A 3D multi-cellular tissue model of the human omentum to study the formation of ovarian cancer metastasis.

Reliable and predictive experimental models are urgently needed to study metastatic mechanisms of ovarian cancer cells in the omentum. Although models for ovarian cancer cell adhesion and invasion were previously investigated, the lack of certain omental cell types, which influence the metastatic behavior of cancer cells, limits the application of these tissue models. Here, we describe a 3D multi-cellular human omentum tissue model, which considers the spatial arrangement of five omental cell types. Reproducible tissue models were fabricated combining permeable cell culture inserts and bioprinting technology to mimic metastatic processes of immortalized and patient-derived ovarian cancer cells. The implementation of an endothelial barrier further allowed studying the interaction between cancer and endothelial cells during hematogenous dissemination and the impact of chemotherapeutic drugs. This proof-of-concept study may serve as a platform for patient-specific investigations in personalized oncology in the future.

Site-specific N-glycosylation of integrin α2 mediates collagen-dependent cell survival.

Integrin alpha 2 (ITGA2) promotes cancer metastasis through selective adhesion to ECM proteins; however, the specific contribution of integrin glycosylation remains uncertain. We provide evidence that ITGA2 is a highly glycosylated transmembrane protein expressed in ovarian cancer tissue and cell lines. In-depth glycoproteomics identified predominant N- and O-glycosylation sites harboring substantially divergent ITGA2 glycosylation profiles. Generated putative ITGA2 N-glycosite mutants halted collagen and laminin binding and cells lacking N-glycosylated ITGA2 were marginally adherent to collagen, likely associated with its enhanced proteasome degradation through poly-ubiquitination. Proteomic and enrichment pathway analysis revealed increased cellular apoptosis and collagen organization in non-glycosylated ITGA2 mutant cells. Moreover, we provide evidence that ITGA2-specific sialylation is involved in selective cell-ECM binding. These results highlight the importance of glycans in regulating ITGA2 stability and ligand binding capacity which in turn modulates downstream focal adhesion and promotes cell survival in a collagen environment.

Design of Perfused PTFE Vessel-Like Constructs for In Vitro Applications.

Tissue models mimic the complex 3D structure of human tissues, which allows the study of pathologies and the development of new therapeutic strategies. The introduction of perfusion overcomes the diffusion limitation and enables the formation of larger tissue constructs. Furthermore, it provides the possibility to investigate the effects of hematogenously administered medications. In this study, the applicability of hydrophilic polytetrafluoroethylene (PTFE) membranes as vessel-like constructs for further use in perfused tissue models is evaluated. The presented approach allows the formation of stable and leakproof tubes with a mean diameter of 654.7 µm and a wall thickness of 84.2 µm. A polydimethylsiloxane (PDMS) chip acts as a perfusion bioreactor and provides sterile conditions. As proof of concept, endothelial cells adhere to the tube's wall, express vascular endothelial cadherin (VE-cadherin) between neighboring cells, and resist perfusion at a shear rate of 0.036 N m-2 for 48 h. Furthermore, the endothelial cell layer delays significantly the diffusion of fluorescently labeled molecules into the surrounding collagen matrix and leads to a twofold reduced diffusion velocity. This approach represents a cost-effective alternative to introduce stable vessel-like constructs into tissue models, which allows adapting the surrounding matrix to the tissue properties in vivo.

Patient-derived and artificial ascites have minor effects on MeT-5A mesothelial cells and do not facilitate ovarian cancer cell adhesion.

The presence of ascites in the peritoneal cavity leads to morphological and functional changes of the peritoneal mesothelial cell layer. Cells loose cell-cell interactions, rearrange their cytoskeleton, activate the production of fibronectin, and change their cell surface morphology in a proinflammatory environment. Moreover, ovarian cancer cell adhesion has been shown to be facilitated by these changes due to increased integrin- and CD44-mediated binding sites. In this study, the biological responsiveness of the human pleural mesothelial cell line MeT-5A to patient-derived and artificial ascites was studied in vitro and adhesion of ovarian cancer cells, i.e. SKOV-3 cells, investigated. Changes were mainly observed in cells exposed to artificial ascites containing higher cytokine concentrations than patient-derived ascites. Interestingly, reduced cell-cell interactions were already observed in untreated MeT-5A cells and effects on tight junction protein expression and permeability upon exposure to ascites were minor. Ascites induced upregulation of CDC42 effector protein 2 expression, which affects stress fiber formation, however significant F-actin reorganization was not observed. Moreover, fibronectin production remained unchanged. Analysis of mesothelial cell surface characteristics showed upregulated expression of intercellular adhesion molecule 1, slightly increased hyaluronic acid secretion and decreased microvillus expression upon exposure to ascites. Nevertheless, the observed changes were not sufficient to facilitate adhesion of SKOV-3 cells on MeT-5A cell layer. This study revealed that MeT-5A cells show a reduced biological responsiveness to the presence of ascites, in contrast to published studies on primary human peritoneal mesothelial cells.

Collagen-rich omentum is a premetastatic niche for integrin α2-mediated peritoneal metastasis.

The extracellular matrix (ECM) plays critical roles in tumor progression and metastasis. However, the contribution of ECM proteins to early metastatic onset in the peritoneal cavity remains unexplored. Here, we suggest a new route of metastasis through the interaction of integrin alpha 2 (ITGA2) with collagens enriched in the tumor coinciding with poor outcome in patients with ovarian cancer. Using multiple gene-edited cell lines and patient-derived samples, we demonstrate that ITGA2 triggers cancer cell adhesion to collagen, promotes cell migration, anoikis resistance, mesothelial clearance, and peritoneal metastasis in vitro and in vivo. Mechanistically, phosphoproteomics identify an ITGA2-dependent phosphorylation of focal adhesion kinase and mitogen-activated protein kinase pathway leading to enhanced oncogenic properties. Consequently, specific inhibition of ITGA2-mediated cancer cell-collagen interaction or targeting focal adhesion signaling may present an opportunity for therapeutic intervention of metastatic spread in ovarian cancer.

Bioprinting for Human Respiratory and Gastrointestinal In Vitro Models.

Increasing ethical and biological concerns require a paradigm shift toward animal-free testing strategies for drug testing and hazard assessments. To this end, the application of bioprinting technology in the field of biomedicine is driving a rapid progress in tissue engineering. In particular, standardized and reproducible in vitro models produced by three-dimensional (3D) bioprinting technique represent a possible alternative to animal models, enabling in vitro studies relevant to in vivo conditions. The innovative approach of 3D bioprinting allows a spatially controlled deposition of cells and biomaterial in a layer-by-layer fashion providing a platform for engineering reproducible models. However, despite the promising and revolutionizing character of 3D bioprinting technology, standardized protocols providing detailed instructions are lacking. Here, we provide a protocol for the automatized printing of simple alveolar, bronchial, and intestine epithelial cell layers as the basis for more complex respiratory and gastrointestinal tissue models. Such systems will be useful for high-throughput toxicity screening and drug efficacy evaluation.

Polydopamine/Transferrin Hybrid Nanoparticles for Targeted Cell-Killing.

Polydopamine can form biocompatible particles that convert light into heat. Recently, a protocol has been optimized to synthesize polydopamine/protein hybrid nanoparticles that retain the biological function of proteins, and combine it with the stimuli-induced heat generation of polydopamine. We have utilized this novel system to form polydopamine particles, containing transferrin (PDA/Tf). Mouse melanoma cells, which strongly express the transferrin receptor, were exposed to PDA/Tf nanoparticles (NPs) and, subsequently, were irradiated with a UV laser. The cell death rate was monitored in real-time. When irradiated, the melanoma cells exposed to PDA/Tf NPs underwent apoptosis, faster than the control cells, pointing towards the ability of PDA/Tf to mediate UV-light-induced cell death. The system was also validated in an organotypic, 3D-printed tumor spheroid model, comprising mouse melanoma cells, and the exposure and subsequent irradiation with UV-light, yielded similar results to the 2D cell culture. The process of apoptosis was found to be targeted and mediated by the lysosomal membrane permeabilization. Therefore, the herein presented polydopamine/protein NPs constitute a versatile and stable system for cancer cell-targeting and photothermal apoptosis induction.