Identification of GB3 as a Novel Biomarker of Tumor-Derived Vasculature in Neuroblastoma Using a Stiffness-Based Model

Neuroblastoma (NB) is a childhood cancer in sympathetic nervous system cells. NB exhibits cellular heterogeneity, with adrenergic and mesenchymal states displaying distinct tumorigenic potentials. NB is highly vascularized, and blood vessels can form through various mechanisms, including endothelial transdifferentiation, leading to the development of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs. Therefore, identifying new TEC biomarkers is vital for effective NB therapies. A stiffness-based platform simulating human arterial and venous stiffness was developed to study NB TECs in vitro. Adrenergic cells cultured on arterial-like stiffness transdifferentiated into TECs, while mesenchymal state cells did not. The TECs derived from adrenergic cells served as a model to explore new biomarkers, with a particular focus on GB3, a glycosphingolipid receptor implicated in angiogenesis, metastasis, and drug resistance. Notably, the TECs unequivocally expressed GB3, validating its novelty as a marker. To explore targeted therapeutic interventions, nanoparticles functionalized with the non-toxic subunit B of the Shiga toxin were generated, because they demonstrated a robust affinity for GB3-positive cells. Our results demonstrate the value of the stiffness-based platform as a predictive tool for assessing NB aggressiveness, the discovery of new biomarkers, and the evaluation of the effectiveness of targeted therapeutic strategies

Decellularized extracellular matrix-based 3D nanofibrous scaffolds functionalized with polydopamine-reduced graphene oxide for neural tissue engineering

Publisher Copyright: © 2023One of the exciting prospects of using decellularized extracellular matrices (ECM) lies in their biochemical profile of preserved components, many of which are regeneration-permissive. Herein, a decellularized ECM from adipose tissue (adECM) was explored to design a scaffolding strategy for the challenging repair of the neural tissue. Targeting the recreation of the nano-scaled architecture of native ECM, adECM was first processed into nanofibers by electrospinning to produce bidimensional platforms. These were further shaped into three-dimensional (3D) nanofibrous constructs by gas foaming. The conversion into a 3D microenvironment of nanofibrous walls was assisted by blending the adECM with lactide-caprolactone copolymers, wherein tuning the adECM/copolymer ratio along with the amount of caprolactone in the copolymer led to modulating the mechanical properties towards soft, yet structurally stable, 3D constructs. In view of boosting their performance to guide neural stem cell fate, adECM-based platforms were doped with a bioinspired surface modification relying on polydopamine-functionalized reduced graphene oxide (PDA-rGO). These adECM-based 3D constructs revealed a permissive microenvironment for neural stem cells (NSCs) to adhere, grow, and migrate throughout the microporosity, owing to the synergy between the unique biochemical features of the adECM and the nanofibrous architecture. NSC responded differently depending on the adECM-based architecture–nanofibrous bidimensional, or 3D design. The 3D spatial arrangement of the nanofibers – induced by the gas foaming – exhibited a remarkable effect on NSCs’ phenotype determination and neurite formation, thereby reinforcing the critical importance of engineering scaffolds with multiple length-scale architecture. Furthermore, PDA-rGO promoted the differentiation of NSC towards the neuronal lineage. Specifically in 3D, it significantly increases the levels of Tuj1 and MAP2 a/b isoforms, confirming its effectiveness in boosting neuronal differentiation and neuritogenesis.This work was supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No 829060 (FETOPEN project: NeuroStimSpinal), and the Portuguese following funding: UIDB/00481/2020 and UIDP/00481/2020, iBiMED (UIDB/4501/2020 and UIDP/4501/2020), the LiM Bioimaging Facility (a PPBI node, POCI-01-0145-FEDER-022122) - Fundação para a Ciência e a Tecnologia (FCT) and CENTRO-01-0145-FEDER-022083 - Centro Portugal Regional Operational Programme (Centro2020) under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund. B.M.S. acknowledges financial support from FCT through the doctoral scholarship 2020.06525.BD. This work was supported by the European Union's Horizon 2020 research and innovation programme under grant agreement No 829060 (FETOPEN project: NeuroStimSpinal), and the Portuguese following funding: UIDB/00481/2020 and UIDP/00481/2020, iBiMED (UIDB/4501/2020 and UIDP/4501/2020), the LiM Bioimaging Facility (a PPBI node, POCI-01-0145-FEDER-022122) - Fundação para a Ciência e a Tecnologia (FCT) and CENTRO-01-0145-FEDER-022083 - Centro Portugal Regional Operational Programme (Centro2020) under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund. B.M.S. acknowledges financial support from FCT through the doctoral scholarship 2020.06525.BD.Peer reviewe