High‐Resolution Multiparameter DNA Flow Cytometry for the Detection and Sorting of Tumor and Stromal Subpopulations from Paraffin‐Embedded Tissues

Willem E. Corver1, Natalja T. ter Haar1

1 Leiden University Medical Centre, Leiden, The Netherlands
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.37
DOI:  10.1002/0471142956.cy0737s55
Online Posting Date:  January, 2011
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This unit contains a detailed protocol for the simultaneous flow cytometric measurement of tumor cells, stromal cells, and DNA content of formalin‐fixed, paraffin‐embedded (FFPE) tissues. The vimentin‐positive stromal cell fraction can be used as an internal reference for DNA content assessments. This allows clear detection of keratin‐positive tumor cells with a DNA index lower than 1.0 and of keratin‐positive tumor cells with a DNA close to 1.0 in overall DNA aneuploid samples, thus improved DNA ploidy assessment in FFPE carcinomas. Furthermore, the protocol is useful for studying molecular genetic alterations and intratumor heterogeneity in archival FFPE samples. Keratin‐positive tumor cell fractions can be flow‐sorted for further molecular genetic analysis, while DNA from the sorted vimentin‐positive stromal cells can serve as a reference when normal tissue of the patient is not available. Curr. Protoc. Cytom. 55:7.37.1‐7.37.21. © 2010 by John Wiley & Sons, Inc.

Keywords: flow cytometry; FFPE tissues; DNA content; stromal cells; carcinoma cells; vimentin; keratin; sorting; genomic alterations

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Table of Contents

  • Introduction
  • Basic Protocol 1: Multiparameter DNA Content Analysis of FFPE Tissues
  • Alternate Protocol 1: Immunocytochemistry for Keratin and Vimentin, and DNA Labeling for Blue and Red Excitation
  • Alternate Protocol 2: Immunocytochemistry for Keratin and Vimentin, and DNA Labeling for Blue Excitation Only
  • Support Protocol 1: Sorting Cell Populations from FFPE Carcinomas
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Multiparameter DNA Content Analysis of FFPE Tissues

  • Formalin‐fixed, paraffin‐embedded tissue sample (carcinoma)
  • 100% xylene
  • 100% (v/v), 70% (v/v), and 50% (v/v) ethanol in distilled water
  • Milli‐Q water
  • Heat‐induced antigen retrieval (HIAR) buffer (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • RPMI 1640 medium (can be obtained ready‐to‐use from: e.g., Invitrogen Life Sciences, http://www.invitrogen.com/site/us/en/home.html)
  • Dissociation solution (see recipe)
  • Hematoxylin (e.g., Polysciences)
  • Crushed ice
  • PBS/BSA/Tween (PBATw buffer; see recipe)
  • Primary reagents (see recipe): anti‐keratin antibodies (IgG1) and anti‐vimentin antibodies: 4B2 IgG2a or V9‐2b IgG2b
  • Secondary reagents (see recipe): goat anti–mouse (GaM)IgG1‐FITC, GaMIgG2a‐RPE, or GaMIgG2b‐RPE
  • DNA staining solution containing DAPI (see recipe)
  • Microtome
  • Nylon biopsy bags (3 × 5–cm, mesh 184‐µm; normally used for fixing and dehydration of biopsies; e.g., Klinipath, http://www.klinipath.nl/)
  • Pair of tweezers (steel)
  • Cigarette lighter
  • 15 × 100–mm glass tubes
  • Tube rack
  • Safety cabinet with an air‐controlled hood
  • Nitril examination gloves
  • 80° and 37°C water baths
  • Vortex
  • 1.5‐ml microcentrifuge tubes
  • Bürker (or equivalent) cell count chamber
  • Standard microscope
  • 12 × 75–mm FACS tubes
  • 80‐µm and 50‐µm nylon mesh filters (e.g., Partec)
  • Centrifuge with cooling device (maximum 750 × g)
  • Micropipets (range 1‐ to 1000‐µl)
  • Conventional refrigerator (2° to 8°C)
  • Flow cytometer equipped with UV (355 nm) and blue (488 nm) excitation and filters for collection of blue (BP 450/50 nm), green (BP 530/30 nm), and orange (BP 575/26 nm) fluorescence

Alternate Protocol 1: Immunocytochemistry for Keratin and Vimentin, and DNA Labeling for Blue and Red Excitation

  • Secondary reagents GaMIgG2a‐APC or GaMIgG2b‐APC instead of GaMIgG2a‐RPE or GaMIgG2b‐RPE
  • DNA staining solution with PI (see recipe)
  • Flow cytometer equipped with blue (488 nm) and red (633 nm) excitation and filters for collection of green, red, and deep‐red fluorescence

Alternate Protocol 2: Immunocytochemistry for Keratin and Vimentin, and DNA Labeling for Blue Excitation Only

  • DNA staining solution containing PI (see recipe) instead of DAPI
  • Flow cytometer equipped with blue (488 nm) excitation and filters for collection of green (FITC), orange (RPE) and red (PI) fluorescence

Support Protocol 1: Sorting Cell Populations from FFPE Carcinomas

  • Proteinase K solution (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Flow cytometer instrument equipped with sorting capabilities (i.e., ability to simultaneously sort four different cell populations), e.g., MoFlow, InFlux, or FACSAria cell sorter
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Literature Cited

Literature Cited
   Abeln, E.C.A., Corver, W.E., Kuipers‐Dijkshoorn, N.J., Fleuren, G.J., and Cornelisse, C.J. 1994. Molecular genetic analysis of flow‐sorted ovarian tumour cells: Improved detection of loss of heterozygosity. Br. J. Cancer 70:255‐262.
   Bagwell, C.B., Clark, G.M., Spyratos, F., Chassevent, A., Bendahl, P.O., Stal, O., Killander, D., Jourdan, M.L., Romain, S., Hunsberger, B., and Baldetorp, B. 2001. Optimizing flow cytometric DNA ploidy and S‐phase fraction as independent prognostic markers for node‐negative breast cancer specimens. Cytometry 46:121‐135.
   Bauer, K.D. 1993. Quality control issues in DNA content flow cytometry. Ann. N.Y. Acad. Sci. 677:59‐77.
   Bonsing, B.A., Corver, W.E., Fleuren, G.J., Cleton‐Jansen, A.M., Devilee, P., and Cornelisse, C.J. 2000. Allelotype analysis of flow‐sorted breast cancer cells demonstrates genetically related diploid and aneuploid subpopulations in primary tumors and lymph node metastasis. Genes Chrom. Cancer 28:173‐183.
   Brown, R.D., Linden, M.D., Mackowiak, P., Kubus, J.J., Zarbo, R.J., and Rabinovitch, P.S. 1996. The effect of number of histogram events on reproducibility and variation of flow cytometric proliferation measurement. Am. J. Clin. Pathol. 105:696‐704.
   Carvalho, R., Milne, A.N., Polak, M., Corver, W.E., Offerhaus, G.J., and Weterman, M.A. 2005. Exclusion of RUNX3 as a tumour‐suppressor gene in early‐onset gastric carcinomas. Oncogene 24:8252‐8258.
   Corver, W.E. and Cornelisse, C.J. 2002. Flow cytometry of human solid tumours: Clinical and research applications. Curr. Diag. Pathol. 8:249‐267.
   Corver, W.E., Cornelisse, C.J., and Fleuren, G.J. 1994. Simultaneous measurement of two cellular antigens and DNA using fluorescein‐isothiocyanate, R‐phycoerythrin, and propidium iodide on a standard FACScan. Cytometry 15:117‐128.
   Corver, W.E., Koopman, L.A., Mulder, A., Cornelisse, C.J., and Fleuren, G.J. 2000a. Distinction between HLA class I positive and negative cervical tumor subpopulations by multiparameter DNA flow cytometry. Cytometry 41:73‐80.
   Corver, W.E., Koopman, L.A., Van der Aa, J., Regensburg, M., Fleuren, G.J., and Cornelisse, C.J. 2000b. Four‐color multiparameter DNA flow cytometric method to study phenotypic intratumor heterogeneity in cervical cancer. Cytometry 39:96‐107.
   Corver, W.E., ter Haar, N.T., Dreef, E.J., Miranda, N.F., Prins, F.A., Jordanova, E.S., Cornelisse, C.J., and Fleuren, G.J. 2005. High‐resolution multi‐parameter DNA flow cytometry enables detection of tumour and stromal cell subpopulations in paraffin‐embedded tissues. J. Pathol. 206:233‐241.
   Corver, W.E., Middeldorp, A., ter Haar, N.T., Jordanova, E.S., van Puijenbroek, M., van Eijk, R., Cornelisse, C.J., Fleuren, G.J., Morreau, H., Oosting, J., and van Wezel, T. 2008. Genome‐wide allelic state analysis on flow‐sorted tumor fractions provides an accurate measure of chromosomal aberrations. Cancer Res. 68:10333‐10340.
   de Boer, M.A., Jordanova, E.S., Kenter, G.G., Peters, A.A., Corver, W.E., Trimbos, J.B., and Fleuren, G.J. 2007. High human papillomavirus oncogene mRNA expression and not viral DNA load is associated with poor prognosis in cervical cancer patients. Clin. Cancer Res. 13:132‐138.
   Douwes Dekker, P.B., Corver, W.E., Hogendoorn, P.C., van der Mey, A.G., and Cornelisse, C.J. 2004. Multiparameter DNA flow‐sorting demonstrates diploidy and SDHD wild‐type gene retention in the sustentacular cell compartment of head and neck paragangliomas: Chief cells are the only neoplastic component. J. Pathol. 202:456‐462.
   Esteban, J.M., Sheibani, K., Owens, M., Joyce, J., Bailey, A., and Battifora, H. 1991. Effects of various fixatives and fixation conditions on DNA ploidy analysis. A need for strict internal DNA standards. Am. J. Clin. Pathol. 95:460‐466.
   Frei, J.V. and Martinez, V.J. 1993. DNA flow cytometry of fresh and paraffin‐embedded tissue using cytokeratin staining [published erratum appears in Mod. Pathol. 1994 Jan;7(1):36]. Mod. Pathol. 6:599‐605.
   Glogovac, J.K., Porter, P.L., Banker, D.E., and Rabinovitch, P.S. 1996. Cytokeratin labeling of breast cancer cells extracted from paraffin‐embedded tissue for bivariate flow cytometric analysis. Cytometry 24:260‐267.
   Hedley, D.W., Friedlander, M.L., Taylor, I.W., Rugg, C.A., and Musgrove, E.A. 1983. Method for analysis of cellular DNA content of paraffin‐embedded pathological material using flow cytometry. J. Histochem. Cytochem. 31:1333‐1335.
   Hedley, D.W., Clark, G.M., Cornelisse, C.J., Killander, D., Kute, T., and Merkel, D. 1993. Consensus review of the clinical utility of DNA cytometry in carcinoma of the breast. Breast Cancer Res. Treat. 28:55‐59.
   Hendrix, M.J.C., Seftor, E.A., Seftor, R.E.B., and Trevor, K.T. 1997. Experimental co‐expression of vimentin and keratin intermediate filaments in human breast cancer cells results in phenotypic interconversion and increased invasive behavior. Am. J. Pathol. 150:483‐495.
   Hirons, G.T., Fawcett, J.J., and Crissman, H.A. 1994. TOTO and YOYO: New very bright fluorochromes for DNA content analyses by flow cytometry. Cytometry 15:129‐140.
   Kloth, J.N., Gorter, A., Haar, N.T., Corver, W.E., Jordanova, E.S., Kenter, G.G., and Fleuren, G.J. 2008. Lack of TNFalpha mRNA expression in cervical cancer is not associated with loss of heterozygosity at 6p21.3, inactivating mutations or promoter methylation. Mol. Immunol. 45:152‐159.
   Koopman, L.A., Corver, W.E., Van der Slik, A.R., Giphart, M.J., and Fleuren, G.J. 2000. Multiple genetic alterations cause frequent and heterogeneous human histocompatibility leukocyte antigen class I loss in cervical cancer. J. Exp. Med. 191:961‐975.
   Leers, M.P.G., Theunissen, P.H.M.H., Schutte, B., and Ramaekers, F.C.S. 1995. Bivariate cytokeratin/DNA flow cytometric analysis of paraffin‐embedded samples from colorectal carcinomas. Cytometry 21:101‐107.
   Leers, M.P.G., Schutte, B., Theunissen, P.H.M.H., Ramaekers, F.C.S., and Nap, M. 1999. Heat pretreatment increases resolution in DNA flow cytometry of paraffin‐embedded tumor tissue. Cytometry 35:260‐266.
   Middeldorp, A., van, P.M., Nielsen, M., Corver, W.E., Jordanova, E.S., ter, H.N., Tops, C.M., Vasen, H.F., Lips, E.H., van, E.R., Hes, F.J., Oosting, J., Wijnen, J., van, W.T., and Morreau, H. 2008. High frequency of copy‐neutral LOH in MUTYH‐associated polyposis carcinomas. J. Pathol. 216:25‐31.
   Morkve, O. and Laerum, O.D. 1991. Flow cytometric measurement of p53 protein expression and DNA content in paraffin‐embedded tissue from bronchial carcinomas. Cytometry 12:438‐444.
   Nylander, K., Stenling, R., Gustafsson, H., and Roos, G. 1994. Application of dual parameter analysis in flow cytometric DNA measurements of paraffin‐embedded samples. J. Oral Pathol. Med. 23:190‐192.
   Overton, W.R. and McCoy, J.P. Jr. 1994. Reversing the effect of formalin on the binding of propidium iodide to DNA. Cytometry 16:351‐356.
   Overton, W.R., Catalano, E., and McCoy, J.P., Jr. 1996. Method to make paraffin‐embedded breast and lymph tissue mimic fresh tissue in DNA analysis. Cytometry 26:166‐171.
   Qiu, W., Hu, M., Sridhar, A., Opeskin, K., Fox, S., Shipitsin, M., Trivett, M., Thompson, E.R., Ramakrishna, M., Gorringe, K.L., Polyak, K., Haviv, I., and Campbell, I.G. 2008. No evidence of clonal somatic genetic alterations in cancer‐associated fibroblasts from human breast and ovarian carcinomas. Nat. Genet. 40:650‐655.
   Rabinovitch, P.S., Torres, R.M., and Engel, D. 1986. Simultaneous cell cycle analysis and two‐color surface immunofluorescence using 7‐amino‐actinomycin D and single laser excitation. Applications to study of cell activation and the cell cycle of murine Ly‐1 B cells. J. Immunol. 136:2769‐2777.
   Shi, S.R., Key, M.E., and Kalra, K.L. 1991. Antigen retrieval in formalin‐fixed, paraffin‐embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 39:741‐748.
   Sieben, N.L., Ter Haar, N.T., Cornelisse, C.J., Fleuren, G.J., and Cleton‐Jansen, A.M. 2000. PCR artifacts in LOH and MSI analysis of microdissected tumor cells. Hum. Pathol. 31:1414‐1419.
   Stephenson, R.A., Gay, H., Fair, W.R., and Melamed, M.R. 1986. Effect of section thickness on quality of flow cytometric DNA content determinations in paraffin‐embedded tissues. Cytometry 7:41‐44.
   Vindelov, L.L., Christensen, I.J., and Nissen, N.I. 1983. A detergent‐trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry 3:323‐327.
   Watson, J.V., Sikora, K., and Evan, G.I. 1985. A simultaneous flow cytometric assay for c‐myc oncoprotein and DNA in nuclei from paraffin embedded material. J. Immunol. Meth. 83:179‐192.
   Yamashita, S. 2007. Heat‐induced antigen retrieval: mechanisms and application to histochemistry. Prog. Histochem. Cytochem. 41:141‐200.
   Zarbo, R.J., Visscher, D., and Crissman, J.D. 1989. Two‐color multiparametric method for flow cytometric DNA analysis of carcinomas using staining for cytokeratin and leukocyte‐common antigen. Anal. Quant. Cytol. Histol. 11:391‐402.
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