Fig. 7: Assessment of RTK ligands in NSCLC tumor tissue.

a–c mRNA was extracted from FFPE tissue from 22 NSCLC patients (n = 13 untreated and n = 9 treated with erlotinib; 12 from UT Southwestern Medical Center, 10 from The Jackson Laboratory). HGF, NRG1, and GDNF mRNA levels were quantified by qPCR. Each dot represents the log2 mRNA level of each patient and median ± interquartile range. *p < 0.05, by Kolmogorov–Smirnov (KS) test. d–f From our A549 RNAseq result andj analyzed by GSEA, there are 237 ligand genes upregulated by erlotinib (positive z-score of Erl/Ctrl) and such upregulation was blocked by prednisolone (negative z-score of prednisolone + erlotinib/erlotinib). The human ligands list generating was described in the “Methods” section. Among these 237 ligands, there are 20 cytokines and 23 receptor tyrosine kinase ligands (RTKLs). These three sets of ligands formed three signatures. g–i RNAseq data of Fragments Per Kilobase of transcript per Million mapped reads (FPKM) among 38 patients with EGFR-activating mutant (exon 19 deletion or L858R) lung adenocarcinoma (LUAD) were downloaded from The Cancer Genome Atlas Program (TCGA)- Lung Adenocarcinoma (LUAD) database: portal.gdc.cancer.gov. 15004 genes out of the whole 60483 genes, with more than 19 (50%) non-zero expression among 38 cases, were selected to perform Single-sample Gene Set Enrichment Analysis (ssGSEA). The 3 signatures’ scores for each of 38 patients were calculated. One score represents how the signature of ligands is coordinately up- or down-regulated in one case. For each signature, patients were equally divided by 50% low (n = 19) and 50% high scores (n = 19). Overall survival analysis were performed on two subgroups of patients, divided by 237 ligands, 20 cytokines, and 23 RTKLs. Kaplan–Meier (KM) plot was drawn, log-rank test was used to validate if these erlotinib-induced and prednisolone-blocked ligands would predict a worse prognosis in EGFR mutant NSCLC patients, who would receive EGFR inhibition treatment. *p < 0.05. by log-rank test. The Kolmogorov–Smirnov tests and survival analyses were performed by GraphPad Prism 9.0.0. Source data are provided as a Source Data file.