Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

Summary

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

Abstract

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

Introduction

Metastasis is the final step in the progression of breast cancer ¹. It involves a series of steps in which malignant cells are released from the primary tumor and disseminated to other organs ². The cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system ³. Each step in tumorigenesis is tightly controlled by cells through various genetic and epigenetic changes ⁴. To study the mechanisms of breast cancer tumorigenesis, mouse model systems have been utilized due to their short lifespan, small size and fast breeding time. Some major advantages of using mouse....

Protocol

All mouse experiments were done in accordance with protocols approved by the Institutional Animal Care and Use Committees (IACUC) of LSU School of Medicine. All instruments must be autoclaved prior to dissection.

1. Preparation of Primary Cells from Mouse Breast Tumors

1. Use a mouse model that was genetically engineered with the Mouse Mammary Tumor Virus Polyoma Middle T-Antigen (MMTV-PyMT-transgenic mouse), which spontaneously develop tumors as previously described ⁶.
2. Sacrifice mice with Isoflurane (99.9%) and spray 75% ethanol to ensure a sterile environment (Figure 1A).
3. Open the skin from the area of ....

Results

The tumor progression stage at which to sacrifice the animal depends on the particular study and IACUC guidelines. In Figure 1A, a 100 day old C57BL/6 mouse with the Mouse Mammary Tumor Virus Polyoma Middle T-Antigen (MMTV-PyMT) was sacrificed at three months. The skin was opened from the area of the urethral orifice to the neck with the scissors and the skin was pinned down to allow easy access to organs as shown in Figure 1B. This allowed easy access to the tumor that was obtained in <.......

Discussion

This protocol is for isolating primary cells from a fresh mouse tumor. Lysates prepared from this method are useful for protein, DNA and RNA analysis. Cancerous cells from the primary breast tumor often metastasize to the brain, lung and bone ¹. Included is a technique to extract the lung for detection of metastasis. Staining the lung with an ink stain will allow for the visualization of tumor nodules as seen in Figure 2B. Fixing the lungs in Z-fix solution will allow us to make sections of lu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	HyClone	SH30243.01	Store at 4 °C
0.05% Trypsin-EDTA	Life  technologies	25300-062	Store at – 20 °C
Collagenase Type IV	Sigma-Aldrich	C5138	Store at – 20 °C
Hyaluronidase	Sigma-Aldrich	H3506	Store at – 20 °C
Pen Strep	Life  technologies	15140-122	Store at 4 °C
Fetal Bovine Serum (FBS)	Gemini Bio-Products	100-106	Store at – 80 °C
Z-fix	ANATECH	#174	Store at RT
India Ink	Yasutomo		Store at RT
AERRANE (Isoflurane)	Baxter	NDC 10019-773-60	Store at RT
Ethanol	EMD	AX0441-3	Store at RT