Study on the Heterogeneity of Cell Subpopulations in Immunotherapy and Immune Checkpoints in Non-small Cell Lung Cancer

Abstract

Objective To identify molecular dysregulation mechanisms, immune checkpoint abnormalities, and immune cells responsive to immunotherapy in non-small cell lung cancer (NSCLC). Methods Gene expression data for NSCLC and controls from the GSE81089, GSE120622, and TCGA databases were collected and subjected to differential expression and enrichment analysis. The impact of differentially expressed immune checkpoints on overall survival was identified. Tumor tissues, adjacent control tissues, and blood samples were collected from NSCLC patients who received neoadjuvant therapy at Cancer Hospital Affiliated to Xinjiang Medical University from June 2022 to May 2023, the expression of immune checkpoints in tumor and adjacent control tissues was detected using qRT-PCR. Additionally, in the GSE207422 single-cell dataset, differences in immune cells between major pathological remission (MPR) and non-major pathological remission (NMPR) after neoadjuvant therapy were identified. The abundance differences of immune cells between MPR and NMPR were further detected by flow cytometry. Results In the GSE81089, GSE120622, and TCGA datasets, an intersection of 2 505 differentially expressed genes was identified. Enrichment analysis revealed that these intersecting genes are primarily involved in neuroactive ligand-receptor interaction, the PI3K-Akt signaling pathway, and cytokine-cytokine receptor interaction, among other signaling pathways. Additionally, six immune checkpoints were differentially expressed in NSCLC, with CD40LG, CD160, VTCN1, and TDO2 significantly impacting overall patient survival. Compared to the control group, qRT-PCR confirmed that CD40LG and CD160 were significantly downregulated in NSCLC(P<0.05), while VTCN1 and TDO2 were significantly upregulated (P<0.05). By integrating the GSE207422 single-cell dataset, 32 cell clusters were classified into 13 cell subtypes. Comparisons revealed a significant enrichment of CD4, CD8, B cells, NK cells, and M2 macrophages in MPR. Flow cytometry confirmed that the abundance of these immune cells was higher in MPR than in NMPR. Conclusion Immune checkpoints were significantly associated with the overall survival of NSCLC patients, and the enrichment of immune cells in MPR was confirmed. These findings offer new biological insights and potential therapeutic targets for the immunotherapy of NSCLC.

Keywords

• non-small cell lung cancer
• immune checkpoint
• major pathological remission
• immune cells