Single-cell RNA sequencing elucidated the landscape of breast cancer brain metastases and identified ILF2 as a potential therapeutic target

Abstract

Distant metastasis remains the primary cause of morbidity in patients with breast cancer. Hence, the development of more efficacious strategies and the exploration of potential targets for patients with metastatic breast cancer are urgently needed. The data of six patients with breast cancer brain metastases (BCBrM) from two centres were collected, and a comprehensive landscape of the entire tumour ecosystem was generated through the utilisation of single-cell RNA sequencing. We utilised the Monocle2 and CellChat algorithms to investigate the interrelationships among each subcluster. In addition, multiple signatures were collected to evaluate key components of the subclusters through multi-omics methodologies. Finally, we elucidated common expression programs of malignant cells, and experiments were conducted in vitro and in vivo to determine the functions of interleukin enhancer-binding factor 2 (ILF2), which is a key gene in the metastasis module, in BCBrM progression. We found that subclusters in each major cell type exhibited diverse characteristics. Besides, our study indicated that ILF2 was specifically associated with BCBrM, and experimental validations further demonstrated that ILF2 deficiency hindered BCBrM progression. Our study offers novel perspectives on the heterogeneity of BCBrM and suggests that ILF2 could serve as a promising biomarker or therapeutic target for BCBrM.

References

[1] SungH, FerlayJ, SiegelRL, et al. Global cancer statistics 2020:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209-249.

[2] LinNU, BellonJR, WinerEP. CNS metastases in breast cancer. J Clin Oncol. 2004;22(17):3608-3617.

[3] CortiC, Antonarelli G, CriscitielloC, et al. Targeting brain metastases in breast cancer. Cancer Treat Rev. 2022;103:102324.

[4] SoffiettiR, Pellerino A. Brain metastasis from HER2-positive breast cancer: an evolving landscape. Clin Cancer Res. 2023;29(1):8-10.

[5] WuS, LuJ, ZhuH, et al. A novel axis of circKIF4A-miR-637-STAT3 promotes brain metastasis in triple-negative breast cancer. Cancer Lett. 2024;581:216508.

[6] BrockA, ChangH, HuangS. Non-genetic heterogeneity—a mutation-independent driving force for the somatic evolution of tumours. Nat Rev Genet. 2009;10(5):336-342.

[7] MarusykA, Almendro V, PolyakK. Intra-tumour heterogeneity: a looking glass for cancer? Nat Rev Cancer. 2012;12(5):323-334.

[8] ZhangY, WangD, PengM, et al. Single-cell RNA sequencing in cancer research. J Exp Clin Cancer Res. 2021;40(1):81.

[9] XuL, ZhangJ, HeY, et al. ScRNAPip: a systematic and dynamic pipeline for single-cell RNA sequencing analysis. iMeta. 2023;2(4):e132.

[10] DingS, ChenX, ShenK. Single-cell RNA sequencing in breast cancer: understanding tumor heterogeneity and paving roads to individualized therapy. Cancer Commun (Lond). 2020;40(8):329-344.

[11] OlbrechtS, Busschaert P, QianJ, et al. High-grade serous tubo-ovarian cancer refined with single-cell RNA sequencing: specific cell subtypes influence survival and determine molecular subtype classification. Genome Med. 2021;13(1):111.

[12] PatelAP, TiroshI, TrombettaJJ, et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science. 2014;344(6190):1396-1401.

[13] LiPH, KongXY, HeYZ, et al. Recent developments in application of single-cell RNA sequencing in the tumour immune microenvironment and cancer therapy. Mil Med Res. 2022;9(1):52.

[14] XieJ, DengW, DengX, et al. Single-cell histone chaperones patterns guide intercellular communication of tumor microenvironment that contribute to breast cancer metastases. Cancer Cell Int. 2023;23(1):311.

[15] BiermannJ, MelmsJC, AminAD, et al. Dissecting the treatment-naive ecosystem of human melanoma brain metastasis. Cell. 2022;185(14):2591-2608.e30.

[16] WangZ, WangY, ChangM, et al. Single-cell transcriptomic analyses provide insights into the cellular origins and drivers of brain metastasis from lung adenocarcinoma. Neuro Oncol. 2023;25(7):1262-1274.

[17] MundA, Brunner AD, MannM. Unbiased spatial proteomics with single-cell resolution in tissues. Mol Cell. 2022;82(12):2335-2349.

[18] RaoA, Barkley D, FrancaGS, YanaiI. Exploring tissue architecture using spatial transcriptomics. Nature. 2021;596(7871):211-220.

[19] XieJ, DengX, XieY, et al. Multi-omics analysis of disulfidptosis regulators and therapeutic potential reveals glycogen synthase 1 as a disulfidptosis triggering target for triple-negative breast cancer. MedComm. 2024;5(3):e502.

[20] HaoY, HaoS, Andersen-NissenE, et al. Integrated analysis of multimodal single-cell data. Cell. 2021;184(13):3573-3587.e29.

[21] TiroshI, IzarB, PrakadanSM, et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science. 2016;352(6282):189-196.

[22] WuT, HuE, XuS, et al. clusterProfiler 4.0:a universal enrichment tool for interpreting omics data. Innovation (Camb). 2021;2(3):100141.

[23] WuY, YangS, MaJ, et al. Spatiotemporal immune landscape of colorectal cancer liver metastasis at single-cell level. Cancer Discov. 2022;12(1):134-153.

[24] TrapnellC, Cacchiarelli D, GrimsbyJ, et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat Biotechnol. 2014;32(4):381-386.

[25] JinS, Guerrero-Juarez CF, ZhangL, et al. Inference and analysis of cell-cell communication using CellChat. Nat Commun. 2021;12(1):1088.

[26] ZouY, XieJ, ZhengS, et al. Leveraging diverse cell-death patterns to predict the prognosis and drug sensitivity of triple-negative breast cancer patients after surgery. Int J Surg. 2022;107:106936.

[27] LiuZ, LiuL, WengS, et al. BEST: a web application for comprehensive biomarker exploration on large-scale data in solid tumors. J Big Data. 2023;10(1):165.

[28] RitchieME, Phipson B, WuD, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47.

[29] ShiW, TanzhuG, ChenL, et al. Radiotherapy in preclinical models of brain metastases: a review and recommendations for future studies. Int J Biol Sci. 2024;20(2):765-783.

[30] ZhangH, YueX, ChenZ, et al. Define cancer-associated fibroblasts (CAFs) in the tumor microenvironment: new opportunities in cancer immunotherapy and advances in clinical trials. Mol Cancer. 2023;22(1):159.

[31] FosterDS, Januszyk M, DelittoD, et al. Multiomic analysis reveals conservation of cancer-associated fibroblast phenotypes across species and tissue of origin. Cancer Cell. 2022;40(11):1392-1406.e7.

[32] WinklerEA, KimCN, RossJM, et al. A single-cell atlas of the normal and malformed human brain vasculature. Science. 2022;375(6584):eabi7377. https://pubmed.ncbi.nlm.nih.gov/35084939

[33] ChenZ, WeiX, WangX, et al. NDUFA4L2 promotes glioblastoma progression, is associated with poor survival, and can be effectively targeted by apatinib. Cell Death Dis. 2021;12(4):377.

[34] LaiRK, XuIM, ChiuDK, et al. NDUFA4L2 fine-tunes oxidative stress in hepatocellular carcinoma. Clin Cancer Res. 2016;22(12):3105-3117.

[35] YuanY, GaoH, ZhuangY, et al. NDUFA4L2 promotes trastuzumab resistance in HER2-positive breast cancer. Ther Adv Med Oncol. 2021;13:17588359211027836. https://pubmed.ncbi.nlm.nih.gov/34276814

[36] LiM, YangY, XiongL, Jiang P, WangJ, LiC. Metabolism, metabolites, and macrophages in cancer. J Hematol Oncol. 2023;16(1):80.

[37] ParkSY, KimIS. Harnessing immune checkpoints in myeloid lineage cells for cancer immunotherapy. Cancer Lett. 2019;452:51-58.

[38] NguyenHM, Gaikwad S, OladejoM, AgrawalMY, Srivastava SK, WoodLM. Interferon stimulated gene 15 (ISG15) in cancer: an update. Cancer Lett. 2023;556:216080.

[39] KangJA, KimYJ, JeonYJ. The diverse repertoire of ISG15:more intricate than initially thought. Exp Mol Med. 2022;54(11):1779-1792.

[40] YinJ, YuanJ, LiY, et al. Neoadjuvant adebrelimab in locally advanced resectable esophageal squamous cell carcinoma: a phase 1b trial. Nat Med. 2023;29(8):2068-2078.

[41] ZengQ, Michael IP, ZhangP, et al. Synaptic proximity enables NMDAR signalling to promote brain metastasis. Nature. 2019;573(7775):526-531.

[42] JinY, KangY, WangM, et al. Targeting polarized phenotype of microglia via IL6/JAK2/STAT3 signaling to reduce NSCLC brain metastasis. Signal Transduct Target Ther. 2022;7(1):52.

[43] HuangQ, LiuL, XiaoD, et al. CD44(+) lung cancer stem cell-derived pericyte-like cells cause brain metastases through GPR124-enhanced trans-endothelial migration. Cancer Cell. 2023;41(9):1621-1636.e8.

[44] SunHF, LiLD, LaoIW, et al. Single-cell RNA sequencing reveals cellular and molecular reprograming landscape of gliomas and lung cancer brain metastases. Clin Transl Med. 2022;12(11):e1101.

[45] XieY, HeL, LuganoR, et al. Key molecular alterations in endothelial cells in human glioblastoma uncovered through single-cell RNA sequencing. JCI Insight. 2021;6(15). https://pubmed.ncbi.nlm.nih.gov/34228647

[46] StogsdillJA, KimK, BinanL, Farhi SL, LevinJZ, ArlottaP. Pyramidal neuron subtype diversity governs microglia states in the neocortex. Nature. 2022;608(7924):750-756.

[47] AndersenJ, ThomN, ShadrachJL, et al. Single-cell transcriptomic landscape of the developing human spinal cord. Nat Neurosci. 2023;26(5):902-914.

[48] ToSKY, TangMKS, TongY, et al. A selective beta-catenin-metadherin/CEACAM1-CCL3 axis mediates metastatic heterogeneity upon tumor-macrophage interaction. Adv Sci (Weinh). 2022;9(16):e2103230.

[49] ChanJM, Quintanal-Villalonga A, GaoVR, et al. Signatures of plasticity, metastasis, and immunosuppression in an atlas of human small cell lung cancer. Cancer Cell. 2021;39(11):1479-1496.e18.

[50] ZhangS, FangW, ZhouS, et al. Single cell transcriptomic analyses implicate an immunosuppressive tumor microenvironment in pancreatic cancer liver metastasis. Nat Commun. 2023;14(1):5123.

[51] MassagueJ, GaneshK. Metastasis-initiating cells and ecosystems. Cancer Discov. 2021;11(4):971-994.

[52] QuailDF, JoyceJA. Microenvironmental regulation of tumor progression and metastasis. Nat Med. 2013;19(11):1423-1437.

[53] FaresJ, FaresMY, KhachfeHH, Salhab HA, FaresY. Molecular principles of metastasis: a hallmark of cancer revisited. Signal Transduct Target Ther. 2020;5(1):28.

[54] LeeJ, HyeonDY, HwangD. Single-cell multiomics: technologies and data analysis methods. Exp Mol Med. 2020;52(9):1428-1442.

[55] ZiegenhainC, ViethB, ParekhS, et al. Comparative analysis of single-cell RNA sequencing methods. Mol Cell. 2017;65(4):631-643.e4.

[56] DaiH, LiL, ZengT, Chen L. Cell-specific network constructed by single-cell RNA sequencing data. Nucleic Acids Res. 2019;47(11):e62.

[57] WuSZ, Al-Eryani G, RodenDL, et al. A single-cell and spatially resolved atlas of human breast cancers. Nat Genet. 2021;53(9):1334-1347.

[58] PalB, ChenY, VaillantF, et al. A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast. EMBO J. 2021;40(11):e107333.

[59] HuL, SuL, ChengH, et al. Single-cell RNA sequencing reveals the cellular origin and evolution of breast cancer in BRCA1 mutation carriers. Cancer Res. 2021;81(10):2600-2611.

[60] RajanAM, MaRC, KochaKM, Zhang DJ, HuangP. Dual function of perivascular fibroblasts in vascular stabilization in zebrafish. PLoS Genet. 2020;16(10):e1008800.

[61] SaundersA, Macosko EZ, WysokerA, et al. Molecular diversity and specializations among the cells of the adult mouse brain. Cell. 2018;174(4):1015-1030.e16.

[62] HuangTX, TanXY, HuangHS, et al. Targeting cancer-associated fibroblast-secreted WNT2 restores dendritic cell-mediated antitumour immunity. Gut. 2022;71(2):333-344.

[63] ArpinatiL, Scherz-Shouval R. From gatekeepers to providers: regulation of immune functions by cancer-associated fibroblasts. Trends Cancer. 2023;9(5):421-443.

[64] AkiyamaT, YasudaT, UchiharaT, et al. Stromal reprogramming through dual PDGFRalpha/beta blockade boosts the efficacy of Anti-PD-1 immunotherapy in fibrotic tumors. Cancer Res. 2023;83(5):753-770.

[65] NakamuraK, SmythMJ. Myeloid immunosuppression and immune checkpoints in the tumor microenvironment. Cell Mol Immunol. 2020;17(1):1-12.

[66] DzimianskiJV, Scholte FEM, BergeronE, PeganSD. ISG15:it’s complicated. J Mol Biol. 2019;431(21):4203-4216.

[67] BurksJ, ReedRE, DesaiSD. Free ISG15 triggers an antitumor immune response against breast cancer: a new perspective. Oncotarget. 2015;6(9):7221-7231.

[68] NiT, MaoG, XueQ, et al. Upregulated expression of ILF2 in non-small cell lung cancer is associated with tumor cell proliferation and poor prognosis. J Mol Histol. 2015;46(4–5):325-335.

[69] OlivieroG, Munawar N, WatsonA, et al. The variant polycomb repressor complex 1 component PCGF1 interacts with a pluripotency sub-network that includes DPPA4, a regulator of embryogenesis. Sci Rep. 2015;5:18388.

[70] WanC, GongC, JiL, et al. NF45 overexpression is associated with poor prognosis and enhanced cell proliferation of pancreatic ductal adenocarcinoma. Mol Cell Biochem. 2015;410(1–2):25-35.

[71] LiY, WangM, YangM, et al. Nicotine-induced ILF2 facilitates nuclear mRNA export of pluripotency factors to promote stemness and chemoresistance in human esophageal cancer. Cancer Res. 2021;81(13):3525-3538.

[72] GaneshK, Massague J. Targeting metastatic cancer. Nat Med. 2021;27(1):34-44.

[73] OuX, TanY, XieJ, et al. Methylation of GPRC5A promotes liver metastasis and docetaxel resistance through activating mTOR signaling pathway in triple negative breast cancer. Drug Resist Updat. 2024;73:101063.

RIGHTS & PERMISSIONS

2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.