EGFR and IRE1α–XBP1s Pathways Together Define Distinct Immunomodulatory Profiles in NSCLC Cell Lines with Acquired Resistance to EGFR TKIs

EGFR-TKI-resistant cell lines were established by long-term exposure to gefitinib, afatinib, and osimertinib via the PC9 model. This model helps study EGFR-TKI resistance mechanisms in non-small cell lung cancer (NSCLC) and may offer insights to improve treatment outcomes, particularly for patients unresponsive to anti-PD-1/PD-L1 therapies. We investigated molecular alterations in these resistant cell lines using multi-omics techniques, including genome sequencing, proteomics, and transcriptomics.Differential dependencies on EGFR downstream pathways were observed among resistant cell lines. Immune evasion mechanisms were analyzed to identify alterations in EGFR downstream pathways and unfolded protein response (UPR) elements contributing to immune phenotypes. Afatinib-resistant cells exhibited the most pronounced immunosuppressive traits compared to gefitinib- and osimertinib-resistant cells. This was characterized by elevated PD-L1 expression, minimal changes in MHC class 1 levels, down-regulation of shared immune-modulated gene sets, and a cytokine profile favoring immunosuppression. These findings suggest a poor prognosis and limited efficacy of anti-PD-1/PD-L1 immunotherapy in NSCLC patients with similar resistance profiles. Notably, inhibition of IRE1α signaling was able to reverse this immunosuppressive profile, indicating a potential strategy to restore immune responsiveness in afatinib-resistant NSCLC. The TRIzol-based method was used to extract total RNA, and all samples (from each cell line in triplicate) were stored at −80℃. The prepared were sent to Biotools Co., Ltd. (Taiwan) for RNA-sequencing. A NovaSeq 6000 (RRID:SCR_016387) was used, and the sequencing depth was approximately 6G. The reference genome used was Homo sapiens. (Database version: hsa_GRCh38, ENSMEBL_96).