Primær Tumor og MEF Cell Isolation å studere Lung Metastase

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 models in cancer research are the molecular and physiological similarities they share with humans ⁵.

Established mouse and human cell lines have been used extensively in breast cancer research. Although we can obtain some information about the steps in metastasis using these cells, genetically manipulated animal models with tumor suppressors/oncogenes will give far more information about tumor development and metastasis starting from the embryonic stage. Thus, with these mouse models, we can obtain genetic and developmental regulation of tumor growth. Furthermore, the tumor microenvironment also plays an important role in tumor cell gene expression and it can be difficult to mimic the same scenario with studies using established cell lines. Primary human mammary tumors can also be used to study mouse mammary tumorigenesis. The main disadvantage of using isolated breast tumors is that this newly implanted tumor does not have the mouse’s vasculature and lymphatic tissue. Whereas the genetic mouse model systems for studying mammary tumorigenesis will allow us to monitor the steps of metastasis, analyze the tumor microenvironment, and access to the entire body ⁵.

The overall goal of this protocol is to study breast tumorigenesis in mice. It is known that breast cancers typically metastasize to the brain, lung and bone. This protocol allows for the isolation of primary mammary tumor cells, as well as, studying breast cancer metastasis to the lung. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. Together, these techniques are useful in studying mouse mammary tumorigenesis.

Protocol

Alle mus forsøkene ble gjort i samsvar med protokoller godkjent av Institutional Animal Care og Bruk komiteer (IACUC) av LSU School of Medicine. Alle instrumenter må autoklaveres før disseksjon. 1. Utarbeidelse av primære celler fra mus brystsvulster Bruk en musemodell som ble genmodifisert med musen Mammary Tumor Virus Polyoma Middle T-antigen (MMTV-PyMT-transgene mus), som spontant utvikle svulster som tidligere beskrevet 6. Offer-mus med Isofluran (99,9%) og spray 75% etano…

Representative Results

Den tumorprogresjon på hvilket stadium å ofre dyret avhenger av den spesielle studier og IACUC retningslinjer. I figur 1A, en 100 dager gammel C57BL / 6 mus med muse Mammary Tumor Virus Polyoma Middle T-antigen (MMTV-PyMT) ble ofret på tre måneder. Huden ble åpnet fra området i urinåpningen til halsen med saksen og huden ble låst ned for å gi enkel tilgang til organer som vist i figur 1B. Dette tillot lett tilgang til tumoren som ble oppnådd i figur 1C. Den pr…

Discussion

Denne protokollen er for å isolere primærceller fra en frisk mus tumor. Lysatene fremstilt fra denne fremgangsmåten er nyttige for protein, DNA og RNA-analyse. Kreftceller fra den primære brysttumor ofte metastasere til hjerne, lunge og ben ^en. Inkludert er en teknikk for å ekstrahere lungen for påvisning av metastase. Farging lungen med en blekkflekk vil tillate visualisering av tumorknuter som vist i figur 2B. Fikse lungene i Z-fix-løsning vil gi oss mulighet til å gjøre deler av l…

Materials

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 room temperature
India Ink	Yasutomo		Store at room temperature
AERRANE (Isoflurane)	Baxter	NDC 10019-773-60	Store at room temperature
Ethanol	EMD	AX0441-3	Store at room temperature