The current development of microfluidics-based microphysiological systems (MPSs) will rapidly lead to a paradigm shift from traditional static 2-dimensional cell cultivation towards organized tissue ...culture within a dynamic cellular milieu. Especially organs-on-a-chip (OoCs) can very precisely re-create the mechanical and unique anatomical structures of the oral environment. This review provides an introduction to such technology, from commonly used chip materials and fabrication methods to the application of OoC in in vitro culture. OoCs are advantageous because of their small-scaled culture environment, the highly controlled dynamic experimental conditions, and the likeness to the in vivo structure. We specifically focus on current chip designs in dental, oral, and craniofacial (DOC) research. Also, future perspectives are discussed, like model standardization and the development of integrated platforms with advanced read-out functionality. By doing so, it will be possible for OoCs to serve as an alternative for animal testing and to develop highly predictive human models for clinical experiments and even personalized medicine.
Cancer immunotherapy has emerged as a promising approach in the treatment of diverse cancer types. However, the development of novel immunotherapeutic agents faces persistent challenges due to poor ...translation from preclinical to clinical stages. To address these challenges, the integration of microfluidic models in research efforts has recently gained traction, bridging the gap between in vitro and in vivo systems. This approach enables modeling of the complex human tumor microenvironment and interrogation of cancer-immune interactions. In this review, we analyze the current and potential applications of microfluidic tumor models in cancer immunotherapy development. We will first highlight current trends in the immunooncology landscape. Subsequently, we will discuss recent examples of microfluidic models applied to investigate mechanisms of immune-cancer interactions and for developing and screening cancer immunotherapies in vitro. First steps toward their validation for predicting human in vivo outcomes are discussed. Finally, promising opportunities that microfluidic tumor models offer are highlighted considering their advantages and current limitations, and we suggest possible next steps toward their implementation and integration into the immunooncology drug development process.
Cells with implants: Porous enzyme‐loaded polymersomes were constructed that display the cell‐penetrating peptide tat on their surfaces. These nanoreactors are taken up by mammalian cells through ...macropinocytosis. Inside the cells, the polymersomes are only partially routed to acidic compartments. Polymersomes with horseradish peroxidase as a model cargo enzyme displayed sustained intracellular activity.
The molecular events that contribute to the cellular uptake of cell-penetrating peptides (CPP) are still a matter of intense research. Here, we report on the identification and characterization of a ...22-amino acid CPP derived from the human milk protein, lactoferrin. The peptide exhibits a conformation-dependent uptake efficiency that is correlated with efficient binding to heparan sulfate and lipid-induced conformational changes. The peptide contains a disulfide bridge formed by terminal cysteine residues. At concentrations exceeding 10 μm, this peptide undergoes the same rapid entry into the cytoplasm that was described previously for the arginine-rich CPPs nona-arginine and Tat. Cytoplasmic entry strictly depends on the presence of the disulfide bridge. To better understand this conformation dependence, NMR spectroscopy was performed for the free peptide, and CD measurements were performed for free and lipid-bound peptide. In solution, the peptides showed only slight differences in secondary structure, with a predominantly disordered structure both in the presence and absence of the disulfide bridge. In contrast, in complex with large unilamellar vesicles, the conformation of the oxidized and reduced forms of the peptide clearly differed. Moreover, surface plasmon resonance experiments showed that the oxidized form binds to heparan sulfate with a considerably higher affinity than the reduced form. Consistently, membrane binding and cellular uptake of the peptide were reduced when heparan sulfate chains were removed.
Recent advances in engineering adenoviruses are paving the way for new therapeutic gene delivery approaches in cancer. However, there is limited knowledge regarding the impact of adenoviral ...retargeting on transduction efficiency in more complex tumor architectures, and the role of the RGD loop at the penton base in retargeting is unclear. To address this gap, we used tumor models of increasing complexity to study the role of the receptor and the RGD motif. Employing tumor-fibroblast co-culture models, we demonstrate the importance of the RGD motif for efficient transduction in 2D through the epithelial cell adhesion molecule (EpCAM), but not the epidermal growth factor receptor (EGFR). Via optical clearing of co-culture spheroids, we show that the RGD motif is required for transduction via both receptors in 3D tumor architectures. We subsequently employed a custom-designed microfluidic model containing collagen-embedded tumor spheroids, mimicking the interplay between interstitial flow, extracellular matrix and adenoviral transduction. Image analysis of on-chip cleared spheroids indicated the importance of the RGD motif for on-chip adenoviral transduction. Together, our results show the interrelationship between receptor characteristics, the RGD motif, the 3D tumor architecture and retargeted adenoviral transduction efficiency. The findings are important for the rational design of next-generation therapeutic adenoviruses.
Transfection of cells with a plasmid encoding for the first ten strands of the GFP protein (GFP1‐10) provides the means to detect cytosolic peptide import at low micromolar concentrations. Cytosolic ...import of the eleventh strand of the GFP protein either by electroporation or by cell‐penetrating peptide‐mediated import leads to formation of the full‐length GFP protein and fluorescence. An increase in sensitivity is achieved through structural modifications of the peptide and the expression of GFP1‐10 as a fusion protein with mCherry.
Peptide‐based GFP complementation: Cells expressing the GFP1‐10 GFP fragment (gray circle) are electroporated or penetrated with GFP‐11 (gray squares) conjugated by a linker (blue line) to a cell‐penetrating peptide (orange line), thereby resulting in delivery into the cytosol and GFP complementation and fluorescence (green circle).
Arginine-rich cell-penetrating peptides (CPP) are widely employed as delivery vehicles for a large variety of macromolecular cargos. As a mechanism-of-action for induction of uptake cross-linking of ...heparan sulfates and interaction with lipid head groups have been proposed. Here, we employed a multivalent display of the CPP nona-arginine (R9) on a linear dextran scaffold to assess the impact of heparan sulfate and lipid interactions on uptake and membrane perturbation. Increased avidity through multivalency should potentiate molecular phenomena that may only play a minor role if only individual peptides are used. To this point, the impact of multivalency has only been explored for dendrimers, CPP-decorated proteins and nanoparticles. We reasoned that multivalency on a linear scaffold would more faithfully mimic the arrangement of peptides at the membrane at high local peptide concentrations. On average, five R9 were coupled to a linear dextran backbone. The conjugate displayed a direct cytoplasmic uptake similar to free R9 at concentrations higher than 10μM. However, this uptake was accompanied by an increased membrane disturbance and cellular toxicity that was independent of the presence of heparan sulfates. In contrast, for erythrocytes, the multivalent conjugate induced aggregation, however, showed only limited membrane perturbation. Overall, the results demonstrate that multivalency of R9 on a linear scaffold strongly increases the capacity to interact with the plasma membrane. However, the induction of membrane perturbation is a function of the cellular response to peptide binding.
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•Multivalency of R9 on a linear scaffold imparts novel cell-binding characteristics.•Multivalency induces strong membrane binding and cell aggregation in erythrocytes.•Severe enhancement of toxicity was only observed in cells showing uptake.
Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to ...exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane-cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane-cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions.
Abstract
Study question
Is it possible to eradicate cancer cells from ovarian cortex by using tumour-specific designed ankyrin repeat protein (DARPin)-toxin fusion proteins, without compromising the ...ovarian tissue?
Summary answer
Purging ovarian cortex ex vivo from experimentally induced breast cancer tumour foci is possible by tumour-targeted DARPin-toxin fusion proteins trough inhibition of protein synthesis.
What is known already
Ovarian tissue cryopreservation and autotransplantation is a successful technique for fertility restoration in cancer patients. The procedure is not without risk since malignant cells may still be present in the graft. Procedures to detect cancer cells render the tissue fragment useless for autotransplantation. Strategies to circumvent this problem such as in vitro maturation of follicles or the construction of artificial ovaries are pursued but are still experimental. Alternatively, we have shown ex vivo purging of ovarian cortex is possible by elimination of rhabdomyosarcoma after treatment with verteporfin. This allows treatment of cortex fragments before autotransplantation without compromising ovarian tissue integrity.
Study design, size, duration
Human ovarian cortex fragments harbouring breast cancer tumour foci were exposed for 24 h to DARPins fused to the translocation and catalytic domain of Pseudomonas aeruginosa exotoxin A (DARPin-toxin fusion proteins) targeting EpCAM or HER2. After treatment with the DARPin-toxin fusion proteins the tissue was cultured for an additional 6 days to allow any remaining tumour cells to form foci. In addition, the functional integrity of the ovarian tissue was analysed after purging.
Participants/materials, setting, methods
Breast cancer cell lines expressing different levels of EpCAM and HER2 were introduced in human ovarian tissue to form tumour foci. After purging with DARPin-toxin fusion proteins, the presence of any remaining cancer cells in the tissue was analysed with (immuno)histochemistry and RT-qPCR. Possible detrimental effects on the viability of ovarian cortex and follicles were determined by (immuno)histology, a follicular viability assay and an assay to determine the in vitro growth capacity of small follicles.
Main results and the role of chance
Ovarian cortex harbouring EpCAM-positive breast cancer cells showed a significant decrease in the number of tumour foci after treatment with the EpCAM-targeted DARPin-toxin fusion proteins. Although exposure to the EpCAM-specific DARPin had no effect on morphology or viability of follicles, a decrease in oocyte viability after in vitro growth experiments was observed, presumably due to low level expression of EpCAM on oocytes. In contrast to the EpCAM-specific DARPin-toxin fusion protein, the DARPin-toxin fusion protein targeting HER2 had no detrimental effects on morphology, viability or in vitro growth of follicles while foci of HER2-positive breast cancer cells were severely affected as indicated by the presence of apoptotic bodies, tumour cell remnants and the absence of viable tumour cells. The histological results after purging with the HER2-specific DARPin-toxin fusions proteins were confirmed by RT-qPCR, showing a decrease to basal levels of HER2 mRNA in the ovarian cortex tissue.
Limitations, reasons for caution
The effect of DARPin-toxin fusion proteins depends heavily on the expression of their target on the cancer cell. The target protein should not be expressed by ovarian cortex as this may lead to tissue damage. The functional integrity of ovarian cortex after the treatment requires further investigation in vivo.
Wider implications of the findings
Purging metastases from ovarian cortex without harming ovarian tissue is possible by targeting tumour specific surface expressed antigens with DARPin-toxin fusion proteins. Purging ovarian cortex tissue with DARPin-toxin fusion proteins provides a feasible therapeutic strategy to prevent reintroduction of cancer by autotransplantation in case of malignancies expressing tumour-specific surface markers.
Trial registration number
not applicable
Modes of transport: A leucine‐zipper‐tagged GFP was transported into cells by “zipping” it (red) to it's complementary leucine zipper (blue) functionalized with a cell‐penetrating peptide (CPP). This ...transport system has an inherent modularity as the CPP is “clicked” to the leucine zipper, and then noncovalently bound to the protein, thus making it system particularly useful for targeting studies.
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