Fig. 6: Inhibition of osimertinib-induced deNG-IL6 restores TKI sensitivity in TKI-resistant NSCLC.

a Survival of parental and OR PC9 and HCC827 cells with osimertinib treatment (72 h). n = 6 independent experiments per data point, mean ± SEM. b Levels of IL6 secreted by the parental and OR PC9 and HCC827 cells after 72 h of attachment. n= 3 technical replicates, mean ± SEM, two-tailed unpaired t-test, PC9-OR: ***p < 0.0001; HCC827-OR: ***p < 0.0001. c mRNA expression of IL6 in OR cells transduced with shIL6-expressing VSV-G particles. The samples were derived from the same experiment for IL6 and GAPDH were seperated in the same gel. d Quantity of total secreted IL6 in OR-shIL6 cells as measured by ELISAs. n = 3 technical replicates, mean ± SEM, one-tailed unpaired t-test, PC9-OR: ***p < 0.0001; HCC827-OR: ***p < 0.0001. e Morphology of OR and OR-shIL6 cells treated with osimertinib (5 μM) for 72 h. Scale bar, 100 μm. f Survival of PC9-OR-shIL6 and HCC827-OR-shIL6 cells treated with osimertinib at 5 μM (72 h). n = 3 independent experiments, mean ± SEM, two-tailed unpaired t-test, PC9-OR: ***p < 0.0001; HCC827-OR: ***p < 0.0001. g Pattern of secreted IL6 in the parental and OR PC9 and HCC827 cells. Because the PC9-P and HCC827-P cells secreted little IL6, which prevented analysis of its glycosylation pattern, we established PC9-IL6-WT and HCC827-IL6-WT cells as control cell lines. The proportions of heavy IL6 in these cells were quantified and are shown in the right panel. n = 3 independent experiments, mean ± SEM, two-tailed unpaired t-test, PC9-OR: ***p < 0.0001; HCC827-OR: ***p < 0.0001. h GSEA of N-linked glycosylation pathways and EMT, SRC and YAP activation statuses in the transcriptome dataset GSE106765 from the HCC827-OR-Taka cells. Nominal p-value was one-tailed test on the appropriate side of the null distribution. i Expression of N-OST genes in the parental and OR PC9 and HCC827 cells, as detected by qPCR. n = 3 independent experiments, mean ± SEM, one-tailed unpaired t-test, PC9-OR: **p = 0.0065, *p = 0.0193, n.s. p = 0.1579, ***p < 0.0001, **p = 0.0099, **p = 0.0047; HCC827-OR: 827-OR: ***p < 0.0001, ***p < 0.0001, *p = 0.0160, ***p = 0.004, **p = 0.0025, **p = 0.0014. j N-glycosylated IL6 detected by lectin blotting with wheat germ agglutinin (WGA). The proportions of WGA-detected NG-IL6 in WT-IL6 or OR-IL6 were quantified and are shown in the right panel. n = 3 independent experiments, mean ± SEM, one-tailed unpaired t-test, PC9-OR: ***p < 0.0001; HCC827-OR: ***p < 0.0001. k Quantitative characterization of N-glycans on purified secreted WT-IL6 or OR-IL6 from CM of PC9 or HCC827 cell derivatives by lectin microarray, as assessed with GlycoStation^TM Reader 1200 (GlycoTechnica). The raw data was calculated and output automatically by GlycoStation Tools Pro Suite 1.5 software. n = 3 technical replicates, mean ± S.D. See also Supplementary Table 1. l Characterization of glycosylation in IL6 by MS. See also Supplementary Fig. 22. m Pattern of secreted WT-IL6 or OR-IL6 with or without PNGase F digestion. The percentage proportions of H-IL6 and L-IL6 were quantified in the segmented histogram. n = 3 independent experiments, mean ± SEM. Source data are provided as a Source Data file.