Integrated single-cell transcriptomic analysis of brain and lymph node metastases in non-small cell lung cancer

Abstract

BACKGROUND

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide, and metastasis remains the primary driver of treatment failure and death. Despite recent advances in single-cell RNA sequencing (scRNA-seq), most studies have focused on isolated metastatic sites; there is still a lack of studies that simultaneously focus on the cellular and molecular characteristics of brain and lymph node metastases in NSCLC.

AIM

To explore features of brain and lymph node metastases in NSCLC and to nominate candidate pathways for subsequent functional interrogation.

METHODS

We integrated three public scRNA-seq datasets (GSE131907, GSE186344, and GSE256491) to construct a high-resolution transcriptomic atlas of NSCLC. These scRNA-seq cohorts were heterogeneous and not clinically matched primary-metastasis pairs, comprising a total of 45 samples (including 11 primary tumors, 7 lymph node metastases, 15 brain metastases, and 12 normal controls) and 208506 high-quality cells. We systematically dissected cellular heterogeneity, the distribution of tumor epithelial subpopulations in different metastatic sites, pathway activation, and immune microenvironment remodeling during metastasis. To assess the relationship between the expression of the tumor subclusters and overall survival of lung adenocarcinoma (LUAD) patients, we conducted a multivariate Cox regression analysis using The Cancer Genome Atlas (TCGA) data with ssGSEA gene-signature scores. Multiplex immunohistochemistry (mIHC) was performed on a cohort of 23 patients (Mets1: 12 cases of lymph node metastasis, including 2 with distant metastasis; Mets0: 11 cases of non-metastasis) to preliminarily validate macrophage infiltration and unfolded protein response (UPR)-related protein expression.

RESULTS

Metastatic lesions exhibited marked enrichment of T cells and myeloid cells. Immune landscape analysis revealed a dual immunosuppressive microenvironment comprising exhausted T cells overexpressing PDCD1, HAVCR2, CTLA4, and LAG3, and increased macrophage infiltration. The distribution of tumor epithelial subpopulations varied in different metastatic foci. The UPR was abnormally activated in both metastatic sites. TCGA data analysis revealed that high expression of the tumor epithelial subpopulation (C14_PBK_Tumor) was associated with inferior overall survival in LUAD patients (multivariate Cox regression analysis, hazard ratio = 2.39, 95%CI: 1.15-4.97, adjusted P = 0.02). Consistently, the mIHC results showed that in the metastasis group, the levels of CD68⁺CD163⁺ macrophages and cells triple-positive for UPR markers (XBP1, ATF6, and ATF4) were higher than those in the non-metastasis group. (Mann–Whitney U test; data are presented as medians; CD68⁺CD163⁺ cell rate: Met0: 1.913 vs Met1: 3.338, P = 0.0129; XBP1⁺/ATF6⁺/ATF4⁺ cell rate: Met0: 60.19 vs Met1: 73.26, P = 0.0028).

CONCLUSION

These findings suggest that in brain and lymph node metastatic sites of NSCLC, there is abnormal activation of the UPR, along with a dual immunosuppressive microenvironment and differential distribution of tumor epithelial subpopulations. However, further functional and prospective studies are needed to confirm these observations.

Keywords: Non-small cell lung cancer; scRNA-seq; T cells; Macrophages; Unfolded protein response; Brain metastasis; Lymph node metastasis

Core Tip: Our study offers a panoramic analysis of two distinct metastatic sites within the same disease context. Our main findings are: The distribution of tumor epithelial subpopulations differs across metastatic foci; abnormal activation of unfolded protein response (UPR) occurs in both metastatic sites; and a dual immunosuppressive microenvironment is characterized. Multivariate Cox regression analysis based on The Cancer Genome Atlas data revealed that, among the tumor epithelial subpopulations, the C14_PBK_Tumor subtype was associated with worse prognosis in lung adenocarcinoma patients. Importantly, multiplex immunohistochemistry was performed to assess macrophage infiltration and the expression of UPR-related proteins. These findings still require further verification through functional experiments and prospective studies.