defects. To this end, peptide- and lipid-based mRNA formulations were successfully mixed with colloidal gelatin in microfluidic devices, while translational activity was studied by the expression of a green fluorescent protein. This microfluidic platform enables the testing of mRNA delivery from colloidal biomaterials of relatively high densities, which represents a first important step towards a bone-on-a-chip platform. Collectively, by illustrating the ease of adaptation of our microfluidic platform towards use in distinct applications, we show that our microfluidic chip represents a powerful and flexible way to investigate drug delivery in 3D disease-mimicking culture systems that recapitulate key parameters associated with in vivo drug application.

A Hybrid In Silico and Tumor-on-a-Chip Approach to Model Targeted Protein Behavior in 3D Microenvironments.

To rationally improve targeted drug delivery to tumor cells, new methods combining in silico and physiologically relevant in vitro models are needed. This study combines mathematical modeling with 3D in vitro co-culture models to study the delivery of engineered proteins, called designed ankyrin repeat proteins (DARPins), in biomimetic tumor microenvironments containing fibroblasts and tumor cells overexpressing epithelial cell adhesion molecule (EpCAM) or human epithelial growth factor receptor (HER2). In multicellular tumor spheroids, we observed strong binding-site barriers in combination with low apparent diffusion coefficients of 1 µm2·s-1 and 2 µm2 ·s-1 for EpCAM- and HER2-binding DARPin, respectively. Contrasting this, in a tumor-on-a-chip model for investigating delivery in real-time, transport was characterized by hindered diffusion as a consequence of the lower local tumor cell density. Finally, simulations of the diffusion of an EpCAM-targeting DARPin fused to a fragment of Pseudomonas aeruginosa exotoxin A, which specifically kills tumor cells while leaving fibroblasts untouched, correctly predicted the need for concentrations of 10 nM or higher for extensive tumor cell killing on-chip, whereas in 2D models picomolar concentrations were sufficient. These results illustrate the power of combining in vitro models with mathematical modeling to study and predict the protein activity in complex 3D models.

A Computational Investigation of In Vivo Cytosolic Protein Delivery for Cancer Therapy.

The ability to specifically block or degrade cytosolic targets using therapeutic proteins would bring tremendous therapeutic opportunities in cancer therapy. Over the last few years, significant progress has been made with respect to tissue targeting, cytosolic delivery, and catalytic inactivation of targets, placing this aim within reach. Here, we developed a mathematical model specifically built for the evaluation of approaches towards cytosolic protein delivery, involving all steps from systemic administration to translocation into the cytosol and target engagement. Focusing on solid cancer tissues, we utilized the model to investigate the effects of microvascular permeability, receptor affinity, the cellular density of targeted receptors, as well as the mode of activity (blocking/degradation) on therapeutic potential. Our analyses provide guidance for the rational optimization of protein design for enhanced activity and highlight the importance of tuning the receptor affinity as a function of receptor density as well as the receptor internalization rate. Furthermore, we provide quantitative insights into how enzymatic cargoes can enhance the distribution, extent, and duration of therapeutic activity, already at very low catalytic rates. Our results illustrate that with current protein engineering approaches, the goal of delivery of cytosolic delivery of proteins for therapeutic effects is well within reach.

Assessing the use of tumor-specific DARPin-toxin fusion proteins for ex vivo purging of cancer metastases from human ovarian cortex before autotransplantation.

OBJECTIVE: To assess the use of tumor-specific designed ankyrin repeat proteins (DARPins) fused to a domain of Pseudomonas aeruginosa exotoxin A for purging of cancer metastases from the ovarian cortex. DESIGN: Experimental study. SETTING: University medical center. PATIENT(S): Human ovarian cortex. INTERVENTION(S): Ovarian cortex harboring artificially induced breast cancer metastases was treated with DARPins targeted to epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2). MAIN OUTCOME MEASURE(S): The presence of any remaining cancer cells after purging was analyzed by (immuno)histochemistry and reverse transcriptase polymerase chain reaction. Effects on the viability of the ovarian cortex were determined by (immuno)histology, a follicular viability assay, and an assay to determine the in vitro growth capacity of small follicles. RESULT(S): After purging with EpCAM-targeted DARPin, all EpCAM-positive breast cancer cells were eradicated from the ovarian cortex. Although treatment had no effect on the morphology or viability of small follicles, a sharp decrease in oocyte viability during in vitro growth was observed, presumably due to low-level expression of EpCAM on oocytes. The HER2-targeted DARPins had no detrimental effects on the morphology, viability, or in vitro growth of small follicles. HER2-positive breast cancer foci were fully eliminated from the ovarian cortex, and the reverse transcriptase polymerase chain reaction showed a decrease to basal levels of HER2 mRNA after purging. CONCLUSION(S): Purging cancer metastases from ovarian cortex without impairing ovarian tissue integrity is possible by targeting tumor cell surface proteins with exotoxin A-fused DARPins. By adapting the target specificity of the cytotoxic DARPin fusions, it should be possible to eradicate metastases from all types of malignancies.

EpCAM-Binding DARPins for Targeted Photodynamic Therapy of Ovarian Cancer.

Ovarian cancer is the most lethal gynecological malignancy due to late detection associated with dissemination throughout the abdominal cavity. Targeted photodynamic therapy (tPDT) aimed at epithelial cell adhesion molecule (EpCAM), overexpressed in over 90% of ovarian cancer metastatic lesions, is a promising novel therapeutic modality. Here, we tested the specificity and activity of conjugates of EpCAM-directed designed ankyrin repeat proteins (DARPins) with the photosensitizer IRDye 700DX in in vitro and in vivo ovarian cancer models. EpCAM-binding DARPins (Ec1: Kd = 68 pM; Ac2: Kd = 130 nM) and a control DARPin were site-specifically functionalized with fluorophores or IRDye 700DX. Conjugation of anti-EpCAM DARPins with fluorophores maintained EpCAM-specific binding in cell lines and patient-derived ovarian cancer explants. Penetration of DARPin Ec1 into tumor spheroids was slower than that of Ac2, indicative of a binding site barrier effect for Ec1. DARPin-IRDye 700DX conjugates killed EpCAM-expressing cells in a highly specific and illumination-dependent fashion in 2D and 3D cultures. Furthermore, they effectively homed to EpCAM-expressing subcutaneous OV90 xenografts in mice. In conclusion, the high activity and specificity observed in preclinical ovarian cancer models, combined with a high specificity in patient material, warrant a further investigation of EpCAM-targeted PDT for ovarian cancer.

Metabolic Switching of Tumor Cells under Hypoxic Conditions in a Tumor-on-a-chip Model.

Hypoxia switches the metabolism of tumor cells and induces drug resistance. Currently, no therapeutic exists that effectively and specifically targets hypoxic cells in tumors. Development of such therapeutics critically depends on the availability of in vitro models that accurately recapitulate hypoxia as found in the tumor microenvironment. Here, we report on the design and validation of an easy-to-fabricate tumor-on-a-chip microfluidic platform that robustly emulates the hypoxic tumor microenvironment. The tumor-on-a-chip model consists of a central chamber for 3D tumor cell culture and two side channels for medium perfusion. The microfluidic device is fabricated from polydimethylsiloxane (PDMS), and oxygen diffusion in the device is blocked by an embedded sheet of polymethyl methacrylate (PMMA). Hypoxia was confirmed using oxygen-sensitive probes and the effect on the 3D tumor cell culture investigated by a pH-sensitive dual-labeled fluorescent dextran and a fluorescently labeled glucose analogue. In contrast to control devices without PMMA, PMMA-containing devices gave rise to decreases in oxygen and pH levels as well as an increased consumption of glucose after two days of culture, indicating a rapid metabolic switch of the tumor cells under hypoxic conditions towards increased glycolysis. This platform will open new avenues for testing anti-cancer therapies targeting hypoxic areas.

CDK5 knockdown in astrocytes provide neuroprotection as a trophic source via Rac1.

Astrocytes perform metabolic and structural support functions in the brain and contribute to the integrity of the blood-brain barrier. Astrocytes influence neuronal survival and prevent gliotoxicity by capturing glutamate (Glu), reactive oxygen species, and nutrients. During these processes, astrocytic morphological changes are supported by actin cytoskeleton remodeling and require the involvement of Rho GTPases, such as Rac1. The protein cyclin-dependent kinase 5 (CDK5) may have a dual effect on astrocytes because it has been shown to be involved in migration, senescence, and the dysfunction of glutamate recapture; however, its role in astrocytes remains unclear. Treating a possible deregulation of CDK5 with RNAi is a strategy that has been proposed as a therapy for neurodegenerative diseases. Models of glutamate gliotoxicity in the C6 astroglioma cell line, primary cultures of astrocytes, and co-cultures with neurons were used to analyze the effects of CDK5 RNAi in astrocytes and the role of Rac1 in neuronal viability. In C6 cells and primary astrocytes, CDK5 RNAi prevented the cell death generated by glutamate-induced gliotoxicity, and this finding was corroborated by pharmacological inhibition with roscovitine. This effect was associated with the appearance of lamellipodia, protrusions, increased cell area, stellation, Rac1 activation, BDNF release, and astrocytic protection in neurons that were exposed to glutamate excitotoxicity. Interestingly, Rac1 inhibition in astrocytes blocked BDNF upregulation and the astrocyte-mediated neuroprotection. Actin cytoskeleton remodeling and stellation may be a functional phenotype for BDNF release that promotes neuroprotection. In summary, our findings suggest that CDK5- knockdown in astrocytes acts as a trophic source for neuronal protection in a Rac1-dependent manner.