Relapse and drug resistance are the main causes of mortality in patients with small‑cell lung cancer (SCLC). Intratumoral heterogeneity (ITH) is a key biological mechanism that leads to relapse and drug resistance. Phenotypic plasticity is an important factor that leads to ITH in SCLC, although its mechanisms and key regulatory factors remain to be elucidated. In the present study, cell proliferation and cell switch assay were measured using trypan blue. Alamar Blue was used to test drug sensitivity. Differential genes were screened by RNA sequencing. Reverse transcription‑quantitative PCR and western blotting were performed to assess the expressions of CSF2/p‑STAT3/MYC pathway related molecules, neuroendocrine (NE)/non‑neuroendocrine (non‑NE), transcription factors and drug‑related targets. The present study found that SCLC cell line NCI‑H69 exhibited adherent (H69A) and suspensive (H69S) phenotypes, which could switch back and forth. The two phenotypic cells had significant differences in cellular NE and non‑NE characteristics, drug sensitivity and expression of drug‑related targets. RNA sequencing showed that granulocyte‑macrophage colony‑stimulating factor [i.e., colony‑stimulating factor 2 (CSF2)] was the main differentially expressed gene between the two phenotypes and that H69A cells highly expressed CSF2. The inhibition of CSF2 promoted the transformation from H69A to H69S, increased drug sensitivity and NE marker expression and decreased the non‑NE marker expression in H69A. The STRING, Pathway Commons and Reactome databases showed a potential regulatory relationship between CSF2 and phosphorylated signal transducer and activator of transcription 3 (p‑STAT3)/MYC. p‑STAT3 and MYC expression was higher in H69A cells than in H69S cells and CSF2 silencing inhibited their expression. Taken together, these results indicated that CSF2 may regulate the phenotypic plasticity of SCLC through the phosphorylated STAT3/MYC pathway, thereby limiting the transformation between cell clones with different phenotypes and changing the sensitivity of specific cell clones to targeted drugs. Targeting CSF2 may be a potential therapeutic strategy to overcome drug resistance in SCLC treatment by influencing ITH.

中文翻译：

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集落刺激因子 CSF2 通过调节 p-STAT3/MYC 通路介导小细胞肺癌的表型可塑性。

复发和耐药性是小细胞肺癌（SCLC）患者死亡的主要原因。瘤内异质性（ITH）是导致复发和耐药性的关键生物学机制。表型可塑性是导致 SCLC 中 ITH 的重要因素，尽管其机制和关键调节因素仍有待阐明。在本研究中，使用台盼蓝测量细胞增殖和细胞转换测定。Alamar Blue 用于测试药物敏感性。通过RNA测序筛选差异基因。进行逆转录定量 PCR 和蛋白质印迹以评估 CSF2/p-STAT3/MYC 通路相关分子、神经内分泌 (NE)/非神经内分泌 (non-NE)、转录因子和药物相关靶点的表达。本研究发现 SCLC 细胞系 NCI-H69 表现出贴壁 (H69A) 和悬浮 (H69S) 表型，可以来回切换。两种表型细胞在细胞 NE 和非 NE 特征、药物敏感性和药物相关靶点的表达方面存在显着差异。RNA测序显示粒细胞-巨噬细胞集落刺激因子[即集落刺激因子2（CSF2）]是两种表型之间的主要差异表达基因，H69A细胞高度表达CSF2。CSF2的抑制促进了H69A向H69S的转化，增加了药物敏感性和NE标志物的表达，降低了H69A中非NE标志物的表达。字符串，Pathway Commons 和 Reactome 数据库显示 CSF2 与磷酸化信号转导和转录激活因子 3 (p-STAT3)/MYC 之间存在潜在的调节关系。H69A 细胞中 p-STAT3 和 MYC 的表达高于 H69S 细胞，CSF2 沉默抑制了它们的表达。综上所述，这些结果表明CSF2可能通过磷酸化的STAT3/MYC通路调节SCLC的表型可塑性，从而限制不同表型细胞克隆之间的转化，改变特定细胞克隆对靶向药物的敏感性。靶向 CSF2 可能是通过影响 ITH 来克服 SCLC 治疗中耐药性的潜在治疗策略。H69A 细胞中 p-STAT3 和 MYC 的表达高于 H69S 细胞，CSF2 沉默抑制了它们的表达。综上所述，这些结果表明CSF2可能通过磷酸化的STAT3/MYC通路调节SCLC的表型可塑性，从而限制不同表型细胞克隆之间的转化，改变特定细胞克隆对靶向药物的敏感性。靶向 CSF2 可能是通过影响 ITH 来克服 SCLC 治疗中耐药性的潜在治疗策略。H69A 细胞中 p-STAT3 和 MYC 的表达高于 H69S 细胞，CSF2 沉默抑制了它们的表达。综上所述，这些结果表明CSF2可能通过磷酸化的STAT3/MYC通路调节SCLC的表型可塑性，从而限制不同表型细胞克隆之间的转化，改变特定细胞克隆对靶向药物的敏感性。靶向 CSF2 可能是通过影响 ITH 来克服 SCLC 治疗中耐药性的潜在治疗策略。