effects-on-rna-quality-pbmcs-under-different-cryopreservation-times

Effects on RNA Quality of PBMCs Under Different Cryopreservation Times and Processing Methods in Patients with Advanced Non-Small Cell Lung Cancer

Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in RNA sequencing (RNA-seq), and the quality of their RNA directly influences ...

Jilin Cancer Hospital

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.

Colony‑stimulating factor CSF2 mediates the phenotypic plasticity of small‑cell lung cancer by regulating the p‑STAT3/MYC pathway.

Patients with non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR)-activating mutations can be treated with EGFR-tyrosine kinase inhibitors (TKIs). Although EGFR-TKI-targeted drugs bring survival promotion in patients with EGFR mutations, drug resistance is inevitable, so it is urgent to explore new treatments to overcome drug resistance. In addition, wild-type EGFR lacks targeted drugs, and new targeted therapies need to be explored. Ferroptosis is a key research direction for overcoming drug resistance. However, the role and mechanism of regulating ferroptosis in different EGFR-mutant NSCLC types remains unclear. In the present study, H1975 (EGFR T790M/L858R mutant), A549 (EGFR wild-type) and H3255 (EGFR L858R mutant) NSCLC cell lines were used. The expression of ferroptosis markers in these cell lines was detected using western blotting and reverse transcription-quantitative PCR. Cell viability was determined using the MTT assay and reactive oxygen species (ROS) levels were measured using flow cytometry. The results showed that, compared with EGFR wild-type/sensitive mutant cells, EGFR-resistant mutant cells were more sensitive to the ferroptosis inducer, erastin. Furthermore, the mammalian target of rapamycin (mTOR) inhibitor, everolimus (RAD001), induced cell death in all three cell lines in a dose-dependent manner. The ferroptosis inhibitor, ferrostatin-1, could reverse cell death in EGFR-resistant mutant and EGFR wild-type cells induced by RAD001, but could not reverse cell death in EGFR-sensitive mutant cells. Compared with EGFR wild-type/sensitive mutant cells, EGFR-resistant mutant cells were more sensitive to RAD001 combined with erastin. In addition, a high-dose of RAD001 reduced the expression levels of ferritin heavy-chain polypeptide 1 (FTH1), glutathione peroxidase 4 (GPX4) and ferroportin and significantly increased ROS and malondialdehyde (MDA) levels in EGFR-resistant mutant and EGFR wild-type cells. In the present study, GPX4 inhibitor only or combined with RAD001 inhibited the AKT/mTOR pathway in EGFR-resistant mutant cells. Therefore, the results of the present study suggested that inhibition of the mTOR pathway may downregulate the expression of ferroptosis-related proteins in EGFR-resistant and EGFR wild-type NSCLC cells, increase the ROS and MDA levels and ultimately induce ferroptosis.

Article	Year
Colony‑stimulating factor CSF2 mediates the phenotypic plasticity of small‑cell lung cancer by regulating the p‑STAT3/MYC pathway. Oncology reports\| PubMed ID: 35583004	2022
Mechanism of targeting the mTOR pathway to regulate ferroptosis in NSCLC with different EGFR mutations. Oncology letters\| PubMed ID: 38751752	2024

Chunying He

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