Characterization of immune-modulating metabolites as biomarkers in non-small cell lung cancer : role in brain metastasis and treatment response prediction

Abstract

Brain metastasis (Bramet) is a major cause of death in patients with Non-Small Cell Lung cancer (NSCLC), the second leading cause of cancer death worldwide and in China. Immune checkpoint such as indoleamine 2,3-dioxygenase (IDO) can converse tryptophan to kynurenine and has been known for its effect on distant metastasis and overall survival (OS) in NSCLC. This study hypothesized that the IDO metabolites were associated with Bramet and treatment responses. Specifically, this study aimed to 1) examine whether the IDO metabolites are correlated with immune cell functions, treatment response and subsequent OS in NSCLC patients with Bramet; 2) identify immune-modulating metabolites beyond IDO metabolites as biomarkers associated with Bramet and radiotherapy treatment responses. This is a prospective biomarker study. A total of 195 patients with NSCLC were enrolled. Blood was collected for IDO metabolites measurement. The baseline circulating level of kynurenine was significantly higher in stage IV than in stage I (2.3 vs.1.7 μM, p < 0.001). In Stage IV patients, the kynurenine concentrations and kynurenine: tryptophan (K:T) ratios were significantly higher in patients with Bramet compared to that of patients with extra-cranial metastasis (2.7 vs. 1.9 μM, p < 0.001; 0.12 vs. 0.10, p = 0.028; respectively). K:T ratios were significantly associated with the relative level of circulating regulatory T cells (Tregs) (p < 0.001) and OS (p = 0.039) in Bramet patients. In-vitro, kynurenine also led to the dysfunction of CD4+ and CD8+ T cells by upregulating suppressive molecules such as PD-1. Single-cell analysis validated more IDO1 expression in stage IV tumors or Bramet metastatic lesions, particularly in endothelial and epithelial cells. In 19 NSCLC patients with Bramet treated with surgical resection, the kynurenine level was increased after surgery in those patients with worse survival (p = 0.037). In a pilot of 31 Bramet patients received thoracic RT (n = 22) or brain RT (n = 9), radiotherapy induced a borderline significant decrease in K:T ratios (p = 0.07). The patients with lower post/pre-K:T ratios had better survival than those with higher K:T ratios (p = 0.014). Brain RT induced changes in K:T ratios was associated with OS in Bramet patients (p = 0.004). Metabolomics profiling was performed in 25 stage IV patients with (n = 12) or without Bramet (n = 13). Five circulating immunosuppressive metabolites, including kynurenine and indole-3-carbinol were identified to be increased in Bramet patients. Notably, indole-3-carbinol level was associated with high Tregs percentage and low NK percentage (R = -0.42, p = 0.06). For Bramet patients (n = 8) treated with brain radiotherapy, 4 circulating immunosuppressive metabolites including kynurenine and bilirubin decreased in the circulation, which may partially explain the Radiation immune modulation effects on the tumor systemic immune environment. In summary, the IDO metabolites, and other immune-modulating metabolites, were significantly associated with the presence of Bramet and survival in Bramet patients, likely through immune suppression effects. These immune-modulating metabolites may serve as a basis for biomarker discovery to guide personalized radiation and immune therapies or targeted therapy. Further studies are warranted.