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

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.

Targeted gene silencing of CCL2 inhibits triple negative breast cancer progression by blocking cancer stem cell renewal and M2 macrophage recruitment

“…To recapitulate formation of TNBC with a humanized stroma, MDA-MB-231 cells were co-transplanted with human carcinoma associated fibroblasts in collagen and transplanted in the inguinal mammary glands of nude mice [40, 41]. When tumors grew to approximately 0.5 cm in diameter, the xenografts received injections every three days with Ca-TAT complexed to control siRNA, huCCL2si1 or huCCL2si2, for a total of three injections per experimental group.…”

“…All animal experiments were performed at the University of Kansas Medical Center under an approved IACUC. 250,000 human carcinoma associated fibroblasts were embedded with 100,000 MDA-MB-231 cells in 50 μl of collagen rat tail collagen I (BD Pharmingen), using methods previously described [40]. One collagen plugs was transplanted into each mouse, in the #4-5 inguinal mammary glands.…”

CCR2 Chemokine Receptors Enhance Growth and Cell-Cycle Progression of Breast Cancer Cells through SRC and PKC Activation

“…HCC1937 cells were cultured in RPMI/10% FBS/2 mmol/L L-glutamate/1% penicillin-streptomycin. Human cancer-associated fibroblasts and normal adjacent fibroblasts were isolated from invasive breast carcinoma tissues obtained from University of Kansas Medical Center Biospecimen Repository, using approaches described (18). For CCL2 knockout, fibroblasts were first immortalized by transfection of hTert, using approaches described (19).…”

“…For CCL2 knockout, fibroblasts were first immortalized by transfection of hTert, using approaches described (19). Fibroblasts were isolated from MMTV-PyVmT transgenic mice or tumor-free mice (C57BL/6xFVB) as described (15,18). Cell lines were cultured less than 3 months at a time and checked for mycoplasma after freeze/thaw, using the Mycoplasma Detection Kit (Lonza; cat# LT07-703).…”

“…Female athymic nude mice (FoxN1 nu/nu ) ages 6 to 8 weeks were purchased from Charles River Laboratories. Breast cancer cells and fibroblasts were transplanted as described (18). Briefly, 100,000 cancer cells and 250,000 fibroblasts were seeded into 50 mL Rat Tail Collagen type I overnight.…”