Fig 2: Effects of HCC827R-CSC-EVs loaded with APE1 shRNA on the Erlotinib resistance of NSCLC cells in vitro. (A) The enrichment of APEX1 (APE1) in EVs of different origins, analysed using the ExoRBase database. (B) The mRNA expression of APE1 in HCC827P-CSCs, HCC827R-CSCs, HCC827P-CSC-EVs and HCC827R-CSC-EVs determined by qRT-PCR assay. (C) The mRNA expression of APE1 in HCC827R-CSCs and HCC827R-CSC-EVs in response to shAPE1 treatment, determined by qRT-PCR assay. (D) The mRNA expression of APE1 in HCC827P and PC9P cells in response to co-culture with HCC827R-CSCs EVs shAPE1 determined by qRT-PCR assay. (E) Western blot measurement of the protein expression of p-STAT3/STAT3 in HCC827P and PC9P cells in response to co-culture with HCC827R-CSCs EVs shAPE1. (F) ELISA detection of the content of IL-6 in the supernatant of HCC827P and PC9P cells in response to co-culture with HCC827R-CSCs EVs shAPE1. (G) Cell viability in HCC827P and PC9P cells in response to co-culture with HCC827R-CSCs EVs shAPE1 and further treatment with Erlotinib, detected by CCK-8 assay. Quantification of the migration (H) and invasion (I) in HCC827P and PC9P cells in response to co-culture with HCC827R-CSCs EVs shAPE1 and further treatment with Erlotinib (5 µM), detected by the Transwell assay. (J) Cell apoptosis in HCC827P and PC9P cells in response to co-culture with HCC827R-CSCs EVs shAPE1 and further treatment with Erlotinib (5 µM), detected by flow cytometry. (K) Protein expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax and cleaved caspase-3 in the Western blot of Erlotinib (5 µM)-treated HCC827P and PC9P cells in response to co-culture with HCC827R-CSCs EVs shAPE1. (L) The expression of MDR1, MRP, LRP and ABCG2 in Erlotinib (5 µM)-treated HCC827P and PC9P cells in response to co-culture with HCC827R-CSCs EVs shAPE1 measured by qRT-PCR and the Western blot. * p < .05. Each cell experiment was conducted in triplicate. APE1, apurinic endonuclease 1; CSC, cancer stem cell; EV, extracellular vesicle; IL-6, interleukin-6; NSCLC, non-small cell lung cancer; shRNA, short hairpin RNA

Fig 3: Effects of shAPE1-loaded HCC827R-CSC-EVs on the Erlotinib resistance of NSCLC cells in nude mice. Nude mice were treated with Erlotinib, Erlotinib + HCC827R-CSC-EVs or Erlotinib + HCC827R-CSC-EVs shAPE1. (A) The volume of tumour xenografts at different time points. (B) The weight of isolated tumour xenografts in mice. (C) ELISA detection of IL-6 protein levels in tumour tissues of mice. (D) The Western blot measurement of the protein expression of APE1, p-STAT3 and STAT3 in tumour tissues of mice. (E) The Western blot measurement of anti-apoptotic Bcl-2 and pro-apoptotic Bax and cleaved caspase-3 in tumour tissue of mice. (F) The expression of MDR1, MRP, LRP and ABCG2 in tumour tissues of mice measured by qRT-PCR and Western blot. * p < .05. n = 8. APE1, apurinic endonuclease 1; CSC, cancer stem cell; EV, extracellular vesicle; IL-6, interleukin-6; NSCLC, non-small cell lung cancer

Fig 4: Effect of HCC827P-CSC-EVs and HCC827R-CSC-EVs on the resistance of NSCLC cells to Erlotinib. (A) Expression of Erlotinib resistance-related genes MDR1, MRP, LRP and ABCG2 in HCC827P and PC9P cells following co-culture with HCC827P-CSC-EVs or HCC827R-CSC-EVs, detected by qRT-PCR and Western blot. (B) Viability of HCC827P and PC9P cells co-cultured with HCC827P-CSC-EVs or HCC827R-CSC-EVs and further treated with different concentrations of Erlotinib, detected by CCK-8 assay. Quantification of the migration (C) and invasion (D) of Erlotinib (5 µM)-treated HCC827P and PC9P cells in response to co-culture with HCC827P-CSC-EVs or HCC827R-CSC-EVs, observed by Transwell assay. (E) Apoptosis of Erlotinib (5 µM)-treated HCC827P and PC9P cells in response to co-culture with HCC827P-CSC-EVs or HCC827R-CSC-EVs, detected by flow cytometry. (F) Protein expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax and cleaved caspase-3 in Western blot of Erlotinib (5 µM)-treated HCC827P and PC9P cells in response to co-culture with HCC827P-CSC-EVs or HCC827R-CSC-EVs. * p < .05. Each cell experiment was conducted in triplicate. CSC, cancer stem cell; EV, extracellular vesicle; NSCLC, non-small cell lung cancer

Fig 5: APE1 affects the Erlotinib resistance of NSCLC cells through mediating the IL-6/STAT3 signalling. (A) The protein expression of APE1 and p-STAT3/STAT3 in HCC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, measured by the Western blot. (B) The content of IL-6 in the supernatant of HCC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, detected by ELISA. (C) Cell viability in HCC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, detected by CCK-8 assay. Quantification of the migration (D) and invasion (E) in HCC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, observed in the Transwell assay. (F) The apoptosis of HCC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, tested by flow cytometry. (G) The protein expression of Bcl-2, Bax and cleaved caspase-3 in HCC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, measured by the Western blot. (H) The mRNA and protein expression of MDR1, MRP, LRP and ABCG2 in CC827R and PC9R cells treated by Erlotinib alone or in combination with shAPE1, measured by qRT-PCR and Western blot. *p < .05. Each cell experiment was conducted in triplicate. APE1, apurinic endonuclease 1; IL-6, interleukin-6; NSCLC, non-small cell lung cancer