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.

Together Intra-Tumor Hypoxia and Macrophagic Immunity Are Driven Worst Outcome in Pediatric High-Grade Osteosarcomas.

BACKGROUND: Osteosarcomas (OTS) represent the most common primary bone cancer diagnosed in adolescents and young adults. Despite remarkable advances, there are no objective molecular or imaging markers able to predict an OTS outcome at diagnosis. Focusing on biomarkers contributing broadly to treatment resistance, we examine the interplay between the tumor-associated macrophages and intra-tumor hypoxia. METHODS: Radiological and immunohistochemical (IHC) data were correlated with the outcome in a retrospective and monocentric cohort of 30 pediatric OTS. We studied hypoxic (pS6, phospho-mTor, HIF-1α and carbonic anhydrase IX (CAIX)) and macrophagic (CD68 and CD163) biomarkers. RESULTS: The imaging analyses were based on MRI manual volumetric measures on axial post-contrast T1 weighted images, where, for each tumor, we determined the necrotic volume and its ratio to the entire tumor volume. When they were above 50 cm3 and 20%, respectively, they correlated with a worse overall survival (p = 0.0072 and p = 0.0136, respectively) and event-free survival (p = 0.0059 and p = 0.0143, respectively). IHC assessments enable a significant statistical link between HIF-1α/CAIX hyper-expressions, CD68+ cells and a worse outcome, whereas activation of mTor pathway was linked to a better survival rate and CD163+ cells. CONCLUSIONS: This study evidenced the links between hypoxia and immunity in OTS, as their poor outcome may be related to a larger necrotic volume on diagnostic MRI and, in biopsies, to a specific IHC profile.

Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions.

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to overcome the resistance to current treatments and find a new way of cure, modeling the disease as close as possible in an in vitro setting to test new drugs and therapeutic procedures is highly demanding. Studying their fundamental pathobiological processes, including glutamatergic neuron hyperexcitability, will be a real advance in understanding interactions between the environmental brain and pHGG cells. Therefore, to recreate neurons/pHGG cell interactions, this work shows the development of a functional in vitro model co-culturing human-induced Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons pHGG cells into compartmentalized microfluidic devices and a process to record their electrophysiological modifications. The first step was to differentiate and characterize human glutamatergic neurons. Secondly, the cells were cultured in microfluidic devices with pHGG derived cell lines. Brain microenvironment and neuronal activity were then included in this model to analyze the electrical impact of pHGG cells on these micro-environmental neurons. Electrophysiological recordings are coupled using multielectrode arrays (MEA) to these microfluidic devices to mimic physiological conditions and to record the electrical activity of the entire neural network. A significant increase in neuron excitability was underlined in the presence of tumor cells.

Biological Relevance of RGD-Integrin Subtype-Specific Ligands in Cancer.

Integrins are heterodimeric transmembrane proteins able to connect cells with the micro-environment. They represent a family of receptors involved in almost all the hallmarks of cancer. Integrins recognizing the Arg-Gly-Asp (RGD) peptide in their natural extracellular matrix ligands have been particularly investigated as tumoral therapeutic targets. In the last 30 years, intense research has been dedicated to designing specific RGD-like ligands able to discriminate selectively the different RGD-recognizing integrins. Chemists' efforts have led to the proposition of modified peptide or peptidomimetic libraries to be used for tumor targeting and/or tumor imaging. Here we review, from the biological point of view, the rationale underlying the need to clearly delineate each RGD-integrin subtype by selective tools. We describe the complex roles of RGD-integrins (mainly the most studied αvß3 and α5ß1 integrins) in tumors, the steps towards selective ligands and the current usefulness of such ligands. Although the impact of integrins in cancer is well acknowledged, the biological characteristics of each integrin subtype in a specific tumor are far from being completely resolved. Selective ligands might help us to reconsider integrins as therapeutic targets in specific clinical settings.

Focus on Hypoxia-Related Pathways in Pediatric Osteosarcomas and Their Druggability.

Osteosarcoma is the most frequent primary bone tumor diagnosed during adolescence and young adulthood. It is associated with the worst outcomes in the case of poor response to chemotherapy and in metastatic disease. While no molecular biomarkers are clearly and currently associated with those worse situations, the study of pathways involved in the high level of tumor necrosis and in the immune/metabolic intra-tumor environment seems to be a way to understand these resistant and progressive osteosarcomas. In this review, we provide an updated overview of the role of hypoxia in osteosarcoma oncogenesis, progression and during treatment. We describe the role of normoxic/hypoxic environment in normal tissues, bones and osteosarcomas to understand their role and to estimate their druggability. We focus particularly on the role of intra-tumor hypoxia in osteosarcoma cell resistance to treatments and its impact in its endogenous immune component. Together, these previously published observations conduct us to present potential perspectives on the use of therapies targeting hypoxia pathways. These therapies could afford new treatment approaches in this bone cancer. Nevertheless, to study the osteosarcoma cell druggability, we now need specific in vitro models closely mimicking the tumor, its intra-tumor hypoxia and the immune microenvironment to more accurately predict treatment efficacy and be complementary to mouse models.

Hypoxia Inducible Factors' Signaling in Pediatric High-Grade Gliomas: Role, Modelization and Innovative Targeted Approaches.

The brain tumor microenvironment has recently become a major challenge in all pediatric cancers, but especially in brain tumors like high-grade gliomas. Hypoxia is one of the extrinsic tumor features that interacts with tumor cells, but also with the blood-brain barrier and all normal brain cells. It is the result of a dramatic proliferation and expansion of tumor cells that deprive the tissues of oxygen inflow. However, cancer cells, especially tumor stem cells, can endure extreme hypoxic conditions by rescheduling various genes' expression involved in cell proliferation, metabolism and angiogenesis and thus, promote tumor expansion, therapeutic resistance and metabolic adaptation. This cellular adaptation implies Hypoxia-Inducible Factors (HIF), namely HIF-1α and HIF-2α. In pediatric high-grade gliomas (pHGGs), several questions remained open on hypoxia-specific role in normal brain during gliomagenesis and pHGG progression, as well how to model it in preclinical studies and how it might be counteracted with targeted therapies. Therefore, this review aims to gather various data about this key extrinsic tumor factor in pHGGs.