Single-cell transcriptomic analysis uncovers intratumoral heterogeneity and drug-tolerant persister in ALK-rearranged lung adenocarcinoma

Acquired drug resistance is the major therapeutic obstacle to maintenance treatment of advanced-stage non-small cell lung cancer. Lung adenocarcinoma (ADC) harboring driver mutations also showed poor response to immune checkpoint inhibitors (ICIs). Underlying mechanisms of how drug insensitivity evolves remain unclear. Here we explored the intratumoral heterogeneity of tyrosine kinase inhibitor (TKI)-resistant anaplastic lymphoma kinase (ALK)-rearranged lung ADC organoids using single-cell RNA-sequencing (scRNA-seq) transcriptomic analysis. IL-17 signaling pathway was found highly induced in a subpopulation of pre-existing ALK-TKI-resistant cells. These drug-tolerant persister (DTP) cells, also found to have high surface intracellular adhesion molecule 1 (ICAM-1) expression level, were more resistant towards ALK-TKI and expressed a higher level of cancer-stem cell transcriptional factors. Moreover, tumor cells with high ICAM-1 expression were found spatially correlated with ROR?t+ Th17 infiltration in ALK-rearranged NSCLC resected tumor tissues. In conclusion, our data revealed marked intratumoral heterogeneity in ALK-rearranged tumor, and pre-existing DTP cells may contribute to the development of drug insensitivity in ALK-rearranged lung ADC. Overall design: Single-cell transcriptomic analysis. To investigate the intratumoral heterogeneity in ALK-rearranged lung ADC, we have generated ALK-rearranged LCOs from lung ADC patients (TS485T-O was derived from resected primary tumor, and FA34-O were derived from malignant pleural effusion, -O = organoids). Continuous exposure to escalating concentration of ALK-TKIs led to the emergence of acquired resistance. Single-cell RNA sequencing (scRNA-seq) analysis on TKI-sensitive and -resistant tumor organoids TS485T-O and FA34-O was performed (-C = crizotinib resistant; -A = alectinib resistant).