ObjectiveTo investigate the mechanisms by which calpain small subunit 1 (CAPN4) regulates cisplatin resistance and cancer stem cell (CSC) stemness in lung adenocarcinoma, and to provide experimental evidence for reversing drug resistance through targeting CSC stemness.MethodsTissue samples were collected from 10 lung adenocarcinoma patients who underwent surgical resection at Fujian Cancer Hospital from January 2023 to January 2024. Immunohistochemistry (IHC) stining was used to detect the differential expression of CAPN4 in five cisplatin-resistant and five cisplatin-sensitive lung adenocarcinoma tissues, followed with a histological scoring (H-score). CAPN4 gene expression-related survival analysis in lung cancer patients was conducted using The Cancer Genome Atlas (TCGA) database and Gene Expression Profiling Interactive Analysis (GEPIA) platform. Additionally, tissue samples from two cisplatin-resistant and two cisplatin-sensitive lung adenocarcinoma cases were collected to establish lung adenocarcinoma organoid (PDO) models. H-E and IHC staining were used to assess the morphological consistency between PDOs and the primary tumors. CAPN4 gene expression was silenced using lentivirus-mediated shRNA transduction. The expression levels of stem cell markers ALDH1A1, CD133, Nanog, and SOX9 were detected at both the gene and protein levels using quantitative polymerase chain reaction (qPCR) and Western blotting (WB), respectively. The sensitivity of CAPN4-knockdown PDOs to cisplatin was evaluated using the adenosine triphosphate (ATP) assay, and the apoptosis was assessed using the caspase-3 assay.ResultsIHC results showed that CAPN4 protein expression was significantly upregulated in cisplatin-resistant lung adenocarcinoma tissues (P < 0.05). TCGA cohort analysis revealed that high CAPN4 expression was significantly associated with poor prognosis (reduced OS) in lung adenocarcinoma patients (HR = 1.4, P < 0.05). PDOs derived from cisplatin-resistant patients exhibited significant upregulation in CAPN4 protein and stemness markers at both gene and protein levels (all P < 0.05). Cisplatin sensitivity assays demonstrated that PDOs derived from cisplatin-resistant patients had significantly higher IC₅₀ values than those from cisplatin-sensitive patients(P < 0.05). After CAPN4 knockdown, the expression of stem cell makers in PDOs derived from cisplatin-resistant patients were significantly reduced, and their sensitivity to cisplatin was enhanced (P < 0.05).ConclusionKnockdown of CAPN4 reduces stem cell marker expression and enhances cisplatin sensitivity in lung adenocarcinoma PDOs, providing a potential therapeutic target for reversing cisplatin resistance in lung cancer.