Abstract
Brain metastases frequently develop in patients with non-small cell lung cancer (NSCLC) and are a common cause of cancer-related deaths, yet our understanding of the underlying human biology is limited. Here we performed multimodal single-nucleus RNA and T cell receptor, single-cell spatial and whole-genome sequencing of brain metastases and primary tumors of patients with treatment-naive NSCLC. Chromosomal instability (CIN) is a distinguishing genomic feature of brain metastases compared with primary tumors, which we validated through integrated analysis of molecular profiling and clinical data in 4,869 independent patients, and a new cohort of 12,275 patients with NSCLC. Unbiased analyses revealed transcriptional neural-like programs that strongly enriched in cancer cells from brain metastases, including a recurring, CINhigh cell subpopulation that preexists in primary tumors but strongly enriched in brain metastases, which was also recovered in matched single-cell spatial transcriptomics. Using multiplexed immunofluorescence in an independent cohort of treatment-naive pairs of primary tumors and brain metastases from the same patients with NSCLC, we validated genomic and tumor-microenvironmental findings and identified a cancer cell population characterized by neural features strongly enriched in brain metastases. This comprehensive analysis provides insights into human NSCLC brain metastasis biology and serves as an important resource for additional discovery.
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Data availability
Processed data (snRNA-seq, TCR-seq, spatial transcriptomics, whole-genome sequencing) are deposited at NCB GEO, with accession number GSE223503. Raw data were uploaded to dbGAP, with accession number phs003865.v1.p1. The deidentified DNA-sequencing and RNA-seq data are owned by Caris Life Sciences and cannot be publicly shared owing to the data usage agreement signed by B.I. at Columbia University Irving Medical Center. Qualified researchers can apply for access to these data by contacting J. Xiu ([email protected]), submitting a brief proposal and signing a data usage agreement. Inquiries will be addressed within 2 weeks, and the signing of data usage agreements may take up to 3 months, depending on institutional requirements. Further information and requests for resources should be directed to the corresponding author and will be fulfilled provided that the request complies with the ethical approval of the study. Reference genomes used in the study include the following: hg19, https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001405.13/ and GRCh38 (TCR-seq), https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001405.26/. Data in validation cohorts can be found at the following and are cited in the references section: TCGA, https://portal.gdc.cancer.gov/; https://doi.org/10.1016/j.cell.2022.01.003 (ref. 27); https://doi.org/10.1038/s41467-020-16164-1 (ref. 12); https://doi.org/10.1158/2159-8290.CD-15-0369 (ref. 7) and https://doi.org/10.1038/s41588-020-0592-7 (ref. 28).
Code availability
The full code for genomics analyses and image analysis is available via GitHub at https://github.com/IzarLab/NSCLC_study and https://github.com/kbestak/nf_mcmicro/tree/unstitch_restitch, respectively.
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Acknowledgements
This study was supported by the National Institute of Health (NIH), National Cancer Institute (NCI) grants R37CA258829, R01CA266446, R01CA280414 and U54CA274506 (to B.I.), and additional support came from the Burroughs Wellcome Fund Career Award for Medical Scientists, a Velocity Fellows Award, the Louis V. Gerstner, Jr. Scholars Program, a Tara Miller Young Investigator Award by the Melanoma Research Alliance, a Tara Miller Team Science Award for Brain Metastasis Research by the Melanoma Research Alliance and the Pershing Square Sohn Cancer Research Alliance Award (to B.I.). B.I. is a CRI Lloyd J. Old STAR (CRI5579). This study was also supported by the Herbert Irving Comprehensive Cancer Center Human Tissue Immunology and Immunotherapy Initiative (to B.I.). L.C. is supported by NIH NCI fellowship F30CA281104. L.C., K.L., E.D’S., P.H., Z.H.W. and D.B. are supported by Medical Scientist Training Program grant T32GM145440. Y.W. was supported by NIH, National Institute of Allergy and Infectious Disease training grant T32AI148099. K.B., M.A.I.A., and D.S. are supported by the German Federal Ministry of Education and Research (BMBF 01ZZ2004) and the state of Baden-Württemberg through bwHPC and the German Research Foundation (DFG) through grant INST 35/1597-1 FUGG. This work was supported by NIH NCI Cancer Center Support grant P30CA013696, the Molecular Pathology Shared Resource and its Tissue Bank and the Human Immune Monitoring Core at Columbia University. Biospecimens and/or data used for this study were obtained from the Columbia University Biobank, which is partially supported by Columbia University’s Clinical and Translational Science Award funded through grant number UL1TR001873.
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Contributions
B.I. conceived of the study and provided overall supervision. S.T., L.C., K.L., E.D’S. Y.W., A.J., C.G., J.B. and N.R. performed analyses of single-cell genomic, spatial and whole-genome sequencing data. L.C., A.D.A., J.C.M., I.B., N.S., P.R., Y.G., P.H., Z.H.W., M.R. and P.S. generated sequencing data. K.B., S.A., P.K., D.Z.B., M.A.I.-.A., D.S. and P.S. analyzed multiplexed imaging data. S.A., V.J., B.B., L.E. and P.S. generated multiplexed imaging data. G.G.L., C.A.S., B.H., N.A.R., M.G.P., D.B., M.I.E., P.C., J.N.B., A.C., A.S., H.H., G.K.S. and B.S.H. provided clinical specimens, clinical annotation and/or histopathological evaluation. S.W., S.K.D. and G.S. contributed DNA and RNA sequencing data. N.A., E.Z., A.M.T. and F.C. provided supervision for specific analyses. D.S. and P.S. provided additional overall supervision. S.T., L.C., P.S. and B.I. wrote the paper and revision. All authors reviewed, contributed to and approved of the paper.
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Competing interests
B.I. has received consulting fees and honoraria from Volastra Therapeutics Inc, Merck, AstraZeneca, Novartis, Eisai and Janssen Pharmaceuticals and has received research funding to Columbia University from Alkermes, Arcus Biosciences, Checkmate Pharmaceuticals, Compugen, Immunocore, Merck, Regeneron and Synthekine. B.I. is a founder of Basima Therapeutics, Inc. C.G. has received consulting fees from Watershed Informatics. B.H. received consulting fees and honoraria from Amgen, Eisai and MJH Life Sciences. A.M.T. received research funding from Ono Pharmaceuticals. N.A.R. is currently an employee and shareholder of Synthekine Inc. B.S.H. has received consulting fees from AstraZeneca, Ideaya, Jazz Pharmaceuticals, Sorrento Therapeutics, Genentech-Roche, OncLive, Veeva, Athenium, Boxer, Dava Oncology and SAI-Med and research funding to Columbia University from Neximmune, Inc, Janssen and Genentech-Roche. A.D.A. is now an employee of Adaptimmune. J.B. is now an employee of Pfizer. S.W., .S.K.D, and G.S. are employees of Caris Life Sciences. A.S. received consulting fees and honoraria from Abbvie, Bristol Myers Squibb, Veracyte, Genentech, Medscape and Physician Education Resource, and research support from Boehringer Ingelheim. All remaining authors report no competing interests.
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Extended data
Extended Data Fig. 1 snRNA-seq quality control and refinement of malignant cell assignment.
a, Basic quality control measures of samples (n = 43; PTs = 12, BMs = 31) processed for snRNA-seq and b, expression of a variety of different artifactual stress signatures, each separated by tissue origin. Upper and lower edges of boxplot indicate 75th and 25th percentiles, respectively, and middle line indicates median. One-sided Wilcoxon tests were performed, with p values as indicated on each graph. c, Representative schematic of refinement of malignant cell identity using URBAN (see Methods). An exemplary case is shown with initial cell type assignment (malignant vs. non-malignant) projected in two UMAP dimensions, a schematic summary of URBAN integrating inferred copy number variants (CNV) in single-cell transcriptome data, and refinement using matched low-pass whole-genome sequencing (lpWGS), that enables more accurate determination of malignant cells. This approach is performed in each sample individually and in an iterative fashion across the cohort. d, Exemplary UMAP embedding indicating PTPRC/CD45 and EPCAM expression across individual samples. e, ichorCNA plots for selected samples. X axis indicates chromosome number and y axis log2 ratio for ploidy, where 0 indicates diploidy, +1 gain, and –1 loss.
Extended Data Fig. 2 Chromosomal instability in NSCLC BMs.
a-b, Enrichment of CNVs per genomic ___location profiled from each individual PT and BM sample, with an upper consensus plot which totals all the CNVs observed at a given genomic ___location. The color strip at the top of the plot represents the plurality consensus at each genomic ___location (red = amp, grey = loh, blue = del, orange = bamp, skyblue = bdel) across the chromosomal landscape (columns) for all samples (rows) from a, primary tumors and b, brain metastases. c, Kaplan Meier survival curve of lung adenocarcinoma patient survival, stratified by CINhigh (upper quartile, red line) and CINlow (lower quartile, blue line) status as estimated by measurement of fraction of genome altered (FGA) from whole-exome sequencing data in the cancer genome atlas (TCGA) lung adenocarcinoma cohort. d, Pearson’s correlation of Caris’ panel DNA sequence of ~700 genes with TCGA WES data based on FGA (R = 0.99, p = 0). One-sided Wilcoxon test of significance performed., e-f, CIN70 signature25 expression (z-score) (e) and STING signature expression (z-score) (f) in LUAD RNAseq data from Caris Life Sciences across disease site (PTs = 5867, BMs = 617, ECMs = 2612), g-h, CIN70 signature25 expression (z-score) (g) and STING signature expression (z-score) (h) in LUSC RNAseq data from Caris Life Sciences across disease site (PTs = 2641, BMs = 91, ECMs = 747). Upper and lower edges of boxplots in e-h indicate 75th and 25th percentiles, respectively, and middle line indicates median. Tails extend to the 95% confidence interval. Statistical test and significance level as indicated on top of each boxplot.
Extended Data Fig. 3 NMF metaprograms.
a, Gene set enrichment analysis indicating cancer hallmarks and gene ontology signatures enriched in each of the 8 metaprograms identified by NMF. Q score is FDR adjusted p-value. b, Expression of mixed PT/BM MP signatures (MPs 1, 2, 3, 6) across healthy and malignant tissues (normal lymph node, normal lung, pleural effusion, early/late stage lung cancer, lymph node metastases, and BMs) profiled in Kim et al 202012. Upper and lower edges of boxplots indicate 75th and 25th percentiles, respectively, and middle line indicates median. Tails extend to the 95% confidence interval. Statistical test and significance level as indicated on each boxplot.
Extended Data Fig. 4 Quality control of rare malignant cell population (Cluster 21).
a-f, Quality control measures of rare, malignant cell population (Cluster 21). Box plots depicting a, gene counts, b, mitochondrial reads, c, cluster 21 signature expression across non-malignant groups, d, doublet score, and e-f, processing-related stress signature expression. g, Copy number alteration (CNA) patterns of cluster 21 cells compared to remaining malignant cells in the patient’s original cluster. Columns represent chromosomes, rows individual cell transcriptomes with inferred CNAs (blue = deletion, red = amplification). Left bar indicates rows occupied by cluster 21 cells (green) or other malignant cells from the representative patient (yellow). h, Gene set enrichment analysis (GSEA) of the cluster 21 population based on differential gene expression, Q score is FDR adjusted p-value. i, Stacked bar plots denoting percentage of PT or BM cells that comprise cluster 21 compared to the proportion of non-cluster 21 cells. Two-sided Fisher’s exact test, significance level as indicated. j, CIN70 signature expression in cancer cells from cluster 21 compared to all others. k-l, Boxplots indicating cluster 21 signature expression across matched sample pairs, PA060 (BM): N254 (PT) (k) and PA067 (BM): N586 (PT) (l). m, Cluster 21 signature expression in 44 snRNA-seq profiles in Kim et al12 dataset. n, Box plots indicating the expression of the cluster 21 signature in cell lines derived from NSCLC primary tumors (n = 91) or metastatic lesions (n = 101) of the cancer cell line encyclopedia (CCLE). o, Kaplan Meier survival curve of lung adenocarcinoma patients, stratified by expression of the cluster 21 signature. Data derived from TCGA. p, UMAP embedding of healthy lung epithelial cell types coupled with density gradients to highlight distinct cellular neighborhoods. q, Computation of probability of random walks starting from mixed lineage cells (ML1-ML15) reaching a class of labeled cells (AT1, AT2-Main, AT2-high MT, Airway Ciliated, Other), followed by assigning each unlabeled cell in ML group a cell state label based maximum probability. Upper and lower edges of boxplots indicate 75th and 25th percentiles, respectively, and middle line indicates median. Tails extend to the 95% confidence interval. Statistical test and significance level as indicated on each boxplot. One-side Wilcoxon tests were performed when utilized.
Extended Data Fig. 5 The tumor microenvironment of NSCLC PTs and BMs.
a, Dot plot of representative T cell marker genes, separated by disease site. Rows indicate selected genes, while columns indicate refined cell type assignment. b, UMAP embedding of CD8 + T cell clusters indicating expression of TCF7 and TOX. c-d, Diffusion component (D.C) analysis of CD8 + T cells colored by TCF7 and TOX expression ratio (c), separated by disease site (d). e, Dot plot of representative Myeloid marker genes, separated by disease site. Rows indicate selected genes, while columns indicate refined cell type assignment. f, Dot plot of representative CNS marker genes. Rows indicate selected genes, while columns indicate refined cell type assignment. g, Gene set enrichment analysis indicating cancer hallmarks and gene ontology signatures enriched in CNS: cancer cell, CNS: myeloid, and CNS:T cells interactions identified in the ContactTracing31 analysis. Q score is FDR adjusted p-value.
Extended Data Fig. 6 The spatial landscape of NSCLC PTs and BMs.
a-f, Correlation of TME cell fraction discovered in spatial transcriptomics (x axis) and snRNA-seq (y axis) analysis. Pearson’s correlation and significance as indicated on plot, Shaded areas surrounding line of best fit indicate 95% confidence interval. One-sided Wilcoxon tests of significance were performed. g, Recurrent drivers of spatial variability (selected genes indicated on the left) across samples profiled by spatial transcriptomics (columns). Top bar indicates cell types (in this case, including only malignant cells), and the bottom bar tissue origin (BM vs. PT), h-q, Spatial cell-type annotation across all other samples profiled, with colors indicating cell type assignment.
Supplementary information
Supplementary Information
Supplementary Figs. 1 and 2.
Supplementary Table 1
snRNA-seq cohort information.
Supplementary Table 2
Data related to metaprogram analyses.
Supplementary Table 3
Cluster 21 DEG analysis.
Supplementary Table 4
DEGs of nonmalignant cell types.
Supplementary Table 5
List of consumables.
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Tagore, S., Caprio, L., Amin, A.D. et al. Single-cell and spatial genomic landscape of non-small cell lung cancer brain metastases. Nat Med 31, 1351–1363 (2025). https://doi.org/10.1038/s41591-025-03530-z
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DOI: https://doi.org/10.1038/s41591-025-03530-z
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