Preparation of High‐Density Fibrillar Collagen Matrices That Mimic Desmoplastic Tumor Stroma

Vira V. Artym1

1 Laboratory of Cell and Developmental Biology, NIDCR, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.19
DOI:  10.1002/0471143030.cb1019s70
Online Posting Date:  March, 2016
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Abstract

The stroma of invasive tumors becomes enriched in dense fibrillar collagen as a result of the desmoplastic reaction. This desmoplastic collagen exerts profound effects on tumor and normal cells. In view of these findings, it is important to develop novel in vitro cell systems that mimic this desmoplastic extracellular matrix in order to permit cell studies under in vivo−like conditions. This unit provides a protocol and troubleshooting guide for preparation of high‐density fibrillar collagen (HDFC) matrices that closely model the desmoplastic collagenous matrix of malignant tumors. It then describes the use of this matrix for in vitro cell studies of invadopodia formation and function in extracellular matrix invasion. In addition, it provides a detailed protocol for immunolabeling of invadopodial proteins and detection of HDFC matrix degradation associated with invadopodia to permit visualization of invadopodia using fluorescence microscopy. © 2016 by John Wiley & Sons, Inc.

Keywords: high‐density fibrillar collagen; HDFC matrix; desmoplastic stroma; fibrosis; collagen type I; invadopodia; invasion

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of High‐Density Fibrillar Collagen Matrices
  • Basic Protocol 2: Using HDFC as an In Vitro Model to Study Invadopodia
  • Reagents And Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of High‐Density Fibrillar Collagen Matrices

  Materials
  • Rat tail collagen type I (Corning; formerly BD Biosciences)
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4, sterile
  • Fixative (see recipe)
  • High‐glucose DMEM (HyQ‐DMEM, HyClone), sterile
  • 35‐mm dish with 20‐mm glass bottom (MatTek, cat. no. P35G‐0‐20‐C)
  • 37°C incubator
  • Phase‐contrast inverted microscope with 10× objective and stage insert to accommodate 35‐mm tissue culture dishes
  • Custom MatTek dish holder assembled from two covers of Corning Costar six‐well cell culture plates and two strips of Time tape (Fig.  )
  • Centrifuge with swinging‐bucket rotors (e.g., Sorvall Legend RT or Eppendorf 5810R) and microplate adapters, set to 20°C one day in advance
  • Parafilm
  • Additional reagents and equipment for preparation of collagen solution (unit 10.18; Artym and Matsumoto, )
NOTE: The collagen solution can be prepared from acid‐extracted rat tail or mouse tail collagen. Both rat and mouse tail collagen are suitable for immunostaining of HDFC degradation sites using the anti– collagen type I fragment antibody described in protocol 2.

Basic Protocol 2: Using HDFC as an In Vitro Model to Study Invadopodia

  Materials
  • HyQ‐DMEM, sterile, 37°C
  • Complete cell culture medium: HyQ‐DMEM with 10% FBS, sterile, 37°C
  • Trypsin‐EDTA, sterile, 37°C
  • MatTek dish with HDFC matrix (see protocol 1), not treated with chemical cross‐linker
  • 75‐cm2 cell culture flask of MDA‐MB‐231 breast carcinoma cells (or other cells of interest) at ∼60% to 70% confluence
  • Fixative (see recipe), 37°C
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • 0.5% (v/v) Triton X‐100 in PBS (see recipe)
  • 2% bovine serum albumin (BSA) in PBS (see recipe)
  • Anti‐cortactin primary antibody (rabbit monoclonal; Epitomics, cat. no. 2067‐1)
  • Anti‐collagen type I ¼ cleavage site (mouse polyclonal raised in‐house; Artym et al., )
  • Rhodamine‐phalloidin (Invitrogen)
  • Secondary antibodies, e.g., Cy2‐conjugated goat anti‐rabbit and Cy5‐conjugated goat anti‐mouse (Jackson ImmunoResearch Laboratories)
  • Humidified 37°C tissue culture incubator with 10% CO 2
  • Tissue culture phase‐contrast microscope
  • 50‐ml polypropylene centrifuge tube
  • Cell culture tabletop centrifuge
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Figures

Videos

Literature Cited

Literature Cited
  Artym, V.V. and Matsumoto, K. 2010. Imaging cells in three‐dimensional collagen matrix. Curr. Protoc. Cell. Biol. 48:10.18:1‐10.18.20. doi: 10.1002/0471143030.cb1018s48.
  Artym, V.V., Yamada, K.M., and Mueller, S.C. 2009. ECM degradation assays for analyzing local cell invasion. Methods Mol. Biol. 522:211‐219. doi: 10.1007/978‐1‐59745‐413‐1_15.
  Artym, V.V., Swatkoski, S., Matsumoto, K., Campbell, C.B., Petrie, R.J., Dimitriadis, E.K., Li, X., Mueller, S.C., Bugge, T.H., Gucek, M., and Yamada, K.M. 2015. Dense fibrillar collagen is a potent inducer of invadopodia via a specific signaling network. J. Cell. Biol. 208:331‐350. doi: 10.1083/jcb.201405099.
  Campbell, C.B., Cukierman, E., and Artym, V.V. 2014. 3‐D extracellular matrix from sectioned human tissues. Curr. Protoc. Cell. Biol. 62:19.16:1‐19.16.20. doi: 10.1002/0471143030.cb1916s62.
  Conklin, M. and Keely, P. 2012. Why the stroma matters in breast cancer: Insights into breast cancer patient outcomes through the examination of stromal biomarkers. Cell. Adh. Migr. 6:249‐260. doi: 10.4161/cam.20567.
  Provenzano, P.P., Inman, D.R., Eliceiri, K.W., Knittel, J.G., Yan, L., Rueden, C.T., White, J.G., and Keely, P.J. 2008. Collagen density promotes mammary tumor initiation and progression. BMC Med. 6:11. doi: 10.1186/1741‐7015‐6‐11.
  Weaver, A.M., Page, J.M., Guelcher, S.A., and Parekh, A. 2013. Synthetic and tissue‐derived models for studying rigidity effects on invadopodia activity. Methods Mol. Biol. 1046:171‐189. doi: 10.1007/978‐1‐62703‐538‐5_10.
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