Background Treatment resistance limits long-term benefit to both immune checkpoint inhibitors (ICI) and targeted tyrosine kinase inhibitors (TKIs) in NSCLC. Resistance is thought to emerge from surviving persister cells, which to-date have been most studied in the context of TKI resistance but remain poorly understood. Persister cell phenotypes in ICI-treated patients, the associated immune cell states, and how these compare to TKI-treated samples, have not been characterized.

Methods We performed single-cell RNA-sequencing (10x 5’ sequencing) on NSCLC samples from patients treated with TKIs or ICI-regimens. Samples were obtained prior to initiation of therapy (pre-treatment) and/or at treatment nadir, typically after 3 months of therapy. Samples were grouped by treatment (TKI vs ICI-combo) and by oncogenic driver, and differential expression and immune composition at treatment nadir vs pre-treatment was compared.

Results A total of 54 samples with > 160,000 cells after quality control and filtering were included, 22 at pre-treatment and 32 at nadir. After identification of tumor and immune cells, hierarchical clustering of immune cell type proportions identified five immune phenotypes consisting of macrophage predominant, stromal, B cell predominant, CD4 lymphocyte predominant, and CD8/CD4 predominant. EGFR mutant pre-treatment samples had a higher proportion of stromal dominant samples compared to the other samples, which had a higher proportion of CD8/CD4 predominant samples. At nadir vs pre-treatment, TKI-treated samples had a relative increase in endothelial cells and a trend toward increased CD8 T cells, consisting primarily in effector memory T cells. ICI-treated samples were predominantly stromal, with non-significant increases in CD8 and CD4 T cells, with a relative decrease in exhausted T cells and increase in cytotoxic T cells. TKI persister cells were characterized by upregulation of class I and class II HLAs, proteasome, and chemokines including CXCL5, which has been shown to recruit myeloid-derived suppressor cells, and down-regulation of NKX2 and neuronal cell markers. ICI samples were characterized by upregulation of PI3K/PTEN signaling and down-regulation of peptide and steroid metabolic pathways.

Conclusions NSCLC therapies induce treatment-specific effects on the tumor and immune microenvironment at treatment nadir, with upregulation of immune and immunosuppressive chemokine signaling to TKI, and upregulation of PI3K/PTEN signaling to ICIs, which has been previously implicated in ICI resistance. Further work to translate these findings into therapeutic vulnerabilities may help improve long-term treatment outcomes.

Acknowledgements This work was supported by the generous philanthropic contributions to The University of Texas MD Anderson Lung Moon Shot Program, the MD Anderson Cancer Center Support Grant P30 CA016672, R01CA276178 (NV), Rexanna’s Foundation for Fighting Lung Cancer.

Ethics Approval This study was approved by the institutional review board at MDACC and all patients provided written informed consent under protocols PA13-0589, PA16-0061 or PA14-0531.