Published: June 12th, 2016
This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.
In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.
Dissemination of tumor cells from the primary mammary tumor has been shown to involve not only tumor cells, but host stromal cells including macrophages and endothelial cells. Furthermore, tumor vasculature is abnormal with increased permeability1. Thus, determining how tumor cells, macrophages and endothelial cells interact to mediate vascular permeability and tumor cell intravasation in the primary tumor microenvironment is important for understanding metastasis. Understanding the kinetics of vascular permeability, tumor cell intravasation and the underlying signaling mechanism of multicellular interactions in the tumor microenvironment can provide crucia....
All procedures described must be performed in accordance with guidelines and regulations for the use of vertebrate animals, including prior approval by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee.
1. Generating Fluorescently Labeled Tumors and Tumor-associated Macrophages
Extended time-lapse intravital microscopy enables single cell resolution imaging of multicellular processes in the tumor microenvironment. By fluorescently labeling tumor cells, macrophages, the vascular space, and visualizing the collagen fiber network using the second harmonic generation signal, multiple compartments in the tumor microenvironment are simultaneously tracked during imaging. Tumor cells labeled with fluorescent proteins can be generated in transgenic mice as has been done .......
Cellular interactions that occur spontaneously in the tumor microenvironment can lead to changes in tumor cell motility and intravasation. High-resolution intravital imaging of live tumor tissue permits the visualization of multi-cellular dynamics that can be highly transient10,13,24. End-point in vivo assays or time-lapse images acquired with discrete time points can provide essential information on molecular mechanisms of processes in the tumor microenvironment. Intravital imaging studies have been .......
|155 kDa dextran-tetramethylrhodamine isothiocyanate
|reconstitute at 20 mg/mL in 1 X PBS
|70 kDa dextran-Texas Red
|reconstitute at 10 mg/mL in 1 X PBS
|10 kDa dextran-fluorescein isothyocyanate
|reconstitute at 20 mg/mL in 1 X PBS
|Qdot 705 ITK Amino (PEG) Quantum Dots
|Dilute 25 uL in 175 uL of 1 X PBS for injection
|Csf1r-ECFP mice (Csf1r-Gal4/VP16,UAS-ECFP)
|1 x PBS
|Henry Schein Animal Health
|1 mL syringe, tuberculin slip tip
|30G x 1 (0.3 mm X 25 mm) needle
|Polyethylene micro medical tubing
|Scientific Commodities Inc
|0.28 mm I.D. X 0.64 mm O.D.
|thickness 1.5, 22 X 50 mm
|MouseOx oximeter, software and sensors
|STARR Life Sciences
|soft rubber pad
|Ultra-Soft Polyurethane Film, 3/16” Thick, 12" x 12", 40 Oo Durometer, Plain Back
|hard rubber pad
|High-Strength Neoprene Rubber Sheet 1/4" Thick, 12" X 12", 50A Durometer,
|The microscope is a custom built two laser multiphoton microscope based on an Olympus IX-71 stand utilizing a 20x 1.05NA objective lens.
|, 1/4" to 1" Hole Diameter, for Hammer-Driven Hole Punch,
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