Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

1. Generate fluorescently labeled tumor cells by crossing the spontaneous, autochthonous, genetically engineered mouse mammary cancer model where the mouse mammary tumor virus long terminal repeat drives the polyoma middle T antigen (MMTV-PyMT) with transgenic mice with fluorescent reporters [i.e., enhanced green fluorescent protein (EG....

Results

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 .......

Discussion

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 transient^10,13,24. 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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
155 kDa dextran-tetramethylrhodamine isothiocyanate	Sigma Aldrich	T1287	reconstitute at 20 mg/ml in 1x PBS
70 kDa dextran-Texas Red	Life Technologies	D-1830	reconstitute at 10 mg/ml in 1x PBS
10 kDa dextran-fluorescein isothyocyanate	Sigma Aldrich	FD10S	reconstitute at 20 mg/ml in 1x PBS
Qdot 705 ITK Amino (PEG) Quantum Dots	Life Technologies	Q21561MP	Dilute 25 μl in 175 μl of 1x PBS for injection
MMTV-PyMT mice	Jackson Laboratory	2374
Csf1r-ECFP mice (Csf1r-Gal4/VP16,UAS-ECFP)	Jackson Laboratory	26051
Csf1r-EGFP mice	Jackson Laboratory	18549
1x PBS	Life Technologies
Isoethesia (isoflurane)	Henry Schein Animal Health	50033	250 ml
Oxygen	AirTech
1 ml syringe, tuberculin slip tip	BD	309659
30 G x 1 (0.3 mm x 25 mm) needle	BD	305128
Polyethylene micro medical tubing	Scientific Commodities Inc	BB31695-PE/1	0.28 mm I.D. x 0.64 mm O.D.
Microscope coverglass	Corning	2980-225	thickness 1.5, 22 x 50 mm
PhysioSuite MouseSTAT pulse oximeter, software and sensors	Kent Scientific
Laboratory tape	Fisher Scientific	159015R
soft rubber pad	McMaster-Carr	8514K62	Ultra-Soft Polyurethane Film, 3/16” Thick, 12" x 12", 40 Oo Durometer, Plain Back
hard rubber pad	McMaster-Carr	8568K615	High-Strength Neoprene Rubber Sheet 1/4" Thick, 12" x 12", 50 A Durometer
Microscope	Olympus		The microscope is a custom built two laser multiphoton microscope based on an Olympus IX-71 stand utilizing a 20X 1.05NA objective lens.
7-Punch set	McMaster-Carr	3429A12	1/4" to 1" Hole Diameter, for Hammer-Driven Hole Punch