Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Summary

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Abstract

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death ¹. Common sites of metastatic spread include lung, lymph node, brain, and bone ². Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level ^3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level ⁷, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

Introduction

QRT-PCR analysis is proposed as a method for assessing tumor metastasis. This method is proposed as an alternative for users that are interested in evaluating metastasis but may not have access to specific equipment such as in vivo imaging equipment or a fluorescence capable stereoscope. A discussion of commonly used methods is presented followed by a demonstration for how QRT-PCR analysis can be used either as a separate or as a companion method to evaluate metastasis. This procedure has the potential to provide a quantitative analysis of metastatic burden.

Standard methods of gross analysis, including visualization of lungs under....

Protocol

The protocol follows the guidelines and animal care standards of the Medical University of South Carolina (MUSC) and its Institutional Animal Care and Use Committee (IACUC).

1. Lung Dissection

1. Mammary tumor dissection (optional depending on study goals and whether primary tumor analysis or tail vein injection was performed).
   1. Euthanize mouse using a lethal dose of isoflurane anesthesia according to IACUC and university regulation. Confirm euthanization by surgical dislocation. 
   2. On foam board, pin with dissection pins by placing mouse on its back with limbs spread.
   3. Spray mouse with 70% ethanol.
   4. Using f....

Results

Beyond the time it takes to perform the initial inoculation of tumor cells into the experimental animal and if performing, primary in vivo tumor analysis, the tissue harvest, RNA isolation, and QRT-PCR analysis is a 1-2 day procedure (Figure 1).

An example of gross analysis is bioluminescent imaging to evaluate tumor cells within the lung tissue. Here, a mammary tumorigenesis experiment was performed to.......

Discussion

This protocol describes use of QRT-PCR to evaluate mammary tumor cell metastasis to the lung using a xenograft mouse model. It is acknowledged that other techniques, including bioluminescent imaging, are available and are also effective for detecting metastasis despite having their own values and limitations. The data presented suggests that QRT-PCR provides an effective measure of detecting tumor-derived mRNA within lung tissue for quantitation of total metastasis. It is proposed that QRT-PCR analysis provides a seconda.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-mercaptoethanol	Fisher	BP176-100
DNAse	Thermo Scientific	EN0521
GeneJet RNA Isolation Kit	Thermo Scientific	K0732
iScript Reverse Transcription Supermix for QRT-PCR	Bio-Rad	1708841
iTaq Universal SYBR Green Supermix	Bio-Rad	172-5121
PrimePCR SYBR Green Assay: ERBB2, Human	Bio-Rad	100-25636
PrimePCR Template for SYBR Green Assay: ERBB2, Human	Bio-Rad	100-25716
gapdh Primer Sequence			For-ATGGTGAAGGTCGGTGTGAACG Rev-CGCTCCTGGAAGATGGTGATGG