Long-term intravital immunofluorescence imaging of tissue matrix components with epifluorescence and two-photon microscopy

Esra Güç, Manuel Fankhauser, Amanda Lund, Melody A. Swartz, Witold W. Kilarski

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

Original languageEnglish (US)
Article numbere51388
JournalJournal of Visualized Experiments
Issue number86
DOIs
StatePublished - Apr 22 2014

Fingerprint

Photons
Fluorescent Antibody Technique
Tumors
Microscopy
Microscopic examination
Tissue
Imaging techniques
Neoplasms
Proteins
Tumor Microenvironment
Extracellular Matrix Proteins
Cell Movement
Extracellular Matrix
Cells
Harmonic generation
Collagen
Fibrillar Collagens
Tenascin
Skin
Lymphatic Vessels

Keywords

  • Bioengineering
  • Epifluorescence
  • Intravital imaging
  • Issue 86
  • Matrix remodeling
  • Tumor matrix
  • Two-photon imaging

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

Long-term intravital immunofluorescence imaging of tissue matrix components with epifluorescence and two-photon microscopy. / Güç, Esra; Fankhauser, Manuel; Lund, Amanda; Swartz, Melody A.; Kilarski, Witold W.

In: Journal of Visualized Experiments, No. 86, e51388, 22.04.2014.

Research output: Contribution to journalArticle

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