Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

Summary

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

Abstract

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

Protocol

1. Isolation and extraction of donor collagen from C57BL/6J mice

1. Sacrifice mature normal female C57BL/6J mice using approved IACUC methods.
2. Harvest the tails and soak in 70% ethanol for 45 minutes to sterilize tissues. Dry the tails with tissue paper, wrap in aluminum foil. Store the tails in -20°C until needed.
3. Place the tails in a sterile environment such as a laminar flow hood. Using scissors, split the skin at the tail root and peel away from the tail. Remove 0.5 to 1 cm from both ends of the tail and divide the remaining tail up into three or four pieces. Dissect the tendons from the tails and separate the tendons into indiv....

Discussion

The functional contribution of fibroblasts in tumor progression has been demonstrated through transplantation models, in which carcinoma associated fibroblasts co-transplanted with benign mammary epithelial cells results in increased tumor growth and invasiveness⁵. Conventional transplantation approaches have involved the use of SCID or nude mice to co-transplant stromal and epithelial cells from different genetic mouse backgrounds or different species. Immunocompromised mice lack functional T cells, which pl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6N mice	Harlan Laboratories	N/A
MMTV-PyVmT transgenic mice	Jackson Laboratory	002374
Fetal Bovine Serum	Fisher Scientific	SH3039603PR
DMEM	VWR international	10000113873
Penicillin/streptomycin	Fisher Scientific	MT-30-001
amphotericin	Fisher Scientific	BP2645-20
Amicon filtration columns ultracel 50k	EMD Millipore	UFC905008
Tubes for Beckman TI rotor	Beckman Coulter Inc.	355618
Rat tail collagen	Fisher Scientific	CB 40236
10x EBSS	Sigma-Aldrich	E7510-100ML
Trypsin 1X, 0.25% in HBSS w/o Calcium and Magnesium	Fisher Scientific	MT-25-050-CI
Glacial acetic acid	Fisher Scientific	A491-212
Coomasie blue	Fisher Scientific	BP101 25
Trypsin	Sigma-Aldrich	T3924-100ml
Collagenase A	Sigma-Aldrich
hyalronidase	Sigma-Aldrich	H3884
DNase	Sigma-Aldrich	D5025
Kaleidoscope Protein standard	Bio-Rad	1610375
Glass slides	Fisher Scientific	12545-78
Glass coverslips	VWR international	101400-042
Vimentin antibody S-20	Santa Cruz Biotechnology, Inc.	SC-7558
α-smooth muscle actin antibody	Abcam	ab5694
CK14 antibody	Santa Cruz Biotechnology, Inc.	sc-53253
CK18 antibody	Abcam	ab668
DAPI	Sigma-Aldrich	D9542
Anti-mouse biotinylated	Vector Laboratories	BA9200	Distributed through Fisher
Anti-mouse-alexa-568	Invitrogen	A10037
Anti-mouse- alexa-488	Invitrogen	A11001
Streptavidin- alexa-488	Invitrogen	S11226
DAPI	Invitrogen	D21490
Prolong antifade	Invitrogen	P-36930
Surgical scissors	Fine Science Tools	91400-12
Fine spring scissors	Fine Science Tools	15000-02
Blunt forceps	Fine Science Tools	11002-12
# 5 fine forceps	Fine Science Tools	11251-10
Gut chromic suture	Fisher Scientific	NC9326254
Glass Pasteur pipet	Fisher Scientific	22-042-815
Ethanol	Fisher Scientific	A406P 4
betadine	Fisher Scientific	NC9386574
Wound clips	Fisher Scientific	12032-07
Wound staple	Fisher Scientific	12031-07