Single-cell transcriptome analysis reveals evolving tumour microenvironment induced by immunochemotherapy in nasopharyngeal carcinoma

Abstract

	•Immunochemotherapy remodeled T cell phenotypes.
	•For the patients achieving complete response, more interferon gamma was provided by CD8+ T cells after therapy, which would be the key for TAMs pro-inflammatory repolarization and eventually promote the CD8+ T cells maturation in turns.
	•Among patients who did not reach complete response, malignant cells exhibited higher level of immune checkpoint genes before therapy, and decreased tumor antigen presentation activity, which may underlie the resistance mechanism to therapy.

Keywords

immunochemotherapy effect / nasopharyngeal carcinoma / scRNA-seq / tumour microenvironment

References

[1] ChangET, YeW, ZengY-X, Adami H-O. The evolving epidemiology of nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev. 2021; 30: 1035-1047.

[2] ZhangL, HuangY, HongS, et al. Gemcitabine plus cisplatin versus fluorouracil plus cisplatin in recurrent or metastatic nasopharyngeal carcinoma: a multicentre, randomised, open-label, phase 3 trial. Lancet. 2016; 388: 1883-1892.

[3] ChenY-P, ChanATC, LeQ-T, et al. Nasopharyngeal carcinoma. Lancet. 2019; 394: 64-80.

[4] LiW-Z, LvX, HuD, et al. Effect of induction chemotherapy with paclitaxel, cisplatin, and capecitabine vs cisplatin and fluorouracil on failure-free survival for patients with stage IVA to IVB nasopharyngeal carcinoma: a multicenter phase 3 randomized clinical trial. JAMA Oncol. 2022; 8: 706-714.

[5] Guo-YingL, Yan-Fang Y, Yao-FeiJ, et al. Nab-paclitaxel, cisplatin, and capecitabine versus cisplatin and gemcitabine as first line chemotherapy in patients with recurrent or metastatic nasopharyngeal carcinoma: randomised phase 3 clinical trial. BMJ. 2024; 385: e077890.

[6] ZhaoC, MiaoJ, ShenG, et al. Anti-epidermal growth factor receptor (EGFR) monoclonal antibody combined with cisplatin and 5-fluorouracil in patients with metastatic nasopharyngeal carcinoma after radical radiotherapy: a multicentre, open-label, phase II clinical trial. Ann Oncol. 2019; 30: 637-643.

[7] LiL, KongF, ZhangL, et al. Apatinib, a novel VEGFR-2 tyrosine kinase inhibitor, for relapsed and refractory nasopharyngeal carcinoma: data from an open-label, single-arm, exploratory study. Invest New Drugs. 2020; 38: 1847-1853.

[8] LvJ-W, LiJ-Y, LuoL-N, Wang Z-X, ChenY-P. Comparative safety and efficacy of anti-PD-1 monotherapy, chemotherapy alone, and their combination therapy in advanced nasopharyngeal carcinoma: findings from recent advances in landmark trials. J Immunother Cancer. 2019; 7: 159.

[9] LiangH, JiangY-F, LiuG-Y, et al. Camrelizumab and apatinib plus induction chemotherapy and concurrent chemoradiotherapy in stage N3 nasopharyngeal carcinoma: a phase 2 clinical trial. Nat Commun. 2024; 15: 1029.

[10] WangF-H, WeiX-L, FengJ, et al. Efficacy, safety, and correlative biomarkers of toripalimab in previously treated recurrent or metastatic nasopharyngeal carcinoma: a phase II clinical trial (POLARIS-02). J Clin Oncol. 2021; 39: 704-712.

[11] MereuE, LafziA, MoutinhoC, et al. Benchmarking single-cell RNA-sequencing protocols for cell atlas projects. Nat Biotechnol. 2020; 38: 747-755.

[12] ChenY-P, YinJ-H, LiW-F, et al. Single-cell transcriptomics reveals regulators underlying immune cell diversity and immune subtypes associated with prognosis in nasopharyngeal carcinoma. Cell Res. 2020; 30: 1024-1042.

[13] JinS, LiR, ChenM-Y, et al. Single-cell transcriptomic analysis defines the interplay between tumor cells, viral infection, and the microenvironment in nasopharyngeal carcinoma. Cell Res. 2020; 30: 950-965.

[14] LiuY, HeS, WangX-L, et al. Tumour heterogeneity and intercellular networks of nasopharyngeal carcinoma at single cell resolution. Nat Commun. 2021; 12: 741.

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

[16] MiloševićD, MedeirosAS, Stojković Piperac M, et al. The application of Uniform Manifold Approximation and Projection (UMAP) for unconstrained ordination and classification of biological indicators in aquatic ecology. Sci Total Environ. 2022; 815: 152365.

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

[18] KorsunskyI, Millard N, FanJ, et al. Fast, sensitive and accurate integration of single-cell data with Harmony. Nat Methods. 2019; 16: 1289-1296.

[19] YuG, WangL-G, HanY, HeQ-Y. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012; 16: 284-287.

[20] LiberzonA, Subramanian A, PinchbackR, et al. Molecular signatures database (MSigDB) 3.0. Bioinformatics. 2011; 27: 1739-1740.

[21] BagaevA, KotlovN, NomieK, et al. Conserved pan-cancer microenvironment subtypes predict response to immunotherapy. Cancer Cell. 2021; 39: 845-865.

[22] QiuX, MaoQ, TangY, et al. Reversed graph embedding resolves complex single-cell trajectories. Nat Methods. 2017; 14: 979-982.

[23] AibarS, González-Blas CB, MoermanT, et al. SCENIC: single-cell regulatory network inference and clustering. Nat Methods. 2017; 14: 1083-1086.

[24] SuoS, ZhuQ, SaadatpourA, et al. Revealing the critical regulators of cell identity in the mouse cell atlas. Cell Rep. 2018; 25: 1436-1445.

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

[26] LiR, LiY, KristiansenK, WangJ. SOAP: short oligonucleotide alignment program. Bioinformatics. 2008; 24: 713-714.

[27] KimD, Langmead B, SalzbergSL. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015; 12: 357-360.

[28] LangmeadB, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9: 357-359.

[29] LiB, DeweyCN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011; 12: 323.

[30] ZhangL, MacIsaac KD, ZhouT, et al. Genomic analysis of nasopharyngeal carcinoma reveals TME-based subtypes. Mol Cancer Res. 2017; 15: 1722-1732.

[31] HumphriesMP, Maxwell P, QuPathSalto-TellezM. The global impact of an open source digital pathology system. Comput Struct Biotechnol J. 2021; 19: 852-859.

[32] ZhaoJ, GuoC, XiongF, et al. Single cell RNA-seq reveals the landscape of tumor and infiltrating immune cells in nasopharyngeal carcinoma. Cancer Lett. 2020; 477: 131-143.

[33] MillerBC, SenDR, Al AbosyR, et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat Immunol. 2019; 20: 326-336.

[34] D’AliseAM, BrasuN, De IntinisC, et al. Adenoviral-based vaccine promotes neoantigen-specific CD8+ T cell stemness and tumor rejection. Sci Transl Med. 2022; 14: eabo7604.

[35] CollinM, BigleyV. Human dendritic cell subsets: an update. Immunology. 2018; 154: 3-20.

[36] LatchmanY, WoodCR, ChernovaT, et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol. 2001; 2: 261-268.

[37] YangR, SunL, LiC-F, et al. Galectin-9 interacts with PD-1 and TIM-3 to regulate T cell death and is a target for cancer immunotherapy. Nat Commun. 2021; 12: 832.

[38] DasM, ZhuC, KuchrooVK. Tim-3 and its role in regulating anti-tumor immunity. Immunol Rev. 2017; 276: 97-111.

[39] LinesJL, Pantazi E, MakJ, et al. VISTA is an immune checkpoint molecule for human T cells. Cancer Res. 2014; 74: 1924-1932.

[40] HallBM, Gleiberman AS, StromE, et al. Immune checkpoint protein VSIG4 as a biomarker of aging in murine adipose tissue. Aging Cell. 2020; 19: e13219.

[41] BiK, HeMX, BakounyZ, et al. Tumor and immune reprogramming during immunotherapy in advanced renal cell carcinoma. Cancer Cell. 2021; 39: 649-661.

[42] LiY, HanS, WuB, et al. CXCL11 correlates with immune infiltration and impacts patient immunotherapy efficacy: a pan-cancer analysis. Front Immunol. 2022; 13: 951247.

[43] MaL, WangL, KhatibSA, et al. Single-cell atlas of tumor cell evolution in response to therapy in hepatocellular carcinoma and intrahepatic cholangiocarcinoma. J Hepatol. 2021; 75: 1397-1408.

[44] QiJ, SunH, ZhangY, et al. Single-cell and spatial analysis reveal interaction of FAP+ fibroblasts and SPP1+ macrophages in colorectal cancer. Nat Commun. 2022; 13: 1742.

[45] LocatiM, Curtale G, MantovaniA. Diversity, mechanisms, and significance of macrophage plasticity. Annu Rev Pathol. 2020; 15: 123-147.

[46] WuK, LinK, LiX, et al. Redefining tumor-associated macrophage subpopulations and functions in the tumor microenvironment. Front Immunol. 2020; 11: 1731.

[47] CoradaM, Orsenigo F, MoriniMF, et al. Sox17 is indispensable for acquisition and maintenance of arterial identity. Nat Commun. 2013; 4: 2609.

[48] VanlandewijckM, HeL, MäeMA, et al. A molecular atlas of cell types and zonation in the brain vasculature. Nature. 2018; 554: 475-480.

[49] SuT, Stanley G, SinhaR, et al. Single-cell analysis of early progenitor cells that build coronary arteries. Nature. 2018; 559: 356-362.

[50] YouL-R, LinF-J, LeeCT, et al. Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity. Nature. 2005; 435: 98-104.

[51] ThiriotA, Perdomo C, ChengG, et al. Differential DARC/ACKR1 expression distinguishes venular from non-venular endothelial cells in murine tissues. BMC Biol. 2017; 15: 45.

[52] dela PazNG, D’Amore PA. Arterial versus venous endothelial cells. Cell Tissue Res. 2009; 335: 5-16.

[53] KaluckaJ, de Rooij LPMH, GoveiaJ, et al. Single-cell transcriptome atlas of murine endothelial cells. Cell. 2020; 180: 764-779.

[54] GillichA, ZhangF, FarmerCG, et al. Capillary cell-type specialization in the alveolus. Nature. 2020; 586: 785-789.

[55] SchuppJC, AdamsTS, CosmeC, et al. Integrated single-cell atlas of endothelial cells of the human lung. Circulation. 2021; 144: 286-302.

[56] KfouryY, Baryawno N, SevereN, et al. Human prostate cancer bone metastases have an actionable immunosuppressive microenvironment. Cancer Cell. 2021; 39: 1464-1478.

[57] BagatiA, KumarS, JiangP, et al. Integrin αvβ6-TGFβ-SOX4 pathway drives immune evasion in triple-negative breast cancer. Cancer Cell. 2021; 39: 54-67.

[58] YuW-L, YuG, DongH, et al. Proteomics analysis identified TPI1 as a novel biomarker for predicting recurrence of intrahepatic cholangiocarcinoma. J Gastroenterol. 2020; 55: 1171-1182.

[59] NallasamyP, Nimmakayala RK, KarmakarS, et al. Pancreatic tumor microenvironment factor promotes cancer stemness via SPP1-CD44 axis. Gastroenterology. 2021; 161: 1998-2013.

[60] LiH, YangF, HuA, et al. Therapeutic targeting of circ-CUX1/EWSR1/MAZ axis inhibits glycolysis and neuroblastoma progression. EMBO Mol Med. 2019; 11: e10835.

[61] LeiS, CaoW, ZengZ, et al. JUND/linc00976 promotes cholangiocarcinoma progression and metastasis, inhibits ferroptosis by regulating the miR-3202/GPX4 axis. Cell Death Dis. 2022; 13: 967.

[62] XiongY, FengY, ZhaoJ, et al. TFAP2A potentiates lung adenocarcinoma metastasis by a novel miR-16 family/TFAP2A/PSG9/TGF-β signaling pathway. Cell Death Dis. 2021; 12: 352.

[63] KarimRM, YangL, ChenL, et al. Discovery of Dual TAF1-atr inhibitors and ligand-induced structural changes of the TAF1 tandem bromodomain. J Med Chem. 2022; 65: 4182-4200.

[64] CamineroA, Comabella M, MontalbanX. Role of tumour necrosis factor (TNF)-α and TNFRSF1A R92Q mutation in the pathogenesis of TNF receptor-associated periodic syndrome and multiple sclerosis. Clin Exp Immunol. 2011; 166: 338-345.

[65] ZhangC, XiaoC, DangE, et al. CD100-Plexin-B2 promotes the inflammation in psoriasis by activating NF-κB and the inflammasome in keratinocytes. J Invest Dermatol. 2018; 138: 375-383.

[66] JeongH, LeeS-Y, SeoH, KimB-J. Recombinant Mycobacterium smegmatis delivering a fusion protein of human macrophage migration inhibitory factor (MIF) and IL-7 exerts an anticancer effect by inducing an immune response against MIF in a tumor-bearing mouse model. J Immunother Cancer. 2021; 9: e003180.

[67] AqbiHF, Wallace M, SappalS, PayneKK, Manjili MH. IFN-γ orchestrates tumor elimination, tumor dormancy, tumor escape, and progression. J Leukoc Biol.2018.

[68] PietrasA, KatzAM, EkströmEJ, et al. Osteopontin-CD44 signaling in the glioma perivascular niche enhances cancer stem cell phenotypes and promotes aggressive tumor growth. Cell Stem Cell. 2014; 14: 357-369.

[69] HeZ, XinZ, YangQ, et al. Mapping the single-cell landscape of acral melanoma and analysis of the molecular regulatory network of the tumor microenvironments. Elife. 2022; 11: e78616.

[70] JhunjhunwalaS, HammerC, DelamarreL. Antigen presentation in cancer: insights into tumour immunogenicity and immune evasion. Nat Rev Cancer. 2021; 21: 298-312.

[71] AnsariJ, Senchenkova EY, VitalSA, et al. Targeting the AnxA1/Fpr2/ALX pathway regulates neutrophil function, promoting thromboinflammation resolution in sickle cell disease. Blood. 2021; 137: 1538-1549.

[72] AustG, ZhengL, QuaasM. To detach, migrate, adhere, and metastasize: CD97/ADGRE5 in cancer. Cells. 2022; 11: 1538.

[73] de MassonA, Darbord D, DobosG, et al. Macrophage-derived CXCL9 and CXCL11, T-cell skin homing, and disease control in mogamulizumab-treated CTCL patients. Blood. 2022; 139: 1820-1832.

[74] YeungJ, Yaghoobi V, MiyagishimaD, et al. Targeting the CSF1/CSF1R axis is a potential treatment strategy for malignant meningiomas. Neuro Oncol. 2021; 23: 1922-1935.

[75] KakogiannosN, Ferrari L, GiampietroC, et al. JAM-A acts via C/EBP-α to promote claudin-5 expression and enhance endothelial barrier function. Circ Res. 2020; 127: 1056-1073.

[76] TuragaSM, SilverDJ, BayikD, et al. JAM-A functions as a female microglial tumor suppressor in glioblastoma. Neuro Oncol. 2020; 22: 1591-1601.

[77] BejaranoL, Jordāo MJC, JoyceJA. therapeutic targeting of the tumor microenvironment. Cancer Discov. 2021; 11: 933-959.

[78] ChenY-P, LvJ-W, MaoY-P, et al. Unraveling tumour microenvironment heterogeneity in nasopharyngeal carcinoma identifies biologically distinct immune subtypes predicting prognosis and immunotherapy responses. Mol Cancer. 2021; 20: 14.

[79] van der LeunAM, Thommen DS, SchumacherTN. CD8+ T cell states in human cancer: insights from single-cell analysis. Nat Rev Cancer. 2020; 20: 218-232.

[80] GuoX, ZhangY, ZhengL, et al. Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing. Nat Med. 2018; 24: 978-985.

[81] BassezA, VosH, Van DyckL, et al. A single-cell map of intratumoral changes during anti-PD1 treatment of patients with breast cancer. Nat Med. 2021; 27: 820-832.

[82] SenDR, Kaminski J, BarnitzRA, et al. The epigenetic landscape of T cell exhaustion. Science. 2016; 354: 1165-1169.

[83] LvJ, WeiY, YinJ-H, et al. The tumor immune microenvironment of nasopharyngeal carcinoma after gemcitabine plus cisplatin treatment. Nat Med. 2023; 29: 1424-1436.

[84] WangD, FangJ, WenS, et al. A comprehensive profile of TCF1+ progenitor and TCF1-terminally exhausted PD-1+CD8+ T cells in head and neck squamous cell carcinoma: implications for prognosis and immunotherapy. Int J Oral Sci. 2022; 14: 8.

[85] GuerrieroJL. Macrophages: the road less traveled, changing anticancer therapy. Trends Mol Med. 2018; 24: 472-489.

[86] MantovaniA, Allavena P, MarchesiF, GarlandaC. Macrophages as tools and targets in cancer therapy. Nat Rev Drug Discov. 2022; 21: 799-820.

[87] BarkalAA, BrewerRE, MarkovicM, et al. CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy. Nature. 2019; 572: 392-396.

[88] XiaY, RaoL, YaoH, et al. Engineering macrophages for cancer immunotherapy and drug delivery. Adv Mater. 2020; 32: e2002054.

[89] BenciJL, Johnson LR, ChoaR, et al. Opposing functions of interferon coordinate adaptive and innate immune responses to cancer immune checkpoint blockade. Cell. 2019; 178: 933-948.

[90] GocherAM, Workman CJ, VignaliDAA. Interferon-γ: teammate or opponent in the tumour microenvironment?. Nat Rev Immunol. 2022; 22: 158-172.

[91] HuB, YuM, MaX, et al. IFNα potentiates anti-PD-1 efficacy by remodeling glucose metabolism in the hepatocellular carcinoma microenvironment. Cancer Discov. 2022; 12: 1718-1741.

[92] ZhangM, HuangL, DingG, et al. Interferon gamma inhibits CXCL8-CXCR2 axis mediated tumor-associated macrophages tumor trafficking and enhances anti-PD1 efficacy in pancreatic cancer. J Immunother Cancer. 2020; 8: e000308.

[93] CuriglianoG, Gelderblom H, MachN, et al. Phase I/Ib clinical trial of sabatolimab, an anti-TIM-3 antibody, alone and in combination with spartalizumab, an anti-PD-1 antibody, in advanced solid tumors. Clin Cancer Res. 2021; 27: 3620-3629.

[94] HellmannMD, BiviN, CalderonB, et al. Safety and immunogenicity of LY3415244, a bispecific antibody against TIM-3 and PD-L1, in patients with advanced solid tumors. Clin Cancer Res. 2021; 27: 2773-2781.

RIGHTS & PERMISSIONS

2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.