DataSheet_1_Effects of osteoblast-derived extracellular vesicles on aggressiveness, redox status and mitochondrial bioenergetics of MNNG/HOS osteosarcoma cells.pdf

Osteosarcoma is the most common primary bone malignancy. The crosstalk between osteosarcoma and the surrounding tumour microenvironment (TME) drives key events that lead to metastasization, one of the main obstacles for definitive cure of most malignancies. Extracellular vesicles (EVs), lipid bilayer nanoparticles used by cells for intercellular communication, are emerging as critical biological mediators that permit the interplay between neoplasms and the tumour microenvironment, modulating re-wiring of energy metabolism and redox homeostatic processes. We previously showed that EVs derived from the human osteosarcoma cells influence bone cells, including osteoblasts. We here investigated whether the opposite could also be true, studying how osteoblast-derived EVs (OB-EVs) could alter tumour phenotype, mitochondrial energy metabolism, redox status and oxidative damage in MNNG/HOS osteosarcoma cells.These were treated with EVs obtained from mouse primary osteoblasts, and the following endpoints were investigated: i) cell viability and proliferation; ii) apoptosis; iii) migration and invasive capacity; iv) stemness features; v) mitochondrial function and energy metabolism; vi) redox status, antioxidant capacity and oxidative molecular damage. OB-EVs decreased MNNG/HOS metabolic activity and viability, which however was not accompanied by impaired proliferation nor by increased apoptosis, with respect to control. In addition, OB-EV-treated cells exhibited a significant reduction of motility and in vitro invasion as compared to untreated cells. Although the antioxidant N-acetyl-L-cysteine reverted the cytotoxic effect of OB-EVs, no evidence of oxidative stress was observed in treated cells. However, the redox balance of glutathione was significantly shifted towards a pro-oxidant state, even though the major antioxidant enzymatic protection did not respond to the pro-oxidant challenge. We did not find strong evidence of mitochondrial involvement or major energy metabolic switches induced by OB-EVs, but a trend of reduction in seahorse assay basal respiration was observed, suggesting that OB-EVs could represent a mild metabolic challenge for osteosarcoma cells. In summary, our findings suggest that OB-EVs could serve as important means through which TME and osteosarcoma core cross-communicate. For the first time, we proved that OB-EVs reduced osteosarcoma cells’ aggressiveness and viability through redox-dependent signalling pathways, even though mitochondrial dynamics and energy metabolism did not appear as processes critically needed to respond to OB-EVs.

骨肉瘤是骨原发恶性肿瘤中最常见的一种。骨肉瘤与周围肿瘤微环境（TME）之间的交互作用推动了导致转移的关键事件，这成为大多数恶性肿瘤治愈的主要障碍之一。细胞间通讯中使用的细胞外囊泡（EVs），即脂质双层纳米颗粒，正成为关键的生物介质，允许肿瘤与肿瘤微环境之间的相互作用，调节能量代谢和氧化还原稳态过程的重新配置。我们先前表明，源自人类骨肉瘤细胞的EVs可影响骨细胞，包括成骨细胞。本研究旨在探讨反之亦然的可能性，研究成骨细胞来源的EVs（OB-EVs）如何改变MNNG/HOS骨肉瘤细胞的肿瘤表型、线粒体能量代谢、氧化还原状态和氧化损伤。这些细胞被来自小鼠原代成骨细胞的EVs处理，并研究了以下终点：i）细胞活力和增殖；ii）细胞凋亡；iii）迁移和侵袭能力；iv）干细胞特征；v）线粒体功能和能量代谢；vi）氧化还原状态、抗氧化能力和氧化分子损伤。OB-EVs降低了MNNG/HOS的代谢活性和活力，然而，这种降低并未伴随增殖受损或细胞凋亡增加，与对照相比。此外，与未处理细胞相比，OB-EV处理的细胞表现出显著降低的移动性和体外侵袭性。尽管抗氧化剂N-乙酰半胱氨酸逆转了OB-EVs的细胞毒性作用，但治疗细胞中未观察到氧化应激的证据。然而，谷胱甘肽的氧化还原平衡显著向促氧化状态偏移，尽管主要的抗氧化酶保护作用并未对促氧化挑战做出反应。我们没有发现OB-EVs引起的线粒体参与或主要能量代谢转换的强有力证据，但观察到海马式基础呼吸的降低趋势，这表明OB-EVs可能代表对骨肉瘤细胞的一种轻微代谢挑战。总之，我们的研究结果提示OB-EVs可能作为TME与骨肉瘤核心之间交叉沟通的重要途径。首次，我们证明了OB-EVs通过氧化还原依赖性信号通路减少了骨肉瘤细胞的侵袭性和活力，尽管线粒体动力学和能量代谢似乎并非响应OB-EVs的必要过程。