Analysis of the Stromal Cellular Components of the Solid Tumor Microenvironment Using Flow Cytometry

Michael Timaner1, Ofrat Beyar‐Katz1, Yuval Shaked1

1 Department of Cell Biology and Cancer Science, Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, Haifa
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.18
DOI:  10.1002/0471143030.cb1918s70
Online Posting Date:  March, 2016
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Abstract

The tumor microenvironment consists of a variety of cell types. The contribution of each cell type to the tumor is an emerging subject in the field of cancer research. Here, we describe protocols for dissociating tumor tissues and Matrigel plugs into single cells for further analysis by flow cytometry. These protocols can be used for evaluating the cellular component of solid tumors from human or mouse origin or Matrigel plugs implanted in mice. The protocols describe the dissociation of tumor tissue with or without dissociation automatic devices. Subsequently, the use of flow cytometry for immunophenotypic analysis of host cells found in the tumor microenvironment, including myeloid derived suppressor cells, endothelial cells, and macrophages is provided. These methods can be used to broaden our understanding of the cross‐talk between tumor and host cells in the tumor microenvironment. © 2016 by John Wiley & Sons, Inc.

Keywords: bone marrow derived cells; host; cancer cells; cell dissociation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Extraction of Single Cells from Solid Tumor Mass
  • Basic Protocol 2: Extraction of Host Cells Colonizing Matrigel Plugs
  • Basic Protocol 3: Extraction of Single Cells from a Tumor Mass Using gentleMACS Device
  • Support Protocol 1: Identification of Cells by Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Extraction of Single Cells from Solid Tumor Mass

  Materials
  • Solid tumors
  • 1× sterile Dulbecco's phosphate‐buffered saline (PBS; Sigma, cat. no. D8537)
  • Enzymatic cocktail mix (see recipe)
  • Working medium (see recipe)
  • DNase I solution (see recipe)
  • Sterile surgical tools including:
    • Tweezers
    • Blunt scissors
    • Scalpel
  • 10‐cm2 petri dishes (Corning)
  • 15‐ml centrifuge tubes
  • Vortex mixer
  • 37°C incubator
  • Shaker
  • 40‐μm cell strainer (BD Falcon)
  • Centrifuge
  • Additional reagents and equipment for counting cells using a hemacytometer and trypan blue (Phelan, )

Basic Protocol 2: Extraction of Host Cells Colonizing Matrigel Plugs

  Materials
  • Mice implanted with Matrigel plugs (see Gingis‐Velitski, )
  • Enzymatic cocktail mix (see recipe)
  • Dispase II solution (see recipe)
  • 1× sterile Dulbecco's phosphate‐buffered saline (PBS; Sigma, cat. no. D8537)
  • DNase I solution (see recipe)
  • Sterile surgical tools including:
    • Tweezers
    • Blunt scissors
    • Scalpel
  • Analytical scale
  • 1.7‐ml microcentrifuge tubes
  • 37°C incubator
  • Shaker
  • Pipet tips
  • 70‐μm cell strainer (BD Falcon)
  • Centrifuge
  • Additional reagents and equipment for counting cells using a hemacytometer and trypan blue (Phelan, )

Basic Protocol 3: Extraction of Single Cells from a Tumor Mass Using gentleMACS Device

  Materials
  • Mice bearing tumors
  • 1× sterile Dulbecco's phosphate‐buffered saline (PBS; Sigma, cat. no. D8537)
  • Working medium (see recipe)
  • Collagenase I solution (see recipe)
  • Dispase II solution (see recipe)
  • DNase I solution (see recipe)
  • Sterile surgical tools including:
    • Tweezers
    • Blunt scissors
  • 10‐cm2 petri dishes (Corning)
  • GentleMACS C tubes (MiltenyiBiotec, cat. no. 130‐096‐334)
  • GentleMACSdissociator (MiltenyiBiotec, cat. no. 130‐093‐235) or gentleMACSOcto dissociator (MiltenyiBiotec, cat. no. 130‐095‐937)
  • 37°C incubator
  • Shaker
  • 40‐μm cell strainer (BD Falcon)
  • 50‐ml centrifuge tube for each tumor sample
  • Centrifuge
  • Additional reagents and equipment for counting cells using a hemacytometer and trypan blue (Phelan, ).

Support Protocol 1: Identification of Cells by Flow Cytometry

  Materials
  • Single‐cell suspension of tumor cells (see Basic Protocols protocol 11, protocol 22, and protocol 33)
  • Lysis buffer (see recipe)
  • Ice
  • Flow cytometry buffer (see recipe)
  • Antibodies (all antibodies are mouse‐specific):
    • FITC conjugated anti‐CD31
    • PE conjugated anti‐ VEGFR2
    • APC conjugated anti‐CD117
    • APC‐Cy7 conjugated anti‐CD45
    • PE conjugated anti‐Gr1
    • FITC conjugated anti‐CXCR4
    • PerCPconjugated anti‐CD11b
    • APC conjugated anti‐VEGFR1
    • APC Cy7 conjugated anti‐CD45
    • PE conjugated anti‐F4\80
    • Bv421 conjugated anti‐CD11c
    • APC conjugated anti‐CD206
  • 15‐ml centrifuge tubes
  • Centrifuge
  • Vortex mixer
  • Flow cytometry microcentrifuge tubes
  • Flow cytometry system: e.g., CyAn ADP(Beckman Coulter), equipped with 405‐nm, 488‐nm, and 635‐nm lasers
  • Summit Version 3.4 software (Beckman Coulter) or equivalent software for flow cytometry analysis
  • Additional reagents and equipment for counting cells using hemacytometer and trypan blue (Phelan, )
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Figures

Videos

Literature Cited

Literature Cited
  Adini, A., Fainaru, O., Udagawa, T., Connor, K.M., Folkman, J., and D'Amato, R.J. 2009. Matrigel cytometry: A novel method for quantifying angiogenesis in vivo. J. Immunol. Methods 342:78‐81. doi: 10.1016/j.jim.2008.11.016.
  Benayoun, L. and Shaked, Y. 2013. In vitro enrichment of tumor‐initiating cells from human established cell lines. Curr. Protoc. Stem Cell Biol. 24:3.7.1‐3.7.15. doi: 10.1002/9780470151808.sc0307s24.
  Cavallo, F., De Giovanni, C., Nanni, P., Forni, G., and Lollini, P.L. 2011. 2011: The immune hallmarks of cancer. Cancer Immunol. Immunother. 60:319‐326. doi: 10.1007/s00262‐010‐0968‐0.
  De Palma, M. and Lewis, C.E. 2013. Macrophage regulation of tumor responses to anticancer therapies. Cancer Cell 23:277‐286. doi: 10.1016/j.ccr.2013.02.013.
  Giesen, C., Wang, H.A., Schapiro, D., Zivanovic, N., Jacobs, A., Hattendorf, B., Schuffler, P.J., Grolimund, D., Buhmann, J.M., Brandt, S., Varga, Z., Wild, P.J., Gunther, D., and Bodenmiller, B. 2014. Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat. Methods 11:417‐422. doi: 10.1038/nmeth.2869.
  Gingis‐Velitski, S., Loven, D., Benayoun, L., Munster, M., Bril, R., Voloshin, T., Alishekevitz, D., Bertolini, F., and Shaked, Y. 2011. Host response to short‐term, single‐agent chemotherapy induces matrix metalloproteinase‐9 expression and accelerates metastasis in mice. Cancer Res. 71:6986‐6996. doi: 10.1158/0008‐5472.CAN‐11‐0629.
  Hanahan, D. and Coussens, L.M. 2012. Accessories to the crime: Functions of cells recruited to the tumor microenvironment. Cancer Cell 21:309‐322. doi: 10.1016/j.ccr.2012.02.022.
  Hanahan, D. and Weinberg, R.A. 2000. The hallmarks of cancer. Cell 100:57‐70. doi: 10.1016/S0092‐8674(00)81683‐9.
  Hanahan, D. and Weinberg, R.A. 2011. Hallmarks of cancer: The next generation. Cell 144:646‐674. doi: 10.1016/j.cell.2011.02.013.
  Jin, D.K., Shido, K., Kopp, H.G., Petit, I., Shmelkov, S.V., Young, L.M., Hooper, A.T., Amano, H., Avecilla, S.T., Heissig, B., Hattori, K., Zhang, F., Hicklin, D.J., Wu, Y., Zhu, Z., Dunn, A., Salari, H., Werb, Z., Hackett, N.R., Crystal, R.G., Lyden, D., and Rafii, S. 2006. Cytokine‐mediated deployment of SDF‐1 induces revascularization through recruitment of CXCR4+ hemangiocytes. Nat. Med. 12:557‐567. doi: 10.1038/nm1400.
  Katz, O.B. and Shaked, Y. 2015. Host effects contributing to cancer therapy resistance. Drug Resist. Update 19:33‐42. doi: 10.1016/j.drup.2014.12.002.
  Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
  Roodhart, J.M., Daenen, L.G., Stigter, E.C., Prins, H.J., Gerrits, J., Houthuijzen, J.M., Gerritsen, M.G., Schipper, H.S., Backer, M.J., van Amersfoort, M., Vermaat, J.S., Moerer, P., Ishihara, K., Kalkhoven, E., Beijnen, J.H., Derksen, P.W., Medema, R.H., Martens, A.C., Brenkman, A.B., and Voest, E.E. 2011. Mesenchymal stem cells induce resistance to chemotherapy through the release of platinum‐induced fatty acids. Cancer Cell 20:370‐383. doi: 10.1016/j.ccr.2011.08.010.
  Shaked, Y. and Voest, E.E. 2009. Bone marrow derived cells in tumor angiogenesis and growth: Are they the good, the bad or the evil? Biochim. Biophys. Acta 1796:1‐4.
  Shaked, Y., Ciarrocchi, A., Franco, M., Lee, C.R., Man, S., Cheung, A.M., Hicklin, D.J., Chaplin, D., Foster, F.S., Benezra, R., and Kerbel, R.S. 2006. Therapy‐induced acute recruitment of circulating endothelial progenitor cells to tumors. Science 313:1785‐1787. doi: 10.1126/science.1127592.
  Shaked, Y., Henke, E., Roodhart, J.M., Mancuso, P., Langenberg, M.H., Colleoni, M., Daenen, L.G., Man, S., Xu, P., Emmenegger, U., Tang, T., Zhu, Z., Witte, L., Strieter, R.M., Bertolini, F., Voest, E.E., Benezra, R., and Kerbel, R.S. 2008. Rapid chemotherapy‐induced acute endothelial progenitor cell mobilization: Implications for antiangiogenic drugs as chemosensitizing agents. Cancer Cell 14:263‐273. doi: 10.1016/j.ccr.2008.08.001.
  Shevchenko, I., Karakhanova, S., Soltek, S., Link, J., Bayry, J., Werner, J., Umansky, V., and Bazhin, A.V. 2013. Low‐dose gemcitabine depletes regulatory T cells and improves survival in the orthotopic Panc02 model of pancreatic cancer. Int. J. Cancer 133:98‐107. doi: 10.1002/ijc.27990.
  Shojaei, F., Wu, X., Malik, A.K., Zhong, C., Baldwin, M.E., Schanz, S., Fuh, G., Gerber, H.P., and Ferrara, N. 2007a. Tumor refractoriness to anti‐VEGF treatment is mediated by CD11b(+)Gr1(+) myeloid cells. Nat. Biotechnol. 25:911‐920. doi: 10.1038/nbt1323.
  Shojaei, F., Wu, X., Zhong, C., Yu, L., Liang, X.H., Yao, J., Blanchard, D., Bais, C., Peale, F.V., van Bruggen, N., Ho, C., Ross, J., Tan, M., Carano, R.A., Meng, Y.G., and Ferrara, N. 2007b. Bv8 regulates myeloid‐cell‐dependent tumour angiogenesis. Nature 450:825‐831. doi: 10.1038/nature06348.
  Shree, T., Olson, O.C., Elie, B.T., Kester, J.C., Garfall, A.L., Simpson, K., Bell‐McGuinn, K.M., Zabor, E.C., Brogi, E., and Joyce, J.A. 2011. Macrophages and cathepsin proteases blunt chemotherapeutic response in breast cancer. Genes Dev. 25:2465‐2479. doi: 10.1101/gad.180331.111.
  Voloshin, T., Voest, E.E., and Shaked, Y. 2013. The host immunological response to cancer therapy: An emerging concept in tumor biology. Exp. Cell Res. 319:1687‐1695. doi: 10.1016/j.yexcr.2013.03.007.
  Voloshin, T., Fremder, E., and Shaked, Y. 2014. Small but mighty: Microparticles as mediators of tumor progression. Cancer Microenviron. 7:11‐21. doi: 10.1007/s12307‐014‐0144‐8.
  Voloshin, T., Alishekevitz, D., Kaneti, L., Miller, V., Isakov, E., Kaplanov, I., Voronov, E., Fremder, E., Benhar, M., Machluf, M., Apte, R.N., and Shaked, Y. 2015. Blocking IL‐1beta pathway following paclitaxel chemotherapy slightly inhibits primary tumor growth but promotes spontaneous metastasis. Mol. Cancer Ther. 14:1385. doi: 10.1158/1535‐7163.MCT‐14‐0969.
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