Skip to main content
SpringerLink
Log in

In Vivo Interrogation of the Hypoxic Transcriptome of Solid Tumors: Optimizing Hypoxic Probe Labeling with Laser Capture Microdissection for Isolation of High-Quality RNA for Deep Sequencing Analysis

  • Conference paper
  • First Online:
Tumor Microenvironment

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 899))

Abstract

Global gene expression analysis is a powerful method for identifying biological networks and regulatory mechanisms that govern cellular or tissue-level responses to physiologic stress. In the context of tumor biology, differential gene expression studies have provided information about the growth, aggressiveness, prognosis, and therapeutic response of tumors in patients. Scientists are using these valuable data to investigate pathways that can be targeted therapeutically with the goal of improving patient outcome. RNA sequencing enables nucleotide resolution of expression of whole transcriptomes, but arrives with a new set of challenges surrounding the management and analysis of large datasets. This chapter aims to review technical advancements to current methods for isolating high-quality RNA for sequencing studies directly from hypoxic tissues and introduces select widely used applications for gene expression analyses of next-generation sequencing data.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Koumenis C, Maxwell PH. Low oxygen stimulates the intellect. Symposium on hypoxia and development, physiology and disease. EMBO Rep. 2006;7(7):679–84.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Komar G, Lehtiö K, Seppänen M, Eskola O, Levola H, Lindholm P, Sipilä H, Seppälä J, Grénman R, Solin O, Minn H. Prognostic value of tumour blood flow, [18F]EF5 and [18F]FDG PET/CT imaging in patients with head and neck cancer treated with radiochemotherapy. Eur J Nucl Med Mol Imaging. 2014;41:2042–50.

    Article  CAS  PubMed  Google Scholar 

  3. Moller-Levet CS, Betts GNJ, Harris AL, Homer JJ, West CML, Miller CJ. Exon array analysis of head and neck cancers identifies a hypoxia related splice variant of LAMA3 associated with a poor prognosis. PLoS Comput Biol. 2009;5(11):1–13.

    Article  Google Scholar 

  4. Oltean S, Bates DO. Hallmarks of alternative splicing in cancer. Oncogene. 2013;33(46):5311–8.

    Article  PubMed  Google Scholar 

  5. Brizel DM, Scully SP, Harrelson JM, Layfield LJ, Bean JM, Prosnitz LR, Dewhirst MW. Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res. 1996;56(5):941–3.

    CAS  PubMed  Google Scholar 

  6. Nordsmark M, Overgaard M, Overgaard J. Pretreatment oxygenation predicts radiation response in advanced squamous cell carcinoma of the head and neck. Radiother Oncol. 1996;41(1):31–9.

    Article  CAS  PubMed  Google Scholar 

  7. Rampling R, Cruickshank G, Lewis AD, Fitzsimmons SA, Workman P. Direct measurement of pO2 distribution and bioreductive enzymes in human malignant brain tumors. Int J Radiat Oncol Biol Phys. 1994;29(3):427–31.

    Article  CAS  PubMed  Google Scholar 

  8. Vaupel P, Mayer A, Höckel M. Tumor hypoxia and malignant progression. In: Chandan KS, Gregg LS, editors. Methods in enzymology, vol. 381. New York: Academic; 2004. p. 335–54.

    Google Scholar 

  9. Rofstad EK, Gaustad JV, Egeland TA, Mathiesen B, Galappathi K. Tumors exposed to acute cyclic hypoxic stress show enhanced angiogenesis, perfusion and metastatic dissemination. Int J Cancer. 2010;127(7):1535–46.

    Article  CAS  PubMed  Google Scholar 

  10. Maftei CA, Bayer C, Shi K, Astner ST, Vaupel P. Quantitative assessment of hypoxia subtypes in microcirculatory supply units of malignant tumors using (immuno-)fluorescence techniques. Strahlenther Onkol. 2011;187(4):260–6.

    Article  PubMed  Google Scholar 

  11. Vaupel P, Mayer A. Hypoxia in tumors: pathogenesis-related classification, characterization of hypoxia subtypes, and associated biological and clinical implications. Adv Exp Med Biol. 2014;812:19–24.

    Article  CAS  PubMed  Google Scholar 

  12. Laderoute KR, Alarcon RM, Brody MD, Calaoagan JM, Chen EY, Knapp AM, Yun Z, Denko NC, Giaccia AJ. Opposing effects of hypoxia on expression of the angiogenic inhibitor thrombospondin 1 and the angiogenic inducer vascular endothelial growth factor. Clin Cancer Res. 2000;6(7):2941–50.

    CAS  PubMed  Google Scholar 

  13. Semenza GL. Hydroxylation of HIF-1: oxygen sensing at the molecular level. Physiology (Bethesda). 2004;19:176–82.

    Article  CAS  Google Scholar 

  14. Tatum JL, Kelloff GJ, Gillies RJ, Arbeit JM, Brown JM, Chao KS, Chapman JD, Eckelman WC, Fyles AW, Giaccia AJ, Hill RP, Koch CJ, Krishna MC, Krohn KA, Lewis JS, Mason RP, Melillo G, Padhani AR, Powis G, Rajendran JG, Reba R, Robinson SP, Semenza GL, Swartz HM, Vaupel P, Yang D, Croft B, Hoffman J, Liu G, Stone H, Sullivan D. Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy. Int J Radiat Biol. 2006;82(10):699–757.

    Article  CAS  PubMed  Google Scholar 

  15. Weinmann M, Belka C, Guner D, Goecke B, Muller I, Bamberg M, Jendrossek V. Array-based comparative gene expression analysis of tumor cells with increased apoptosis resistance after hypoxic selection. Oncogene. 2005;24(38):5914–22.

    Article  CAS  PubMed  Google Scholar 

  16. Pawlus MR, Wang L, Hu CJ. STAT3 and HIF1[alpha] cooperatively activate HIF1 target genes in MDA-MB-231 and RCC4 cells. Oncogene. 2014;33(13):1670–9.

    Article  CAS  PubMed  Google Scholar 

  17. Bristow RG, Hill RP. Hypoxia and metabolism. Hypoxia, DNA repair and genetic instability. Nat Rev Cancer. 2008;8(3):180–92.

    Article  CAS  PubMed  Google Scholar 

  18. Sena JA, Wang L, Heasley LE, Hu C-J. Hypoxia regulates alternative splicing of HIF and non-HIF target genes. Mol Cancer Res. 2014;12:1233–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sena JA, Wang L, Pawlus MR, Hu C-J. HIFs enhance the transcriptional activation and splicing of adrenomedullin. Mol Cancer Res. 2014;12(5):728–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Denko N, Wernke-Dollries K, Johnson AB, Hammond E, Chiang CM, Barton MC. Hypoxia actively represses transcription by inducing negative cofactor 2 (Dr1/DrAP1) and blocking preinitiation complex assembly. J Biol Chem. 2003;278(8):5744–9.

    Article  CAS  PubMed  Google Scholar 

  21. Denko NC, Fontana LA, Hudson KM, Sutphin PD, Raychaudhuri S, Altman R, Giaccia AJ. Investigating hypoxic tumor physiology through gene expression patterns. Oncogene. 2003;22(37):5907–14.

    Article  CAS  PubMed  Google Scholar 

  22. Tang X, Lucas JE, Chen JL, LaMonte G, Wu J, Wang MC, Koumenis C, Chi JT. Functional interaction between responses to lactic acidosis and hypoxia regulates genomic transcriptional outputs. Cancer Res. 2012;72(2):491–502.

    Article  CAS  PubMed  Google Scholar 

  23. Marotta D, Karar J, Jenkins WT, Kumanova M, Jenkins KW, Tobias JW, Baldwin D, Hatzigeorgiou A, Alexiou P, Evans SM, Alarcon R, Maity A, Koch C, Koumenis C. In vivo profiling of hypoxic gene expression in gliomas using the hypoxia marker EF5 and laser-capture microdissection. Cancer Res. 2011;71(3):779–89.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Koch CJ, Hahn SM, Rockwell Jr K, Covey JM, McKenna WG, Evans SM. Pharmacokinetics of EF5 [2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] in human patients: implications for hypoxia measurements in vivo by 2-nitroimidazoles. Cancer Chemother Pharmacol. 2001;48(3):177–87.

    Article  CAS  PubMed  Google Scholar 

  25. Gross MW, Karbach U, Groebe K, Franko AJ, Mueller-Klieser W. Calibration of misonidazole labeling by simultaneous measurement of oxygen tension and labeling density in multicellular spheroids. Int J Cancer. 1995;61(4):567–73.

    Article  CAS  PubMed  Google Scholar 

  26. Vordermark D, Brown JM. Endogenous markers of tumor hypoxia predictors of clinical radiation resistance? Strahlenther Onkol. 2003;179(12):801–11.

    Article  PubMed  Google Scholar 

  27. Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA. Laser capture microdissection. Science. 1996;274(5289):998–1001.

    Article  CAS  PubMed  Google Scholar 

  28. Suarez-Quian CA, Goldstein SR, Pohida T, Smith PD, Peterson JI, Wellner E, Ghany M, Bonner RF. Laser capture microdissection of single cells from complex tissues. Biotechniques. 1999;26(2):328–35.

    CAS  PubMed  Google Scholar 

  29. Liu A. Laser capture microdissection in the tissue biorepository. J Biomol Tech. 2010;21(3):120–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Vandewoestyne M, Goossens K, Burvenich C, Van Soom A, Peelman L, Deforce D. Laser capture microdissection: should an ultraviolet or infrared laser be used? Anal Biochem. 2013;439(2):88–98.

    Article  CAS  PubMed  Google Scholar 

  31. Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol. 2006;7:3.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Ozsolak F, Milos PM. RNA sequencing: advances, challenges and opportunities. Nat Rev Genet. 2011;12(2):87–98.

    Article  CAS  PubMed  Google Scholar 

  33. Au KF, Underwood JG, Lee L, Wong WH. Improving PacBio long read accuracy by short read alignment. PLoS One. 2012;7(10):e46679.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Quail MA, Smith M, Coupland P, Otto TD, Harris SR, Connor TR, Bertoni A, Swerdlow HP, Gu Y. A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics. 2012;13:341.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Toung JM, Morley M, Li M, Cheung VG. RNA-sequence analysis of human B-cells. Genome Res. 2011;21(6):991–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Liu Y, Zhou J, White KP. RNA-seq differential expression studies: more sequence or more replication? Bioinformatics. 2014;30(3):301–4.

    Article  CAS  PubMed  Google Scholar 

  37. Alberts B, Johnson A, Lewis J, et al. Molecular biology of the cell. 4th ed. New York: Garland Science; 2002.

    Google Scholar 

  38. Levin JZ, Berger MF, Adiconis X, Rogov P, Melnikov A, Fennell T, Nusbaum C, Garraway LA, Gnirke A. Targeted next-generation sequencing of a cancer transcriptome enhances detection of sequence variants and novel fusion transcripts. Genome Biol. 2009;10(10):R115.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Li JB, Levanon EY, Yoon J-K, Aach J, LeProust E, Zhang K, Gao Y, Church GM. Genome-wide identification of human RNA editing sites by parallel DNA capturing and sequencing. Science (New York, NY). 2009;324(5931):1210–3.

    Article  CAS  Google Scholar 

  40. Cock PJ, Fields CJ, Goto N, Heuer ML, Rice PM. The Sanger FASTQ file format for sequences with quality scores, and the Solexa/Illumina FASTQ variants. Nucleic Acids Res. 2010;38(6):1767–71.

    Article  CAS  PubMed  Google Scholar 

  41. Pruitt KD, Tatusova T, Maglott DR. NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 2005;33(Database issue):D501–4.

    Article  CAS  PubMed  Google Scholar 

  42. Soneson C, Delorenzi M. A comparison of methods for differential expression analysis of RNA-seq data. BMC Bioinformatics. 2013;14(91).

    Google Scholar 

  43. Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009;25(9):1105–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wang K, Singh D, Zeng Z, Coleman SJ, Huang Y, Savich GL, He X, Mieczkowski P, Grimm SA, Perou CM, MacLeod JN, Chiang DY, Prins JF, Liu J. MapSplice: accurate mapping of RNA-seq reads for splice junction discovery. Nucleic Acids Res. 2010;38(18):e178.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Wu TD, Nacu S. Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics. 2010;26(7):873–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, Pachter L. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol. 2013;31(1):46–53.

    Article  CAS  PubMed  Google Scholar 

  47. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7(3):562–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28(5):511–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Katz Y, Wang ET, Airoldi EM, Burge CB. Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat Methods. 2010;7(12):1009–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Shiraki T, Kondo S, Katayama S, Waki K, Kasukawa T, Kawaji H, Kodzius R, Watahiki A, Nakamura M, Arakawa T, Fukuda S, Sasaki D, Podhajska A, Harbers M, Kawai J, Carninci P, Hayashizaki Y. Cap analysis gene expression for high-throughput analysis of transcriptional starting point and identification of promoter usage. Proc Natl Acad Sci U S A. 2003;100(26):15776–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Churchman LS, Weissman JS. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature. 2011;469(7330):368–73.

    Article  CAS  PubMed  Google Scholar 

  52. Ingolia NT, Lareau LF, Weissman JS. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell. 2011;147(4):789–802.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Lauren K. Brady, Vladimir Popov & Constantinos Koumenis

  2. Cellular and Molecular Biology Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA

    Lauren K. Brady

Authors
  1. Lauren K. Brady
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vladimir Popov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Constantinos Koumenis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Constantinos Koumenis .

Editor information

Editors and Affiliations

  1. Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Constantinos Koumenis

  2. Cell & Developmental Biology, Oregon Health & Science University, Portland, Oregon, USA

    Lisa M. Coussens

  3. Department of Radiation Oncology, Stanford University School of Medic, Stanford, California, USA

    Amato Giaccia

  4. Department of Oncology, University of Oxford, Oxford, Oxfordshire, United Kingdom

    Ester Hammond

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer International Publishing Switzerland

About this paper

Cite this paper

Brady, L.K., Popov, V., Koumenis, C. (2016). In Vivo Interrogation of the Hypoxic Transcriptome of Solid Tumors: Optimizing Hypoxic Probe Labeling with Laser Capture Microdissection for Isolation of High-Quality RNA for Deep Sequencing Analysis. In: Koumenis, C., Coussens, L., Giaccia, A., Hammond, E. (eds) Tumor Microenvironment. Advances in Experimental Medicine and Biology, vol 899. Springer, Cham. https://doi.org/10.1007/978-3-319-26666-4_4

Download citation

Publish with us

Policies and ethics

Search

Navigation