Engineering Novel 3D Models to Recreate High-Grade Osteosarcoma and its Immune and Extracellular Matrix Microenvironment.

Osteosarcoma (OS) is the most common primary bone cancer, where the overall 5-year surviving rate is below 20% in resistant forms. Accelerating cures for those poor outcome patients remains a challenge. Nevertheless, several studies of agents targeting abnormal cancerous pathways have yielded disappointing results when translated into clinic because of the lack of accurate OS preclinical modeling. So, any effort to design preclinical drug testing may consider all inter-, intra-, and extra-tumoral heterogeneities throughout models mimicking extracellular and immune microenvironment. Therefore, the bioengineering of patient-derived models reproducing the OS heterogeneity, the interaction with tumor-associated macrophages (TAMs), and the modulation of oxygen concentrations additionally to recreation of bone scaffold is proposed here. Eight 2D preclinical models mimicking several OS clinical situations and their TAMs in hypoxic conditions are developed first and, subsequently, the paired 3D models faithfully preserving histological and biological characteristics are generated. It is possible to shape reproducibly M2-like macrophages cultured with all OS patient-derived cell lines in both dimensions. The final 3D models pooling all heterogeneity features are providing accurate proliferation and migration data to understand the mechanisms involved in OS and immune cells/biomatrix interactions and sustained such that engineered 3D preclinical systems will improve personalized medicine.

Pulsed Electric Fields Induce Extracellular Matrix Remodeling through Matrix Metalloproteinases Activation and Decreased Collagen Production.

Impairment of extracellular matrix remodeling is observed in the tumor microenvironment or fibrosis and results in excessive collagen production and/or decreased degradation by matrix metalloproteinases (MMPs). Thanks to their local application and transient effects, physical stimuli appear as attractive tools to remodel the extracellular matrix. We assessed the potential of pulsed electric field technology, classically applied to drug delivery, to induce collagen remodeling at the tissue scale. A sophisticated in vitro tissue-engineered human dermal substitute was used to show that microsecond and millisecond pulsed electric fields induced (i) a rapid modulation (4 hours after electrostimulation) of mRNA genes composing the matrisome, particularly a downregulation of procollagens and extracellular matrix maturation enzymes such as transglutaminase 2 and lysyl oxidase like; (ii) a transient decrease in procollagens production and hydroxyproline tissue content within a week after electrostimulation; (iii) a long-lasting ROS-dependent overactivation of matrix metalloproteinases for at least 48 hours; and (iv) a downregulation of TGFß1. These observations underpin that pulsed electric fields, a technology already approved for clinical use combined with anticancer agents, are particularly promising to provide local and effective treatment of abnormal extracellular matrix.

Electroporation does not affect human dermal fibroblast proliferation and migration properties directly but indirectly via the secretome.

Aesthetic wound healing is often experienced by patients after electrochemotherapy. We hypothesized that pulsed electric fields applied during electrochemotherapy (ECT) or gene electrotransfer (GET) protocols could stimulate proliferation and migration of human cutaneous cells, as described in protocols for electrostimulation of wound healing. We used videomicroscopy to monitor and quantify in real time primary human dermal fibroblast behavior when exposed in vitro to ECT and GET electric parameters, in terms of survival, proliferation and migration in a calibrated scratch wound assay. Distinct electric field intensities were applied to allow gradient in cell electropermeabilization while maintaining reversible permeabilization conditions, in order to mimic in vivo heterogeneous electric field distribution of complex tissues. Neither galvanotaxis nor statistical modification of fibroblast migration were observed in a calibrated scratch wound assay after application of ECT and GET parameters. The only effect on proliferation was observed under the strongest GET conditions, which drastically reduced the number of fibroblasts through induction of mitochondrial stress and apoptosis. Finally, we found that 24 h-conditioned cell culture medium by electrically stressed fibroblasts tended to increase the migration properties of cells that were not exposed to electric field. RT-qPCR array indicated that several growth factor transcripts were strongly modified after electroporation.