Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Summary

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Abstract

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Introduction

The deadliest aspect of cancer is metastasis, which accounts for more than 90% of cancer-related morbidity and mortality¹. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular mechanisms that govern metastatic dissemination and growth are still elusive. To metastasize, tumor cells in the primary tumor must detach from their neighboring cells and basement membrane, cross through the extracellular matrix, intravasate, travel via blood or lymphatic vessels, extravasate at the secondary site, and finally, survive and establish secondary tumors. In addition to the properties of the tumor cells, the contribution from the m....

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Protocol

All procedures described must be performed in accordance with guidelines and regulations for the use of vertebrate animals, including prior approval by the local Institutional Animal Care and Use Committee (IACUC).

1. Generation of Lung Metastases for Ex vivo Live Imaging (Transgenic or Tail Vein Injection)

NOTE: Lung metastases can be generated by utilizing genetically engineered mouse models or by intravenous (i.v) injection of cancer cells.

1. Generate lung metastases for imaging by crossing a genetically engineered tumor mouse model into a transgenic reporter mouse, e.g., c....

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Results

Using spinning-disk confocal microscopy, various mouse model systems and injectables, the metastatic microenvironment can be visualized and tracked over time. Using an MMTV-PyMT; ACTB-ECFP; c-fms-EGFP triple transgenic mouse model, different cellular components are fluorescently labeled (Figure 2A, Movie 1). The typical structure of the lung parenchyma can be visualized in the CFP channel since all cells express ECFP under the β-actin promoter. Larger/multicellular lung met.......

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Discussion

This manuscript describes a detailed method for ex vivo live imaging of lung metastasis in mouse models of metastasis. This imaging protocol provides a direct visualization of the dynamic and spatial tumor cell-stroma interactions within the lung microenvironment. It is a relatively easy and fast method that allows reliable imaging of lung metastasis for at least 4 hr. Movies acquired from these experiments can be used to track dynamic processes as cell motility and cellular interactions.

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Materials

Name	Company	Catalog Number	Comments
MMTV-PyMT/FVB mice	Jackson Laboratory	2374	Female mice
ACTB-ECFP/FVB mice	UCSF Werb lab		Female mice
c-fms-EGFP/FVB mice	UCSF Werb lab		Female mice
FVB mice	Jackson Laboratory	1800	Female mice
GFP+ VO-PyMT cells	UCSF Werb lab
70,000 kDa Dextran, rhodamine-conjugated	Invitrogen	D1818	Dilute to  4mg/ml in 1 x PBS and store at -20  °C. Use 0.4 mg per animal.
10,000 kDa Dextran, Alexa Fluor 647 conjugated	Invitrogen	D22914	Dilute to  4mg/ml in 1 x PBS and store at -20  °C. Use 0.4 mg per animal.
Anti-mouse Gr-1 antibody Alexa Fluor 647	UCSF Monoclonal antibody core		Stock 1mg/ml. Use 7 ug per animal.
Anesthetic			Anesthesia approved by IACUC, used for anesthesia and/or euthanesia
1X PBS	UCSF cell culture facility
PBS, USP sterile	Amresco INC	K813-500ML	Ultra pure grade for i.v. injection
Styrofoam platform			Will be used as dissection board
Fine scissors sharp	Fine Science Tools	14060-11
Forceps	Roboz Surgical Store	RS-5135
Hot bead sterilizer	Fine Science Tools	18000-45	Turn ON 30min before use
Air	UCSF
Oxygen	UCSF
Carbon dioxide	UCSF
1 mL syringe without needle	BD	309659
27 G x 1/2 needle	BD	305109	for i.v. injection
20 G x 1 needle, short bevel	BD	305178
Low-melting-temperature agarose	Lonza	50111	To make 10 ml of solution, weigh 0.2 g of agarose, add to 10 ml 1 x PBS, and heat to dissolve. Agarose will solidify at room temperature, so maintain in a 37 °C water bath until used for inflation.
RPMI-1640 medium without phenol red	Life Technologies	11835-030
24 well Imaging plate	E&K scientific	EK-42892
Glass cover slides, 15 mm	Fisher Scientific	22-031-144
Digital CO2 and temperature controller	Okolab	DGTCO2BX	http://www.oko-lab.com
Climate chamber	Okolab		http://www.oko-lab.com
Cell Observer spinning disk confocal microscope	Zeiss
Zen software	Zeiss
Inverted microscope	Carl Zeiss Inc	Zeiss Axiovert 200M
ICCD camera	Stanford Photonics	XR-Mega-10EX S-30
Spinning disk confocal scan-head	Yokogawa Corporation	CSU-10b
Imaris	Bitplane
mManager	Vale lab, UCSF		Open-source software