肺癌恶性胸腔积液中稀有肿瘤细胞的鉴定与单细胞测序分析
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摘要
肿瘤细胞的异质性是指肿瘤细胞在基因组或表型水平上具有不同特征。在外界环境压力或人为杀伤因素刺激下,肿瘤细胞具有不同的响应方式,从而导致它们在细胞增殖、侵袭转移及耐药能力等方面产生差别,尤其是一部分具有转移能力的肿瘤细胞能脱离原位组织并在远处器官形成转移灶。因此,肿瘤细胞的异质性为其发生转移和耐药提供了可能。传统的肿瘤异质性研究主要基于不同位置原位组织样本中的群体细胞,缺乏单细胞层面的解析,尤其是对具有转移能力的肿瘤细胞的异质性研究。本研究建立了基于肺癌恶性胸腔积液中转移性肿瘤细胞的单细胞研究路线,首先利用代谢标志物鉴定恶性胸腔积液中高代谢活性的肿瘤细胞,其次通过单细胞Sanger测序揭示这些肿瘤细胞具有一致的驱动基因突变特征,然后利用高通量测序技术对肿瘤细胞染色体拷贝数变异进行分析,从而揭示同一患者具有转移能力的肿瘤细胞即使具有相同的驱动基因突变特征,但在基因组层面上仍具有异质性,可进一步细分为若干子群。本研究结果对于更深入地理解肿瘤转移机制具有重要意义。
Tumor heterogeneity refers to distinct genomic or phenotypic characteristics of tumor cells. Under the environmental or drug stress, tumor cells exhibit different responses, corresponding to different properties of cell proliferation, invasion, metastasis and drug resistance. In particular, a small fraction of tumor cells are capable of detaching from primary tumor sites and initiating distant metastases. Thus, tumor heterogeneity sets the basis for tumor resistance andmetastasis. Traditional methods in studying tumor heterogeneity are mainly based on bulk cells from different locations in primary tumors, lacking analysis at the single-cell level and of metastatic tumor cells. This study establishes a single-cell method to study metastatic tumor cells in malignant pleural effusions of lung cancer patients. Metabolically active tumor cells in malignant pleural effusions are firstly identified with a metabolic marker 2-NBDG, a fluorescent glucose analog. These metabolically active tumor cells are confirmed to harbor the same driver oncogenic mutations by Sanger sequencing, followed by high-throughput sequencing to analyze copy number variation profiles. Our results show metastatic tumor cells in pleural effusion have the same driver mutations but different features in copy number variation patterns. The study provides new insights to understand the mechanism of tumor metastasis.
Tumor heterogeneity refers to distinct genomic or phenotypic characteristics of tumor cells. Under the environmental or drug stress, tumor cells exhibit different responses, corresponding to different properties of cell proliferation, invasion, metastasis and drug resistance. In particular, a small fraction of tumor cells are capable of detaching from primary tumor sites and initiating distant metastases. Thus, tumor heterogeneity sets the basis for tumor resistance andmetastasis. Traditional methods in studying tumor heterogeneity are mainly based on bulk cells from different locations in primary tumors, lacking analysis at the single-cell level and of metastatic tumor cells. This study establishes a single-cell method to study metastatic tumor cells in malignant pleural effusions of lung cancer patients. Metabolically active tumor cells in malignant pleural effusions are firstly identified with a metabolic marker 2-NBDG, a fluorescent glucose analog. These metabolically active tumor cells are confirmed to harbor the same driver oncogenic mutations by Sanger sequencing, followed by high-throughput sequencing to analyze copy number variation profiles. Our results show metastatic tumor cells in pleural effusion have the same driver mutations but different features in copy number variation patterns. The study provides new insights to understand the mechanism of tumor metastasis.
引文
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[2]Aparicio S,Mardis E.Tumor heterogeneity:next-generation sequencing enhances the view from the pathologist’s microscope.Genome Biol,2014,15(9):463.
[3]Swanton C.Intratumor Heterogeneity:evolution through Space and Time.Cancer Res,2012,72(19):4875-4882.
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[11]Valastyan S,Weinberg RA.Tumor metastasis:molecular insights and evolving paradigms.Cell,2011,147(2):275-292.
[12]Yang YL,Chu JY,Wang MR.Tumor genetic heterogeneity.Hereditas(Beijing),2013,35(1):1-9.杨壹羚,褚嘉祐,王明荣.肿瘤遗传异质性.遗传,2013,35(1):1-9.
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[15]Tang Y,Wang Z,Li ZM,Kim J,Deng YL,Li Y,Heath JR,Wei W,Lu S,Shi QH.High-throughput screening of rare metabolically active tumor cells in pleural effusion and peripheral blood of lung cancer patients.Proc Natl Acad Sci USA,2017,114(10):2544-2549.
[16]Sun S,Deng YL.Single-cell detection of EGFR gene mutation in circulating tumor cells in lung cancer.Hereditas(Beijing),2015,37(12):1251-1257.孙帅,邓宇亮.肺癌循环肿瘤细胞的单细胞EGFR基因突变检测.遗传,2015,37(12):1251-1257.
[17]Cai X,Evrony GD,Lehmann HS,Elhosary PC,Mehta BK,Poduri A,Walsh CA.Single-Cell,Genome-wide sequencing identifies clonal somatic Copy-Number variation in the human brain.Cell Rep,2015,10(4):645.
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[20]Li H,Handsaker B,Wysoker A,Fennell T,Ruan J,Homer N,Marth G,Abecasis G,Durbin R.The sequence alignment/map format and samtools.Bioinformatics,2009,25(16):2078-2079.
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[22]Maemondo M,Inoue A,Kobayashi K,Sugawara S,Oizumi S,Isobe H,Gemma A,Harada M,Yoshizawa H,Kinoshita I,Fujita Y,Okinaga S,Hirano H,Yoshimori K,Harada T,Ogura T,Ando M,Miyazawa H,Tanaka T,Saijo Y,Hagiwara K,Morita S,Nukiwa T.Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR.N Engl J Med,2010,362(25):2380-2388.
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[24]Asahina H,Yamazaki K,Kinoshita I,Sukoh N,Harada M,Yokouchi H,Ishida T,Ogura S,Kojima T,Okamoto Y,Fujita Y,Dosaka-Akita H,Isobe H,Nishimura M.A phase II trial of gefitinib as first-line therapy for advanced non-small cell lung cancer with epidermal growth factor receptor mutations.Br J Cancer,2006,95(8):998-1004.
[25]Stankiewicz P,Lupski JR.Genome architecture,rearrangements and genomic disorders.Trends Genet,2002,18(2):74-82.
[26]Litt M,Luty JA.A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene.Am J Hum Genet,1989,44(3):397-401.
[27]Saxonov S,Berg P,Brutlag DL.A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters.Proc Natl Acad Sci USA,2006,103(5):1412-1417.
[28]Wang DC,Wang W,Zhu B,Wang X.Lung cancer heterogeneity and new strategies for drug therapy.Annu Rev Pharmacol,2017,58:531-546.
[29]Greaves M,Maley CC.Clonal evolution in cancer.Nature,2012,481(7381):306-313.
[30]He YL,Ying Y,Xu YL,Su JF,Luo H,Wang HF.Effects of Lycium barbarum polysaccharide on tumor microenvironment T-lymphocyte subsets and dendritic cells in H22-bearing mice.J Chin Integr Med,2005,3(5):374-377.何彦丽,应逸,许艳丽,苏俊芳,罗惠,王惠峰.枸杞多糖对荷瘤小鼠肿瘤微环境T淋巴细胞亚群及树突状细胞的影响.中西医结合学报,2005,3(5):374-377.
[31]Liotta LA,Kohn EC.The microenvironment of the tumour-host interface.Nature,2001,411(6835):375-379.
[32]Douma S,van Laar T,Zevenhoven J,Meuwissen R,van Garderen E,Peeper DS.Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB.Nature,2004,430(7003):1034-1039.
[33]Pantel K,Alix-panabières C.Functional studies on viable circulating tumor cells.Clin Chem,2015,62(2):328-334.
[34]Vaupel P,Kallinowski F,Okunieff P.Blood flow,oxygen and nutrient supply,and metabolic microenvironment of human tumors:a review.Cancer Res,1989,49(23):6449-6465.
[35]Kalmus J,Okunieff P,Vaupel P.Effect of intraperitoneal versus intravenous glucose administration on laser Doppler flow in murine FSaII tumors and normal skin.Cancer Res,1989,49(22):6313-6317.
[36]Shackleton M,Quintana E,Fearon ER,Morrison SJ.Heterogeneity in cancer:cancer stem cells versus clonal evolution.Cell,2009,138(5):822-829.
[37]Singh SK,Clarke ID,Terasaki M,Bonn VE,Hawkins C,Squire J,Dirks PB.Identification of a cancer stem cell in human brain tumors.Cancer Res,2003,63(18):5821-5828.
[38]Heidt DG,Li C,Dalerba P,Burant CF,Zhang L,Adsay V,Wicha M,Clarke MF,Simeone DM.Identification of pancreatic cancer stem cells.Cancer Res,2007,67(3):1030-1037.
[2]Aparicio S,Mardis E.Tumor heterogeneity:next-generation sequencing enhances the view from the pathologist’s microscope.Genome Biol,2014,15(9):463.
[3]Swanton C.Intratumor Heterogeneity:evolution through Space and Time.Cancer Res,2012,72(19):4875-4882.
[4]Wang LW,Wang L,Li N.Clinical cancer genomics and personalized treatment.Chin Clin Oncol,2010,15(6):481-486.王理伟,王雷,李宁.临床肿瘤基因组学与肿瘤个体化治疗.临床肿瘤学杂志,2010,15(6):481-486.
[5]Peng QP,Liang HJ.Specific phenotype for glycolysis in malignant tumor cells and its significance.Chin Clin Oncol,2009,14(5):470-473.彭秋平,梁后杰.糖酵解代谢在恶性肿瘤细胞中的特异性表型及其意义.临床肿瘤学杂志,2009,14(5):470-473.
[6]Khoo BL,Grenci G,Lin YB,Lee SC,Han J,Lim CT.Expansion of patient-derived circulating tumor cells from liquid biopsies using a CTC microfluidic culture device.Nat Protoc,2018,13(1):34-58
[7]Gerlinger M,Rowan A,Horswell S,Math M,Larkin J,Endesfelder D,Gronroos E,Martinez P,Matthews N,Stewart A,Tarpey P,Varela I,Phillimore B,Begum S,McDonald NQ,Butler A,Jones D,Raine K,Latimer C,Santos CR,Nohadani M,Eklund AC,Spencer-Dene B,Clark G,Pickering L,Stamp G,Gore M,Szallasi Z,Downward J,Futreal PA,Swanton C.Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.N Engl J Med,2012,366(10):883-892.
[8]Xu X,Hou Y,Yin XY,Bao L,Tang AF,Song LT,Li FQ,Tsang S,Wu K,Wu HJ,He WM,Zeng L,Xing MJ,Wu RH,Jiang H,Liu X,Cao DD,Guo GW,Hu XD,Gui YT,Li ZS,Xie WY,Sun XJ,Shi M,Cai ZM,Wang B,Zhong MM,Li JX,Lu ZH,Gu N,Zhang XQ,Goodman L,Bolund L,Wang J,Yang HM,Kristiansen K,Dean M,Li YR,Wang J.Single-cell exome sequencing reveals single-nucleotide mutation characteristics of a kidney tumor.Cell,2012,148(5):886-895.
[9]Hou Y,Song LT,Zhu P,Zhang B,Tao Y,Xu X,Li FQ,Wu K,Liang J,Shao D,Wu HJ,Ye XF,Ye C,Wu RH,Jian M,Chen Y,Xie W,Zhang RR,Chen L,Liu X,Yao XT,Zheng HC,Yu C,Li QB,Gong ZL,Mao M,Yang X,Yang L,Li JX,Wang W,Lu ZH,Gu N,Wang J.Single-Cell exome sequencing and monoclonal evolution of a JAK2-Negative myeloproliferative neoplasm.Cell,2012,148(5):873-885.
[10]Navin N,Kendall J,Troge J,Andrews P,Rodgers L,McIndoo J,Cook K,Stepansky A,Levy D,Esposito D,Muthuswamy L,Krasnitz A,McCombie WR,Hicks J,Wigler M.Tumour evolution inferred by single-cell sequencing.Nature,2011,472(7341):90-94.
[11]Valastyan S,Weinberg RA.Tumor metastasis:molecular insights and evolving paradigms.Cell,2011,147(2):275-292.
[12]Yang YL,Chu JY,Wang MR.Tumor genetic heterogeneity.Hereditas(Beijing),2013,35(1):1-9.杨壹羚,褚嘉祐,王明荣.肿瘤遗传异质性.遗传,2013,35(1):1-9.
[13]Fenton KN,Richardson JD.Diagnosis and management of malignant pleural effusions.Respirology,1995,170(1):69-74.
[14]Roberts ME,Neville E,Berrisford RG,Antunes G,Ali NJ,BTS Pleural Disease Guideline Group.Management of a malignant pleural effusion:British thoracic society pleural disease guideline 2010.Thorax,2010,65(Suppl.2):ii32-ii40.
[15]Tang Y,Wang Z,Li ZM,Kim J,Deng YL,Li Y,Heath JR,Wei W,Lu S,Shi QH.High-throughput screening of rare metabolically active tumor cells in pleural effusion and peripheral blood of lung cancer patients.Proc Natl Acad Sci USA,2017,114(10):2544-2549.
[16]Sun S,Deng YL.Single-cell detection of EGFR gene mutation in circulating tumor cells in lung cancer.Hereditas(Beijing),2015,37(12):1251-1257.孙帅,邓宇亮.肺癌循环肿瘤细胞的单细胞EGFR基因突变检测.遗传,2015,37(12):1251-1257.
[17]Cai X,Evrony GD,Lehmann HS,Elhosary PC,Mehta BK,Poduri A,Walsh CA.Single-Cell,Genome-wide sequencing identifies clonal somatic Copy-Number variation in the human brain.Cell Rep,2015,10(4):645.
[18]Bolger AM,Lohse M,Usadel B.Trimmomatic:a flexible trimmer for illumina sequence data.Bioinformatics,2014,30(15):2114-2120.
[19]Li H,Durbin R.Fast and accurate short read alignment with Burrows-Wheeler transform.Bioinformatics,2009,25(14):1754-1760.
[20]Li H,Handsaker B,Wysoker A,Fennell T,Ruan J,Homer N,Marth G,Abecasis G,Durbin R.The sequence alignment/map format and samtools.Bioinformatics,2009,25(16):2078-2079.
[21]Venkatraman ES,Olshen AB.A faster circular binary segmentation algorithm for the analysis of array CGH data.Bioinformatics,2007,23(6):657-663.
[22]Maemondo M,Inoue A,Kobayashi K,Sugawara S,Oizumi S,Isobe H,Gemma A,Harada M,Yoshizawa H,Kinoshita I,Fujita Y,Okinaga S,Hirano H,Yoshimori K,Harada T,Ogura T,Ando M,Miyazawa H,Tanaka T,Saijo Y,Hagiwara K,Morita S,Nukiwa T.Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR.N Engl J Med,2010,362(25):2380-2388.
[23]Cappuzzo F,Hirsch FR,Rossi E,Bartolini S,Ceresoli GL,Bemis L,Haney J,Witta S,Danenberg K,Domenichini I,Ludovini V,Magrini E,Gregorc V,Doglioni C,Sidoni A,Tonato M,Franklin WA,Crino L,Bunn PA,Varella-Garcia M.Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer.J Natl Cancer Inst Monograp,2005,97(9):643-655.
[24]Asahina H,Yamazaki K,Kinoshita I,Sukoh N,Harada M,Yokouchi H,Ishida T,Ogura S,Kojima T,Okamoto Y,Fujita Y,Dosaka-Akita H,Isobe H,Nishimura M.A phase II trial of gefitinib as first-line therapy for advanced non-small cell lung cancer with epidermal growth factor receptor mutations.Br J Cancer,2006,95(8):998-1004.
[25]Stankiewicz P,Lupski JR.Genome architecture,rearrangements and genomic disorders.Trends Genet,2002,18(2):74-82.
[26]Litt M,Luty JA.A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene.Am J Hum Genet,1989,44(3):397-401.
[27]Saxonov S,Berg P,Brutlag DL.A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters.Proc Natl Acad Sci USA,2006,103(5):1412-1417.
[28]Wang DC,Wang W,Zhu B,Wang X.Lung cancer heterogeneity and new strategies for drug therapy.Annu Rev Pharmacol,2017,58:531-546.
[29]Greaves M,Maley CC.Clonal evolution in cancer.Nature,2012,481(7381):306-313.
[30]He YL,Ying Y,Xu YL,Su JF,Luo H,Wang HF.Effects of Lycium barbarum polysaccharide on tumor microenvironment T-lymphocyte subsets and dendritic cells in H22-bearing mice.J Chin Integr Med,2005,3(5):374-377.何彦丽,应逸,许艳丽,苏俊芳,罗惠,王惠峰.枸杞多糖对荷瘤小鼠肿瘤微环境T淋巴细胞亚群及树突状细胞的影响.中西医结合学报,2005,3(5):374-377.
[31]Liotta LA,Kohn EC.The microenvironment of the tumour-host interface.Nature,2001,411(6835):375-379.
[32]Douma S,van Laar T,Zevenhoven J,Meuwissen R,van Garderen E,Peeper DS.Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB.Nature,2004,430(7003):1034-1039.
[33]Pantel K,Alix-panabières C.Functional studies on viable circulating tumor cells.Clin Chem,2015,62(2):328-334.
[34]Vaupel P,Kallinowski F,Okunieff P.Blood flow,oxygen and nutrient supply,and metabolic microenvironment of human tumors:a review.Cancer Res,1989,49(23):6449-6465.
[35]Kalmus J,Okunieff P,Vaupel P.Effect of intraperitoneal versus intravenous glucose administration on laser Doppler flow in murine FSaII tumors and normal skin.Cancer Res,1989,49(22):6313-6317.
[36]Shackleton M,Quintana E,Fearon ER,Morrison SJ.Heterogeneity in cancer:cancer stem cells versus clonal evolution.Cell,2009,138(5):822-829.
[37]Singh SK,Clarke ID,Terasaki M,Bonn VE,Hawkins C,Squire J,Dirks PB.Identification of a cancer stem cell in human brain tumors.Cancer Res,2003,63(18):5821-5828.
[38]Heidt DG,Li C,Dalerba P,Burant CF,Zhang L,Adsay V,Wicha M,Clarke MF,Simeone DM.Identification of pancreatic cancer stem cells.Cancer Res,2007,67(3):1030-1037.