骨肉瘤（OS）是一种恶性骨癌类型，在骨骼形成增加时出现。这些肿瘤的治疗策略基本上几十年来没有改变，大多数晚期病例的总体生存率仍然非常低。这在某种程度上是由于存在对药物产生抵抗的癌症干细胞（CSC），具有前体特性，负责肿瘤复发和转移。在寻求OS治疗替代方案时，冷等离子体和等离子体处理液（PTL）已成为能产生对多种癌细胞系具有选择性的反应氧化物和氮化物的来源。然而，至今对它们对CSC亚群和体内肿瘤生长的影响几乎没有研究。在使用生物工程3D肿瘤模型和体内试验的情况下，我们展示了PTL的低剂量增加了前干细胞因子水平和OS细胞的自我更新能力，与增加的体内肿瘤生长潜能相结合。这对该领域具有关键意义。通过提出联合治疗，我们的研究结果表明，在与STAT3抑制剂S3I-201相结合时，PTL介导的有害前干细胞信号可以被消除，从而强烈抑制体内肿瘤生长。总的来说，我们的研究揭示了PTL在癌症中促进干细胞的功能不良，并支持使用与STAT3抑制剂相结合的联合策略作为有效治疗OS的方法，从而避免重要的副作用。我们预计我们的工作将成为使用相关3D肿瘤模型评估等离子体疗法对不同癌症类型的肿瘤亚群影响的更广泛研究的起点。此外，与STAT3抑制或其他适合的癌症类型特异性靶点的结合可能有助于巩固该领域的发展。版权所有©2023作者。由Elsevier B.V.出版。保留所有权利。

Osteosarcoma (OS) is a malignant type of bone cancer that arises in periods of increased bone formation. Curative strategies for these types of tumors have remained essentially unchanged for decades and the overall survival for most advanced cases is still dismally low. This is in part due to the existence of drug resistant Cancer Stem Cells (CSC) with progenitor properties that are responsible for tumor relapse and metastasis. In the quest for therapeutic alternatives for OS, Cold Atmospheric Plasmas and Plasma-Treated Liquids (PTL) have come to the limelight as a source of Reactive Oxygen and Nitrogen Species displaying selectivity towards a variety of cancer cell lines. However, their effects on CSC subpopulations and in vivo tumor growth have been barely studied to date. By employing bioengineered 3D tumor models and in vivo assays, here we show that low doses of PTL increase the levels of pro-stemness factors and the self-renewal ability of OS cells, coupled to an enhanced in vivo tumor growth potential. This could have critical implications to the field. By proposing a combined treatment, our results demonstrate that the deleterious pro-stemness signals mediated by PTL can be abrogated when this is combined with the STAT3 inhibitor S3I-201, resulting in a strong suppression of in vivo tumor growth. Overall, our study unveils an undesirable stem cell-promoting function of PTL in cancer and supports the use of combinatorial strategies with STAT3 inhibitors as an efficient treatment for OS avoiding critical side effects. We anticipate our work to be a starting point for wider studies using relevant 3D tumor models to evaluate the effects of plasma-based therapies on tumor subpopulations of different cancer types. Furthermore, combination with STAT3 inhibition or other suitable cancer type-specific targets can be relevant to consolidate the development of the field.Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.