简介：循环肿瘤细胞 (CTC) 代表脱落到脉管系统中的细胞亚群，能够在血流中存活、粘附到目标血管内皮细胞上，并重新生长到远端器官中。在大多数患有实体瘤的患者的血液中都发现了 CTC，并被用作诊断标记物。尽管 CTC 具有复杂的基因型和表型特征，但在悬浮液中存活的能力构成了最关键的特性，称为抗失巢凋亡，例如抵抗因底物粘附丧失而导致的细胞凋亡的能力。在这里，我们选择了对失巢凋亡具有抵抗力的黑色素瘤细胞，并研究了它们的代谢重编程，旨在确定 CTC 的新代谢靶点。方法：通过悬浮液中连续三次摇动暴露，选择表达高抗失巢凋亡表型的黑色素瘤细胞亚群，并研究其表型和代谢特征。此外，我们还测试了针对糖酵解 (2DG)、LDHA (LDHA-in-3)、线粒体电子传递链复合物 I（鱼藤酮）、谷氨酰胺酶 (BPTES)、脂肪酸转运蛋白 (SSO)、脂肪酸的不同代谢抑制剂的功效。合酶（denifanstat）、CPT1（etomoxir），在摇动条件 24 小时后抑制细胞存活和集落形成能力。结果：与对照细胞相比，失巢凋亡抗性细胞在琼脂糖覆盖的培养皿中悬浮生长的能力更高，并且在进一步摇动条件下后细胞活力和集落形成能力更高。他们还表现出与高侵袭性和干细胞样表型相关的上皮-间质转变。悬浮液中的抗失巢凋亡黑色素瘤细胞表现出代谢重编程，从特征性糖酵解代谢转向基于谷氨酰胺和脂肪酸使用的氧化性代谢，而在培养皿上的重新粘附则将代谢逆转为糖酵解。代谢抑制剂的治疗强调了鱼藤酮、BPTES、SSO 和依托莫克西在降低能够在悬浮液中存活的细胞的活力和集落形成能力方面的有效性，证实了它们的代谢对氧化磷酸化的依赖性，使用谷氨酰胺和脂肪酸作为最重要的燃料。讨论：这一发现开辟了基于谷氨酰胺酶和脂肪酸氧化代谢抑制剂的新治疗策略，用于治疗 CTC 和黑色素瘤转移。版权所有 © 2024 Peppicelli、Kersikla、Menegazzi、Andreucci、Ruzzolini、Nediani、Bianchini 和 Calorini。

Introduction: Circulating tumor cells (CTCs) represent the sub-population of cells shed into the vasculature and able to survive in the bloodstream, adhere to target vascular endothelial cells, and re-growth into the distant organ. CTCs have been found in the blood of most solid tumor-bearing patients and are used as a diagnostic marker. Although a complex genotypic and phenotypic signature characterizes CTCs, the ability to survive in suspension constitutes the most critical property, known as resistance to anoikis, e.g., the ability to resist apoptosis resulting from a loss of substrate adhesion. Here, we selected melanoma cells resistant to anoikis, and we studied their metabolic reprogramming, with the aim of identifying new metabolic targets of CTCs. Methods: Subpopulations of melanoma cells expressing a high anoikis-resistant phenotype were selected by three consecutive rocking exposures in suspension and studied for their phenotypic and metabolic characteristics. Moreover, we tested the efficacy of different metabolic inhibitors targeting glycolysis (2DG), LDHA (LDHA-in-3), the mitochondrial electron transport chain complex I (rotenone), glutaminase (BPTES), fatty acid transporter (SSO), fatty acid synthase (denifanstat), CPT1 (etomoxir), to inhibit cell survival and colony formation ability after 24 h of rocking condition. Results: Anoikis-resistant cells displayed higher ability to grow in suspension on agarose-covered dishes respect to control cells, and higher cell viability and colony formation capability after a further step in rocking condition. They showed also an epithelial-to-mesenchymal transition associated with high invasiveness and a stemness-like phenotype. Anoikis-resistant melanoma cells in suspension showed a metabolic reprogramming from a characteristic glycolytic metabolism toward a more oxidative metabolism based on the use of glutamine and fatty acids, while re-adhesion on the dishes reversed the metabolism to glycolysis. The treatment with metabolic inhibitors highlighted the effectiveness of rotenone, BPTES, SSO, and etomoxir in reducing the viability and the colony formation ability of cells capable of surviving in suspension, confirming the dependence of their metabolism on oxidative phosphorylation, using glutamine and fatty acids as the most important fuels. Discussion: This finding opens up new therapeutic strategies based on metabolic inhibitors of glutaminase and fatty acid oxidation for the treatment of CTCs and melanoma metastases.Copyright © 2024 Peppicelli, Kersikla, Menegazzi, Andreucci, Ruzzolini, Nediani, Bianchini and Calorini.