Pancreatic Cancer pp 43-59

Part of the Methods in Molecular Biology book series (MIMB, volume 980)

A Method for Conducting Highly Sensitive MicroRNA In Situ Hybridization and Immunohistochemical Analysis in Pancreatic Cancer

Protocol

Abstract

Profiling experiments in whole tissue biopsies have linked altered expression of microRNAs (miRNAs) to different types of cancer, including pancreatic ductal adenocarcinoma (PDAC). Emerging evidence indicates that altered miRNA expression can occur in different cellular compartments (cancer and non-cancer cells) in tumor lesions, and thus it is important to ascertain which specific cell type expresses a particulars miRNA in PDAC tissues. Here, we describe a highly sensitive fluorescence-based ISH method to visualize miRNA accumulation within individual cells in formalin-fixed paraffin-embedded (FFPE) tissue sections using 5′ and 3′ terminally fluorescein-labeled locked nucleic acid (LNA)-modified probes. We describe a multicolor ISH/IHC method based on sequential rounds of horseradish peroxidase (HRP)-mediated tyramide signal amplification (TSA) reactions with different in-house synthesized fluorochrome-conjugated substrates that enable co-detection of miRNAs, abundant noncoding RNAs and protein markers for signal quantification, and cell type co-localization studies in FFPE pancreatic tissue sections from clinical specimens and mouse models of PDAC.

Key words

MicroRNA In situ hybridization Locked nucleic acid Fluorescence microscopy Immunohistochemistry Multiplexing Pancreatic cancer Pancreatic adenocarcinoma Genetically engineered mouse models 

References

  1. 1.
    Korc M (2007) Pancreatic cancer-associated stroma production. Am J Surg 194:S84–S86PubMedCrossRefGoogle Scholar
  2. 2.
    Ambros V (2004) The functions of animal microRNAs. Nature 431:350–355PubMedCrossRefGoogle Scholar
  3. 3.
    Bartel DP, Chen CZ (2004) Micromanagers of gene expression: the potentially widespread influence of metazoan microRNAs. Nat Rev Genet 5:396–400PubMedCrossRefGoogle Scholar
  4. 4.
    Kim VN, Han J, Siomi MC (2009) Biogenesis of small RNAs in animals. Nat Rev Mol Cell Biol 10:126–139PubMedCrossRefGoogle Scholar
  5. 5.
    Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136: 215–233PubMedCrossRefGoogle Scholar
  6. 6.
    Sempere LF, Kauppinen S (2009) Translational implications of microRNAs in clinical diagnostics and therapeutics. In: Bradshaw RA, Dennis EA (eds) Handbook of cell signaling, 2nd edn. Academic, Oxford, pp 2965–2981Google Scholar
  7. 7.
    Ventura A, Jacks T (2009) MicroRNAs and cancer: short RNAs go a long way. Cell 136: 586–591PubMedCrossRefGoogle Scholar
  8. 8.
    Garzon R, Marcucci G, Croce CM (2010) Targeting microRNAs in cancer: rationale, strategies and challenges. Nat Rev Drug Discov 9:775–789PubMedCrossRefGoogle Scholar
  9. 9.
    Greither T, Grochola LF, Udelnow A, Lautenschlager C, Wurl P, Taubert H (2010) Elevated expression of microRNAs 155, 203, 210 and 222 in pancreatic tumors is associated with poorer survival. Int J Cancer 126:73–80PubMedCrossRefGoogle Scholar
  10. 10.
    Bloomston M, Frankel WL, Petrocca F, Volinia S, Alder H, Hagan JP, Liu CG, Bhatt D, Taccioli C, Croce CM (2007) MicroRNA expression patterns to differentiate pancreatic adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA 297:1901–1908PubMedCrossRefGoogle Scholar
  11. 11.
    Szafranska AE, Davison TS, John J, Cannon T, Sipos B, Maghnouj A, Labourier E, Hahn SA (2007) MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma. Oncogene 26:4442–4452PubMedCrossRefGoogle Scholar
  12. 12.
    Lee EJ, Gusev Y, Jiang J, Nuovo GJ, Lerner MR, Frankel WL, Morgan DL, Postier RG, Brackett DJ, Schmittgen TD (2007) Expression profiling identifies microRNA signature in pancreatic cancer. Int J Cancer 120:1046–1054PubMedCrossRefGoogle Scholar
  13. 13.
    du Rieu MC, Torrisani J, Selves J, Al ST, Souque A, Dufresne M, Tsongalis GJ, Suriawinata AA, Carrere N, Buscail L, Cordelier P (2010) MicroRNA-21 is induced early in pancreatic ductal adenocarcinoma precursor lesions. Clin Chem 56:603–612PubMedCrossRefGoogle Scholar
  14. 14.
    Hanoun N, Delpu Y, Suriawinata AA, Bournet B, Bureau C, Selves J, Tsongalis GJ, Dufresne M, Buscail L, Cordelier P, Torrisani J (2010) The silencing of microRNA 148a production by DNA hypermethylation is an early event in pancreatic carcinogenesis. Clin Chem 56:1107–1118PubMedCrossRefGoogle Scholar
  15. 15.
    Kauppinen S, Vester B, Wengel J (2006) Locked nucleic acid: high-affinity targeting of complementary RNA for RNomics. Handb Exp Pharmacol 173:405–422PubMedCrossRefGoogle Scholar
  16. 16.
    Kloosterman WP, Wienholds E, de Brujin E, Kauppinen S, Plasterk RH (2006) In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods 3:27–29PubMedCrossRefGoogle Scholar
  17. 17.
    Wienholds E, Kloosterman WP, Miska E, Varez-Saavedra E, Berezikov E, de Brujin E, Horvitz RH, Kauppinen S, Plasterk RH (2005) MicroRNA expression in zebrafish embryonic development. Science 309:310–311PubMedCrossRefGoogle Scholar
  18. 18.
    Nelson PT, Baldwin DA, Kloosterman WP, Kauppinen S, Plasterk RH, Mourelatos Z (2006) RAKE and LNA-ISH reveal microRNA expression and localization in archival human brain. RNA 12:187–191PubMedCrossRefGoogle Scholar
  19. 19.
    Sempere LF, Christensen M, Silahtaroglu A, Bak M, Heath CV, Schwartz G, Wells W, Kauppinen S, Cole CN (2007) Altered MicroRNA expression confined to specific epithelial cell subpopulations in breast cancer. Cancer Res 67:11612–11620PubMedCrossRefGoogle Scholar
  20. 20.
    Yamamichi N, Shimomura R, Inada K, Sakurai K, Haraguchi T, Ozaki Y, Fujita S, Mizutani T, Furukawa C, Fujishiro M, Ichinose M, Shiogama K, Tsutsumi Y, Omata M, Iba H (2009) Locked nucleic acid in situ hybridization analysis of miR-21 expression during colorectal cancer development. Clin Cancer Res 15:4009–4016PubMedCrossRefGoogle Scholar
  21. 21.
    Liu X, Sempere LF, Ouyang H, Memoli VA, Andrew AS, Luo Y, Demidenko E, Korc M, Shi W, Preis M, Dragnev KH, Li H, DiRenzo J, Bak M, Freemantle SJ, Kauppinen S, Dmitrovsky E (2010) MicroRNA-31 functions as an oncogenic microRNA in mouse and human lung cancer cells by repressing specific tumor suppressors. J Clin Invest 120: 1298–1309PubMedCrossRefGoogle Scholar
  22. 22.
    Dillhoff M, Liu J, Frankel W, Croce C, Bloomston M (2008) MicroRNA-21 is overexpressed in pancreatic cancer and a potential predictor of survival. J Gastrointest Surg 12:2171–2176PubMedCrossRefGoogle Scholar
  23. 23.
    Habbe N, Koorstra JB, Mendell JT, Offerhaus GJ, Ryu JK, Feldmann G, Mullendore ME, Goggins MG, Hong SM, Maitra A (2009) MicroRNA miR-155 is a biomarker of early pancreatic neoplasia. Cancer Biol Ther 8: 340–346PubMedCrossRefGoogle Scholar
  24. 24.
    Liu X, Sempere LF, Guo Y, Korc M, Kauppinen S, Freemantle SJ, Dmitrovsky E (2011) Involvement of microRNAs in lung cancer biology and therapy. Transl Res 157:200–208PubMedCrossRefGoogle Scholar
  25. 25.
    Speel EJ, Hopman AH, Komminoth P (2006) Tyramide signal amplification for DNA and mRNA in situ hybridization. Methods Mol Biol 326:33–60PubMedGoogle Scholar
  26. 26.
    Sempere LF, Preis M, Yezefski T, Ouyang H, Suriawinata AA, Silahtaroglu A, Conejo-Garcia JR, Kauppinen S, Wells W, Korc M (2010) Fluorescence-based codetection with protein markers reveals distinct cellular compartments for altered MicroRNA expression in solid tumors. Clin Cancer Res 16:4246–4255PubMedCrossRefGoogle Scholar
  27. 27.
    Garcea G, Neal CP, Pattenden CJ, Steward WP, Berry DP (2005) Molecular prognostic markers in pancreatic cancer: a systematic review. Eur J Cancer 41:2213–2236PubMedCrossRefGoogle Scholar
  28. 28.
    Aguirre AJ, Bardeesy N, Sinha M, Lopez L, Tuveson DA, Horner J, Redston MS, DePinho RA (2003) Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma. Genes Dev 17:3112–3126PubMedCrossRefGoogle Scholar
  29. 29.
    Bardeesy N, Cheng KH, Berger JH, Chu GC, Pahler J, Olson P, Hezel AF, Horner J, Lauwers GY, Hanahan D, DePinho RA (2006) Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer. Genes Dev 20:3130–3146PubMedCrossRefGoogle Scholar
  30. 30.
    Zhang L, Sanderson SO, Lloyd RV, Smyrk TC (2007) Pancreatic intraepithelial neoplasia in heterotopic pancreas: evidence for the progression model of pancreatic ductal adenocarcinoma. Am J Surg Pathol 31:1191–1195PubMedCrossRefGoogle Scholar
  31. 31.
    Funahashi H, Satake M, Dawson D, Huynh NA, Reber HA, Hines OJ, Eibl G (2007) Delayed progression of pancreatic intraepithelial neoplasia in a conditional Kras(G12D) mouse model by a selective cyclooxygenase-2 inhibitor. Cancer Res 67:7068–7071PubMedCrossRefGoogle Scholar
  32. 32.
    Ijichi H, Chytil A, Gorska AE, Aakre ME, Fujitani Y, Fujitani S, Wright CV, Moses HL (2006) Aggressive pancreatic ductal adenocarcinoma in mice caused by pancreas-specific blockade of transforming growth factor-beta signaling in cooperation with active Kras expression. Genes Dev 20:3147–3160PubMedCrossRefGoogle Scholar
  33. 33.
    Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA, Ross S, Conrads TP, Veenstra TD, Hitt BA, Kawaguchi Y, Johann D, Liotta LA, Crawford HC, Putt ME, Jacks T, Wright CV, Hruban RH, Lowy AM, Tuveson DA (2003) Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 4:437–450PubMedCrossRefGoogle Scholar
  34. 34.
    Guerra C, Mijimolle N, Dhawahir A, Dubus P, Barradas M, Serrano M, Campuzano V, Barbacid M (2003) Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell 4: 111–120PubMedCrossRefGoogle Scholar
  35. 35.
    Guerra C, Schuhmacher AJ, Canamero M, Grippo PJ, Verdaguer L, Perez-Gallego L, Dubus P, Sandgren EP, Barbacid M (2007) Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice. Cancer Cell 11: 291–302PubMedCrossRefGoogle Scholar
  36. 36.
    Carriere C, Seeley ES, Goetze T, Longnecker DS, Korc M (2007) The Nestin progenitor lineage is the compartment of origin for pancreatic intraepithelial neoplasia. Proc Natl Acad Sci U S A 104:4437–4442PubMedCrossRefGoogle Scholar
  37. 37.
    Hezel AF, Kimmelman AC, Stanger BZ, Bardeesy N, DePinho RA (2006) Genetics and biology of pancreatic ductal adenocarcinoma. Genes Dev 20:1218–1249PubMedCrossRefGoogle Scholar
  38. 38.
    Heiser PW, Lau J, Taketo MM, Herrera PL, Hebrok M (2006) Stabilization of beta-catenin impacts pancreas growth. Development 133:2023–2032PubMedCrossRefGoogle Scholar
  39. 39.
    Kawaguchi Y, Cooper B, Gannon M, Ray M, MacDonald RJ, Wright CV (2002) The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors. Nat Genet 32:128–134PubMedCrossRefGoogle Scholar
  40. 40.
    Pena JT, Sohn-Lee C, Rouhanifard SH, Ludwig J, Hafner M, Mihailovic A, Lim C, Holoch D, Berninger P, Zavolan M, Tuschl T (2009) miRNA in situ hybridization in formaldehyde and EDC-fixed tissues. Nat Methods 6:139–141PubMedCrossRefGoogle Scholar
  41. 41.
    Nelson PT, Wilfred BR (2009) In situ hybridization is a necessary experimental complement to microRNA (miRNA) expression profiling in the human brain. Neurosci Lett 466:69–72PubMedCrossRefGoogle Scholar
  42. 42.
    Silahtaroglu AN, Nolting D, Dyrskjot L, Berezikov E, Moller M, Tommerup N, Kauppinen S (2007) Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification. Nat Protoc 2:2520–2528PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Department of MedicineDartmouth Hitchcock Medical CenterHanoverUSA
  2. 2.Department of MedicineNorris Cotton Cancer Center, Dartmouth Hitchcock Medical CenterLebanonUSA
  3. 3.Department of Pharmacology and ToxicologyDartmouth Medical SchoolHanoverUSA

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