Intratumor heterogeneity is a primary feature of high-grade gliomas, complicating their therapy. As accumulating evidence suggests that intratumor heterogeneity is a consequence of cellular subsets with different cycling frequencies, we developed a method for transcriptional profiling of gliomas, using a novel technique to dissect the tumors into two fundamental cellular subsets, namely, the proliferating and non-proliferating cell fractions. The tumor fractions were sorted whilst maintaining their molecular integrity, by incorporating the thymidine analog 5-ethynyl-2'-deoxyuridine into actively dividing cells. We sorted the actively dividing versus non-dividing cells from cultured glioma cells, and parental and clonally derived orthotopic tumors, and analyzed them for a number of transcripts. While there was no significant difference in the transcriptional profiles between the two cellular subsets in cultured glioma cells, we demonstrate ∼2-6 fold increase in transcripts of cancer and neuronal stem cell and tumor cell migration/invasion markers, and ∼2-fold decrease in transcripts of markers of hypoxia and their target genes, in the dividing tumor cells of the orthotopic glioma when compared to their non-proliferative counterparts. This suggests the influence of the brain microenvironment in transcriptional regulation and, thereby, the physiology of glioma cells in vivo. When clonal glioma cells were derived from a parental glioma and the resultant orthotopic tumors were compared, their transcriptional profiles were closely correlated to tumor aggression and consequently, survival of the experimental animals. This study demonstrates the resolution of intratumor heterogeneity for profiling studies based on cell proliferation, a defining feature of cancers, with implications for treatment design.

Laboratory of Cancer Gene Therapy National Cancer Centre 169610 Singapore

School of Medical Science Griffith University Southport Qld 4222 Australia

School of Medical Science Griffith University Southport Qld 4222 Australia; Institute of Biotechnology Academy of Sciences of the Czech Republic Prague 142 20 Czech Republic

Zobrazit více v PubMed

Anido, J. , Sáez-Borderías, A. , Gonzàlez-Juncà, A. , Rodón, L. , Folch, G. , Carmona, M.A. , Prieto-Sánchez, R.M. , Barba, I. , Martínez-Sáez, E. , Prudkin, L. , 2010. TGF-β receptor inhibitors target the CD44 high/Id1 high glioma-initiating cell population in human glioblastoma. Cancer Cell. 18, 655–668. PubMed

Annovazzi, L. , Mellai, M. , Caldera, V. , Valente, G. , Schiffer, D. , 2011. SOX2 expression and amplification in gliomas and glioma cell lines. Cancer Genomics-Proteomics. 8, 139–147. PubMed

Antoniou, A. , Hébrant, A. , Dom, G. , Dumont, J. , Maenhaut, C. , 2013. Cancer stem cells, a fuzzy evolving concept: a cell population or a cell property?. Cell Cycle. 12, 3743–3748. PubMed PMC

Badie, B. , Schartner, J. , 2001. Role of microglia in glioma biology. Microsc. Res. Tech. 54, 106–113. PubMed

Baker, M. , 2008. Cancer stem cells, becoming common. Nat. Rep. Stem Cells. 10.1038/stemcells.2008.153 DOI

Beier, D. , Hau, P. , Proescholdt, M. , Lohmeier, A. , Wischhusen, J. , Oefner, P.J. , Aigner, L. , Brawanski, A. , Bogdahn, U. , Beier, C.P. , 2007. CD133+ and CD133− glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles. Cancer Res. 67, 4010–4015. PubMed

Bozic, I. , Antal, T. , Ohtsuki, H. , Carter, H. , Kim, D. , Chen, S. , Karchin, R. , Kinzler, K.W. , Vogelstein, B. , Nowak, M.A. , 2010. Accumulation of driver and passenger mutations during tumor progression. Proc. Natl. Acad. Sci. 107, 18545–18550. PubMed PMC

Buck, S.B. , Bradford, J. , Gee, K.R. , Agnew, B.J. , Clarke, S.T. , Salic, A. , 2008. Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies. BioTechniques. 44, 927–929. PubMed

Camphausen, K. , Purow, B. , Sproull, M. , Scott, T. , Ozawa, T. , Deen, D.F. , Tofilon, P.J. , 2005. Influence of in vivo growth on human glioma cell line gene expression: convergent profiles under orthotopic conditions. Proc. Natl. Acad. Sci. U. S. A. 102, 8287–8292. PubMed PMC

Cappella, P. , Gasparri, F. , Pulici, M. , Moll, J. , 2008. A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining. Cytometry. Part A: J. Int. Soc. Anal. Cytol. 73, 626–636. PubMed

Catalano, V. , Turdo, A. , Di Franco, S. , Dieli, F. , Todaro, M. , Stassi, G. , 2013. Tumor and its microenvironment: a synergistic interplay. Semin. Cancer Biol. 522–532. Elsevier PubMed

Cavanagh, B.L. , Walker, T. , Norazit, A. , Meedeniya, A.C. , 2011. Thymidine analogues for tracking DNA synthesis. Molecules. 16, 7980–7993. PubMed PMC

Chehrehasa, F. , Meedeniya, A.C. , Dwyer, P. , Abrahamsen, G. , Mackay-Sim, A. , 2009. EdU, a new thymidine analogue for labelling proliferating cells in the nervous system. J. Neurosci. Methods. 177, 122–130. PubMed

Chen, R. , Nishimura, M.C. , Bumbaca, S.M. , Kharbanda, S. , Forrest, W.F. , Kasman, I.M. , Greve, J.M. , Soriano, R.H. , Gilmour, L.L. , Rivers, C.S. , 2010. A hierarchy of self-renewing tumor-initiating cell types in glioblastoma. Cancer Cell. 17, 362–375. PubMed

Chou, Y.T. , Lee, C.C. , Hsiao, S.H. , Lin, S.E. , Lin, S.C. , Chung, C.H. , Chung, C.H. , Kao, Y.R. , Wang, Y.H. , Chen, C.T. , 2013. The emerging role of SOX2 in cell proliferation and survival and its crosstalk with oncogenic signaling in lung cancer. Stem Cells. 31, 2607–2619. PubMed

Cooper, L.A. , Gutman, D.A. , Chisolm, C. , Appin, C. , Kong, J. , Rong, Y. , Kurc, T. , Van Meir, E.G. , Saltz, J.H. , Moreno, C.S. , 2012. The tumor microenvironment strongly impacts master transcriptional regulators and gene expression class of glioblastoma. Am. J. Pathol. 180, 2108–2119. PubMed PMC

Deleyrolle, L.P. , Harding, A. , Cato, K. , Siebzehnrubl, F.A. , Rahman, M. , Azari, H. , Olson, S. , Gabrielli, B. , Osborne, G. , Vescovi, A. , 2011. Evidence for label-retaining tumour-initiating cells in human glioblastoma. Brain. 134, 1331–1343. PubMed PMC

Diermeier-Daucher, S. , Clarke, S.T. , Hill, D. , Vollmann-Zwerenz, A. , Bradford, J.A. , Brockhoff, G. , 2009. Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry. Cytometry. Part A: J. Int. Soc. Anal. Cytol. 75, 535–546. PubMed

Farragher, S.M. , Tanney, A. , Kennedy, R.D. , Harkin, D.P. , 2008. RNA expression analysis from formalin fixed paraffin embedded tissues. Histochem. Cell Biol. 130, 435–445. PubMed

Fatoo, A. , Nanaszko, M.J. , Allen, B.B. , Mok, C.L. , Bukanova, E.N. , Beyene, R. , Moliterno, J.A. , Boockvar, J.A. , 2011. Understanding the role of tumor stem cells in glioblastoma multiforme: a review article. J. Neuro-oncol. 103, 397–408. PubMed

Fedorowicz, G. , Guerrero, S. , Wu, T.D. , Modrusan, Z. , 2009. Microarray analysis of RNA extracted from formalin-fixed, paraffin-embedded and matched fresh-frozen ovarian adenocarcinomas. BMC Med. Genomics. 2, 23 PubMed PMC

Furnari, F.B. , Fenton, T. , Bachoo, R.M. , Mukasa, A. , Stommel, J.M. , Stegh, A. , Hahn, W.C. , Ligon, K.L. , Louis, D.N. , Brennan, C. , Chin, L. , DePinho, R.A. , Cavenee, W.K. , 2007. Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes Dev. 21, 2683–2710. PubMed

Greaves, M. , Maley, C.C. , 2012. Clonal evolution in cancer. Nature. 481, 306–313. PubMed PMC

Hatzimichael, E. , Crook, T. , 2013. Cancer epigenetics: new therapies and new challenges. J. Drug Deliv. 2013, PubMed PMC

Hoshino, T. , Ahn, D. , Prados, M.D. , Lamborn, K. , Wilson, C.B. , 1993. Prognostic significance of the proliferative potential of intracranial gliomas measured by bromodeoxyuridine labeling. Int. J. Cancer. 53, 550–555. PubMed

Huang, Z. , Cheng, L. , Guryanova, O.A. , Wu, Q. , Bao, S. , 2010. Cancer stem cells in glioblastoma—molecular signaling and therapeutic targeting. Protein & Cell. 1, 638–655. PubMed PMC

Inoue, A. , Takahashi, H. , Harada, H. , Kohno, S. , Ohue, S. , Kobayashi, K. , Yano, H. , Tanaka, J. , Ohnishi, T. , 2010. Cancer stem-like cells of glioblastoma characteristically express MMP-13 and display highly invasive activity. Int. J. Oncol. 37, 1121–1131. PubMed

Ji, H. , Zhao, X. , Yuza, Y. , Shimamura, T. , Li, D. , Protopopov, A. , Jung, B.L. , McNamara, K. , Xia, H. , Glatt, K.A. , 2006. Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors. Proc. Natl. Acad. Sci. 103, 7817–7822. PubMed PMC

Louis, D.N. , Ohgaki, H. , Wiestler, O.D. , Cavenee, W.K. , Burger, P.C. , Jouvet, A. , Scheithauer, B.W. , Kleihues, P. , 2007. The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol. 114, 97–109. PubMed PMC

Ludyga, N. , Grünwald, B. , Azimzadeh, O. , Englert, S. , Höfler, H. , Tapio, S. , Aubele, M. , 2012. Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses. Virchows Arch. 460, 131–140. PubMed

Mimeault, M. , Batra, S.K. , 2013. Hypoxia-inducing factors as master regulators of stemness properties and altered metabolism of cancer-and metastasis-initiating cells. J. Cell. Mol. Med. 17, 30–54. PubMed PMC

Mischel, P.S. , Nelson, S.F. , Cloughesy, T.F. , 2003. Molecular analysis of glioblastoma: pathway profiling and its implications for patient therapy. Cancer Biol. Ther. 2, 242–247. PubMed

Mischel, P.S. , Shai, R. , Shi, T. , Horvath, S. , Lu, K.V. , Choe, G. , Seligson, D. , Kremen, T.J. , Palotie, A. , Liau, L.M. , Cloughesy, T.F. , Nelson, S.F. , 2003. Identification of molecular subtypes of glioblastoma by gene expression profiling. Oncogene. 22, 2361–2373. PubMed

Omuro, A. , DeAngelis, L.M. , 2013. Glioblastoma and other malignant gliomas: a clinical review. JAMA: J. Am. Med. Assoc. 310, 1842–1850. PubMed

Patru, C. , Romao, L. , Varlet, P. , Coulombel, L. , Raponi, E. , Cadusseau, J. , Renault-Mihara, F. , Thirant, C. , Leonard, N. , Berhneim, A. , 2010. CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors. BMC Cancer. 10, 66 PubMed PMC

Pece, S. , Tosoni, D. , Confalonieri, S. , Mazzarol, G. , Vecchi, M. , Ronzoni, S. , Bernard, L. , Viale, G. , Pelicci, P.G. , Di Fiore, P.P. , 2010. Biological and molecular heterogeneity of breast cancers correlates with their cancer stem cell content. Cell. 140, 62–73. PubMed

Quinn, C.M. , Wright, N.A. , 1990. The clinical assessment of proliferation and growth in human tumours: evaluation of methods and applications as prognostic variables. J. Pathol. 160, 93–102. PubMed

Ross, H.H. , Rahman, M. , Levkoff, L.H. , Millette, S. , Martin-Carreras, T. , Dunbar, E.M. , Reynolds, B.A. , Laywell, E.D. , 2011. Ethynyldeoxyuridine (EdU) suppresses in vitro population expansion and in vivo tumor progression of human glioblastoma cells. J. Neuro-oncol. 105, 485–498. PubMed PMC

Salic, A. , Mitchison, T.J. , 2008. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. U. S. A. 105, 2415–2420. PubMed PMC

Seidel, S. , Garvalov, B.K. , Wirta, V. , von Stechow, L. , Schänzer, A. , Meletis, K. , Wolter, M. , Sommerlad, D. , Henze, A.-T. , Nistér, M. , 2010. A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2α. Brain. 133, 983–995. PubMed

Senner, V. , Sturm, A. , Baur, I. , Schrell, U.H. , Distel, L. , Paulus, W. , 1999. CD24 promotes invasion of glioma cells in vivo. J. Neuropathol. Exp. Neurol. 58, 795–802. PubMed

Siegel, R. , Ma, J. , Zou, Z. , Jemal, A. , 2014. Cancer statistics, 2014. CA: A Cancer J. Clin. 64, 9–29. PubMed

Singh, S.K. , Clarke, I.D. , Terasaki, M. , Bonn, V.E. , Hawkins, C. , Squire, J. , Dirks, P.B. , 2003. Identification of a cancer stem cell in human brain tumors. Cancer Res. 63, 5821–5828. PubMed

Suetsugu, A. , Nagaki, M. , Aoki, H. , Motohashi, T. , Kunisada, T. , Moriwaki, H. , 2006. Characterization of CD133+ hepatocellular carcinoma cells as cancer stem/progenitor cells. Biochem. Biophys. Res. Commun. 351, 820–824. PubMed

Verhaak, R.G. , Hoadley, K.A. , Purdom, E. , Wang, V. , Qi, Y. , Wilkerson, M.D. , Miller, C.R. , Ding, L. , Golub, T. , Mesirov, J.P. , Alexe, G. , Lawrence, M. , O'Kelly, M. , Tamayo, P. , Weir, B.A. , Gabriel, S. , Winckler, W. , Gupta, S. , Jakkula, L. , Feiler, H.S. , Hodgson, J.G. , James, C.D. , Sarkaria, J.N. , Brennan, C. , Kahn, A. , Spellman, P.T. , Wilson, R.K. , Speed, T.P. , Gray, J.W. , Meyerson, M. , Getz, G. , Perou, C.M. , Hayes, D.N. , 2010. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell. 17, 98–110. PubMed PMC

Zbinden, M. , Duquet, A. , Lorente-Trigos, A. , Ngwabyt, S.N. , Borges, I. , Ruiz i Altaba, A. , 2010. NANOG regulates glioma stem cells and is essential in vivo acting in a cross-functional network with GLI1 and p53. EMBO J. 29, 2659–2674. PubMed PMC

Zheng, H. , Ying, H. , Yan, H. , Kimmelman, A. , Hiller, D. , Chen, A.-J. , Perry, S. , Tonon, G. , Chu, G. , Ding, Z. , 2008. Pten and p53 converge on c-Myc to control differentiation, self-renewal, and transformation of normal and neoplastic stem cells in glioblastoma. Cold Spring Harb. Symp. Quant. Biol. Cold Spring Harbor Laboratory Press, p. sqb. 2008.2073. 2047 PubMed