Abstract
Brain tumors are heterogeneous with respect to genetic and histological properties of cells within the tumor tissue. To study subpopulations of cells, we developed a protocol for obtaining viable single cells from freshly isolated human brain tissue for single cell gene expression profiling. We evaluated this technique for characterization of cell populations within brain tumor and tumor penumbra. Fresh tumor tissue was obtained from one astrocytoma grade IV and one oligodendroglioma grade III tumor as well as the tumor penumbra of the latter tumor. The tissue was dissociated into individual cells and the expression of 36 genes was assessed by reverse transcription quantitative PCR followed by data analysis. We show that tumor cells from both the astrocytoma grade IV and oligodendroglioma grade III tumor constituted cell subpopulations defined by their gene expression profiles. Some cells from the oligodendroglioma grade III tumor proper shared molecular characteristics with the cells from the penumbra of the same tumor suggesting that a subpopulation of cells within the oligodendroglioma grade III tumor consisted of normal brain cells. We conclude that subpopulations of tumor cells can be identified by using single cell gene expression profiling.
References
Liu X, Long F, Peng H, Aerni SJ, Jiang M, Sanchez-Blanco A, Murray JI, Preston E, Mericle B, Batzoglou S, Myers EW, Kim SK (2009) Analysis of cell fate from single-cell gene expression profiles in C. elegans. Cell 139(3):623–633. doi:10.1016/j.cell.2009.08.044
Wills QF, Livak KJ, Tipping AJ, Enver T, Goldson AJ, Sexton DW, Holmes C (2013) Single-cell gene expression analysis reveals genetic associations masked in whole-tissue experiments. Nat Biotechnol 31(8):748–752. doi:10.1038/nbt.2642
Wang D, Bodovitz S (2010) Single cell analysis: the new frontier in ‘omics’. Trends Biotechnol 28(6):281–290. doi:10.1016/j.tibtech.2010.03.002
Dalerba P, Kalisky T, Sahoo D, Rajendran PS, Rothenberg ME, Leyrat AA, Sim S, Okamoto J, Johnston DM, Qian D, Zabala M, Bueno J, Neff NF, Wang J, Shelton AA, Visser B, Hisamori S, Shimono Y, van de Wetering M, Clevers H, Clarke MF, Quake SR (2011) Single-cell dissection of transcriptional heterogeneity in human colon tumors. Nat Biotechnol 29(12):1120–1127. doi:10.1038/nbt.2038
Powell AA, Talasaz AH, Zhang H, Coram MA, Reddy A, Deng G, Telli ML, Advani RH, Carlson RW, Mollick JA, Sheth S, Kurian AW, Ford JM, Stockdale FE, Quake SR, Pease RF, Mindrinos MN, Bhanot G, Dairkee SH, Davis RW, Jeffrey SS (2012) Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines. PLoS One 7(5):e33788. doi:10.1371/journal.pone.0033788
Ramsköld D, Luo S, Wang YC, Li R, Deng Q, Faridani OR, Daniels GA, Khrebtukova I, Loring JF, Laurent LC, Schroth GP, Sandberg R (2012) Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells. Nat Biotechnol 30(8):777–782. doi:10.1038/nbt.2282
Ståhlberg A, Andersson D, Aurelius J, Faiz M, Pekna M, Kubista M, Pekny M (2011) Defining cell populations with single-cell gene expression profiling: correlations and identification of astrocyte subpopulations. Nucleic Acids Res 39(4):e24. doi:10.1093/nar/gkq1182
Socialstyrelsen Cancerfonden (2013) Cancer i siffror 2013, vol 2013. Socialstyrelsen, Cancerfonden, Stockholm
Ohgaki H, Kleihues P (2005) Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas. J Neuropathol Exp Neurol 64(6):479–489
Nafe R, Glienke W, Burgemeister R, Gangnus R, Haar B, Pries A, Schlote W (2004) Regional heterogeneity of EGFR gene amplification and nuclear morphology in glioblastomas. An investigation using laser microdissection and pressure catapulting. Anal Quant Cytol Histol 26(2):65–76
Shapiro JR, Yung WK, Shapiro WR (1981) Isolation, karyotype, and clonal growth of heterogeneous subpopulations of human malignant gliomas. Cancer Res 41(6):2349–2359
Walker C, du Plessis DG, Joyce KA, Machell Y, Thomson-Hehir J, Al Haddad SA, Broome JC, Warnke PC (2003) Phenotype versus genotype in gliomas displaying inter- or intratumoral histological heterogeneity. Clin Cancer Res 9(13):4841–4851
Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63(18):5821–5828
Vescovi AL, Galli R, Reynolds BA (2006) Brain tumour stem cells. Nat Rev Cancer 6(6):425–436. doi:10.1038/nrc1889
Bonavia R, Inda MM, Cavenee WK, Furnari FB (2011) Heterogeneity maintenance in glioblastoma: a social network. Cancer Res 71(12):4055–4060. doi:10.1158/0008-5472.CAN-11-0153
Ståhlberg A, Rusnakova V, Forootan A, Anderova M, Kubista M (2012) RT-qPCR work-flow for single-cell data analysis. Methods. doi:10.1016/j.ymeth.2012.09.007
Ringner M (2008) What is principal component analysis? Nat Biotechnol 26(3):303–304. doi:10.1038/nbt0308-303
Kohonen T (1982) Self-Organized Formation of Topologically Correct Feature Maps. Biol Cybern 43(1):59–69. doi:10.1007/Bf00337288
Ward JH (1963) Hierarchical grouping to optimize an objective function. J Am Stat Assoc 58(301):236–244. doi:10.2307/2282967
Mann HB, Whitney DR (1947) On a test of whether one of two random variables is stochastically larger than the other. Ann Math Stat 18(1):50–60
Dunn OJ (1961) Multiple comparisons among means. J Am Stat Assoc 56(293):52–64
Eng LF, Ghirnikar RS, Lee YL (2000) Glial fibrillary acidic protein: GFAP-thirty-one years (1969-2000). Neurochem Res 25(9–10):1439–1451
Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, Xing Y, Lubischer JL, Krieg PA, Krupenko SA, Thompson WJ, Barres BA (2008) A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci 28(1):264–278. doi:10.1523/JNEUROSCI.4178-07.2008
Brunner C, Lassmann H, Waehneldt TV, Matthieu JM, Linington C (1989) Differential ultrastructural localization of myelin basic protein, myelin/oligodendroglial glycoprotein, and 2′,3′-cyclic nucleotide 3′-phosphodiesterase in the CNS of adult rats. J Neurochem 52(1):296–304
Marangos PJ, Schmechel DE (1987) Neuron specific enolase, a clinically useful marker for neurons and neuroendocrine cells. Annu Rev Neurosci 10:269–295. doi:10.1146/annurev.ne.10.030187.001413
Schwartz PH, Nethercott H, Kirov II, Ziaeian B, Young MJ, Klassen H (2005) Expression of neurodevelopmental markers by cultured porcine neural precursor cells. Stem Cells 23(9):1286–1294. doi:10.1634/stemcells.2004-0306
Jinno S (2011) Regional and laminar differences in antigen profiles and spatial distributions of astrocytes in the mouse hippocampus, with reference to aging. Neuroscience 180:41–52. doi:10.1016/j.neuroscience.2011.02.013
Souter S, Lee G (2010) Tubulin-independent tau in Alzheimer’s disease and cancer: implications for disease pathogenesis and treatment. Curr Alzheimer Res 7(8):697–707
Rorive S, Lopez XM, Maris C, Trepant AL, Sauvage S, Sadeghi N, Roland I, Decaestecker C, Salmon I (2010) TIMP-4 and CD63: new prognostic biomarkers in human astrocytomas. Mod Pathol 23(10):1418–1428. doi:10.1038/modpathol.2010.136
Haas K, Staller P, Geisen C, Bartek J, Eilers M, Moroy T (1997) Mutual requirement of CDK4 and Myc in malignant transformation: evidence for cyclin D1/CDK4 and p16INK4A as upstream regulators of Myc. Oncogene 15(2):179–192. doi:10.1038/sj.onc.1201171
Lam PY, Di Tomaso E, Ng HK, Pang JC, Roussel MF, Hjelm NM (2000) Expression of p19INK4d, CDK4, CDK6 in glioblastoma multiforme. Br J Neurosurg 14(1):28–32
Tsutsui T, Hesabi B, Moons DS, Pandolfi PP, Hansel KS, Koff A, Kiyokawa H (1999) Targeted disruption of CDK4 delays cell cycle entry with enhanced p27(Kip1) activity. Mol Cell Biol 19(10):7011–7019
Zorniak M, Clark PA, Leeper HE, Tipping MD, Francis DM, Kozak KR, Salamat MS, Kuo JS (2012) Differential expression of 2′,3′-cyclic-nucleotide 3′-phosphodiesterase and neural lineage markers correlate with glioblastoma xenograft infiltration and patient survival. Clinical Cancer Res 18(13):3628–3636. doi:10.1158/1078-0432.CCR-12-0339
Lahav R (2005) Endothelin receptor B is required for the expansion of melanocyte precursors and malignant melanoma. Int J Dev Biol 49(2–3):173–180
Heimberger AB, Suki D, Yang D, Shi W, Aldape K (2005) The natural history of EGFR and EGFRvIII in glioblastoma patients. J Transl Med 3:38. doi:10.1186/1479-5876-3-38
Soh MA, Garrett SH, Somji S, Dunlevy JR, Zhou XD, Sens MA, Bathula CS, Allen C, Sens DA (2011) Arsenic, cadmium and neuron specific enolase (ENO2, gamma-enolase) expression in breast cancer. Cancer Cell Int 11(1):41. doi:10.1186/1475-2867-11-41
Rouzier R, Rajan R, Wagner P, Hess KR, Gold DL, Stec J, Ayers M, Ross JS, Zhang P, Buchholz TA, Kuerer H, Green M, Arun B, Hortobagyi GN, Symmans WF, Pusztai L (2005) Microtubule-associated protein tau: a marker of paclitaxel sensitivity in breast cancer. Proc Natl Acad Sci USA 102(23):8315–8320. doi:10.1073/pnas.0408974102
Souter S, Lee G (2009) Microtubule-associated protein tau in human prostate cancer cells: isoforms, phosphorylation, and interactions. J Cell Biochem 108(3):555–564. doi:10.1002/jcb.22287
Fassl A, Tagscherer KE, Richter J, Berriel Diaz M, Alcantara Llaguno SR, Campos B, Kopitz J, Herold-Mende C, Herzig S, Schmidt MH, Parada LF, Wiestler OD, Roth W (2012) Notch1 signaling promotes survival of glioblastoma cells via EGFR-mediated induction of anti-apoptotic Mcl-1. Oncogene 31(44):4698–4708. doi:10.1038/onc.2011.615
Ligon KL, Alberta JA, Kho AT, Weiss J, Kwaan MR, Nutt CL, Louis DN, Stiles CD, Rowitch DH (2004) The oligodendroglial lineage marker OLIG2 is universally expressed in diffuse gliomas. J Neuropathol Exp Neurol 63(5):499–509
Ligon KL, Huillard E, Mehta S, Kesari S, Liu H, Alberta JA, Bachoo RM, Kane M, Louis DN, Depinho RA, Anderson DJ, Stiles CD, Rowitch DH (2007) Olig2-regulated lineage-restricted pathway controls replication competence in neural stem cells and malignant glioma. Neuron 53(4):503–517. doi:10.1016/j.neuron.2007.01.009
Cho HS, Hayami S, Toyokawa G, Maejima K, Yamane Y, Suzuki T, Dohmae N, Kogure M, Kang D, Neal DE, Ponder BA, Yamaue H, Nakamura Y, Hamamoto R (2012) RB1 methylation by SMYD2 enhances cell cycle progression through an increase of RB1 phosphorylation. Neoplasia 14(6):476–486
Nakamura M, Yonekawa Y, Kleihues P, Ohgaki H (2001) Promoter hypermethylation of the RB1 gene in glioblastomas. Lab Invest 81(1):77–82
Murphree AL, Benedict WF (1984) Retinoblastoma: clues to human oncogenesis. Science 223(4640):1028–1033
Lu Z, Zhou L, Killela P, Rasheed AB, Di C, Poe WE, McLendon RE, Bigner DD, Nicchitta C, Yan H (2009) Glioblastoma proto-oncogene SEC61gamma is required for tumor cell survival and response to endoplasmic reticulum stress. Cancer Res 69(23):9105–9111. doi:10.1158/0008-5472.CAN-09-2775
Gangemi RM, Griffero F, Marubbi D, Perera M, Capra MC, Malatesta P, Ravetti GL, Zona GL, Daga A, Corte G (2009) SOX2 silencing in glioblastoma tumor-initiating cells causes stop of proliferation and loss of tumorigenicity. Stem Cells 27(1):40–48. doi:10.1634/stemcells.2008-0493
Iliopoulos D, Lindahl-Allen M, Polytarchou C, Hirsch HA, Tsichlis PN, Struhl K (2010) Loss of miR-200 inhibition of Suz12 leads to polycomb-mediated repression required for the formation and maintenance of cancer stem cells. Mol Cell 39(5):761–772. doi:10.1016/j.molcel.2010.08.013
Levallet G, Bergot E, Antoine M, Creveuil C, Santos AO, Beau-Faller M, de Fraipont F, Brambilla E, Levallet J, Morin F, Westeel V, Wislez M, Quoix E, Debieuvre D, Dubois F, Rouquette I, Pujol JL, Moro-Sibilot D, Camonis J, Zalcman G, Intergroupe Francophone de Cancerologie T (2012) High TUBB3 expression, an independent prognostic marker in patients with early non-small cell lung cancer treated by preoperative chemotherapy, is regulated by K-Ras signaling pathway. Mol Cancer Ther 11(5):1203–1213. doi:10.1158/1535-7163.MCT-11-0899
Ahmed Z, Shaw G, Sharma VP, Yang C, McGowan E, Dickson DW (2007) Actin-binding proteins coronin-1a and IBA-1 are effective microglial markers for immunohistochemistry. J Histochem Cytochem 55(7):687–700. doi:10.1369/jhc.6A7156.2007
Harada A, Sekido N, Akahoshi T, Wada T, Mukaida N, Matsushima K (1994) Essential involvement of interleukin-8 (IL-8) in acute inflammation. J Leukoc Biol 56(5):559–564
Woodfin A, Voisin MB, Nourshargh S (2007) PECAM-1: a multi-functional molecule in inflammation and vascular biology. Arterioscler Thromb Vasc Biol 27(12):2514–2523. doi:10.1161/ATVBAHA.107.151456
Ehrlich LC, Hu S, Sheng WS, Sutton RL, Rockswold GL, Peterson PK, Chao CC (1998) Cytokine regulation of human microglial cell IL-8 production. J Immunol 160(4):1944–1948
Raghavan R, Steart PV, Weller RO (1990) Cell proliferation patterns in the diagnosis of astrocytomas, anaplastic astrocytomas and glioblastoma multiforme: a Ki-67 study. Neuropathol Appl Neurobiol 16(2):123–133
Taran K, Sitkiewicz A, Andrzejewska E, Kobos J (2011) Minichromosome maintenance 2 (MCM2) is a new prognostic proliferative marker in Wilms tumour. Pol J Pathol 62(2):84–88
Eliasson C, Sahlgren C, Berthold CH, Stakeberg J, Celis JE, Betsholtz C, Eriksson JE, Pekny M (1999) Intermediate filament protein partnership in astrocytes. J Biol Chem 274(34):23996–24006
Sofroniew MV (2005) Reactive astrocytes in neural repair and protection. Neuroscientist 11(5):400–407. doi:10.1177/1073858405278321
Rogers SD, Peters CM, Pomonis JD, Hagiwara H, Ghilardi JR, Mantyh PW (2003) Endothelin B receptors are expressed by astrocytes and regulate astrocyte hypertrophy in the normal and injured CNS. Glia 41(2):180–190. doi:10.1002/glia.10173
Danovi D, Meulmeester E, Pasini D, Migliorini D, Capra M, Frenk R, de Graaf P, Francoz S, Gasparini P, Gobbi A, Helin K, Pelicci PG, Jochemsen AG, Marine JC (2004) Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity. Mol Cell Biol 24(13):5835–5843. doi:10.1128/MCB.24.13.5835-5843.2004
Francoz S, Froment P, Bogaerts S, De Clercq S, Maetens M, Doumont G, Bellefroid E, Marine JC (2006) Mdm4 and Mdm2 cooperate to inhibit p53 activity in proliferating and quiescent cells in vivo. Proc Natl Acad Sci USA 103(9):3232–3237. doi:10.1073/pnas.0508476103
Kubben FJ, Peeters-Haesevoets A, Engels LG, Baeten CG, Schutte B, Arends JW, Stockbrugger RW, Blijham GH (1994) Proliferating cell nuclear antigen (PCNA): a new marker to study human colonic cell proliferation. Gut 35(4):530–535
Kros JM, Van Eden CG, Stefanko SZ, Waayer-Van Batenburg M, van der Kwast TH (1990) Prognostic implications of glial fibrillary acidic protein containing cell types in oligodendrogliomas. Cancer 66(6):1204–1212
Nakopoulou L, Kerezoudi E, Thomaides T, Litsios B (1990) An immunocytochemical comparison of glial fibrillary acidic protein, S-100p and vimentin in human glial tumors. J Neurooncol 8(1):33–40
van der Meulen JD, Houthoff HJ, Ebels EJ (1978) Glial fibrillary acidic protein in human gliomas. Neuropathol Appl Neurobiol 4(3):177–190
Pusztai L, Jeong JH, Gong Y, Ross JS, Kim C, Paik S, Rouzier R, Andre F, Hortobagyi GN, Wolmark N, Symmans WF (2009) Evaluation of microtubule-associated protein-Tau expression as a prognostic and predictive marker in the NSABP-B 28 randomized clinical trial. J Clin Oncol 27(26):4287–4292. doi:10.1200/JCO.2008.21.6887
Xu P, Yu S, Jiang R, Kang C, Wang G, Jiang H, Pu P (2009) Differential expression of Notch family members in astrocytomas and medulloblastomas. Pathol Oncol Res 15(4):703–710. doi:10.1007/s12253-009-9173-x
Acknowledgments
We would like to thank Dr. Yolanda de Pablo for providing mouse brain tissue for development of the cell dissociation protocols. This work was supported by Swedish Medical Research Council (11548 to MP), ALF Gothenburg (11267 to MP, 11392 to BR, 137241 to AS), Söderbergs Foundations, Hjärnfonden, Amlöv’s Foundation, E. Jacobson’s Donation Fund, NanoNet COST Action (BM1002), EU FP 7 Program EduGlia (237956) to MP, EU FP 7 Program TargetBraIn (279017), AFA Research Foundation, Gothenburg Foundation for Neurological Research, FoU (Västra Götalandsregionen), the Swedish Cancer Society, and Wilhelm and Martina Lundgren foundation.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Special Issue: In Honor of Michael Norenberg.
Rights and permissions
About this article
Cite this article
Möllerström, E., Rydenhag, B., Andersson, D. et al. Classification of Subpopulations of Cells Within Human Primary Brain Tumors by Single Cell Gene Expression Profiling. Neurochem Res 40, 336–352 (2015). https://doi.org/10.1007/s11064-014-1431-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11064-014-1431-y