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.
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.
1. Isolation and extraction of donor collagen from C57BL/6J mice
2. Isolation and culture of donor mammary carcinoma cells and fibroblasts from normal and PyVmT C57BL/6J mice
3. Immunofluorescence staining of cultured cells
4. Preparation of collagen embedded cells for grafting
5. Orthoptic transplantation of collagen embedded cells in C57BL/6J mice
6. Representative Results:
Isolation and extraction of collagen from C57BL/6J mice
These procedures were adapted from1. Extraction of collagen protein from 5-7 mouse tails yields approximately 1-1.5 mg/ml in a 6 ml final volume, or 6- 9 mg of protein. By coommassie stain, bands corresponding to 90 kda and 130 kda are detected in the lanes loaded with samples extracted from mouse tails, indicating the presence of collagen type I and pro-collagen respectively (Figure 1).
Isolation and culture of mammary carcinoma cells and fibroblasts from normal and PyVmT C57BL/6J mice.
The procedures were adapted from2. PyVmT carcinoma cells and fibroblasts can be distinguished by differences in cell morphology and expression of specific epithelial and mesenchymal markers. PyVmT carcinoma cells are identified by cobblestone shape and co-express CK18, a luminal epithelial marker and CK14, a basal epithelial marker, but not express α-sma (Figure 2). Mammary fibroblasts are larger cells with a spindle shaped phenotype and express high levels of α-sma but do not express CK14 or CK 18 (Figure 2). These data indicate over 95% cell purity for fibroblasts and epithelial cells using the outlined procedures.
Orthoptic transplantation of collagen embedded cells in C57BL/6J mice
These procedures were adapted from3, 4. Transplantation recipient mice are sacrificed when tumors in either experimental group reach 1.0 cm in diameter, or approximately 60 days. While transplantation of PyVmT cells alone results in palpable tumors after 30- 40 days, reaching a mean tumor mass of 0.335 grams at 60 days, co-transplantation of PyVmT carcinoma cells with mammary fibroblasts results in a mean tumor mass of 0.630 grams, indicating enhancement of tumor growth by fibroblasts (Figure 3).
Figure 1. Coomassie stain analysis of collagen type I extraction from mouse tails. BSA (˜ 66 kda) is indicated by arrow. Commercial rat tail collagen (20 μg protein), and purified mouse tail collagen (20 μg protein) are indicated by box (˜130 kda, ˜ 90 kda proteins). Std= molecular weight standard.
Figure 2. Immunofluorescence staining of donor PyVmT mammary carcinoma cells and fibroblasts. Panels a and b represent PyVmT mammary carcinoma cells immunostained for antibodies to CK14 and CK18. Panel c represents fibroblasts stained with antibodies to α-sma. Images shown with DAPI overlay at 20x magnification.
Figure 3. Mammary tumor development in C57BL/6J mice in the presence or absence of fibroblasts. Mammary tumors were harvested from mice transplanted with PyVmT carcinoma cells in the presence or absence of mammary fibroblasts and weighed. Mean+standard error of the mean. N=6 per group.
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 play critical roles in mediating anti-tumor immunity through recognition of tumor specific antigens and subsequent targeting of tumor cells, inhibiting metastatic spread 6. Furthermore, recent studies have shown that fibroblasts are important regulators of immune cell recruitment 7, 8, indicating that development of new approaches to study stromal: epithelial cell interactions are necessary in order to more clearly understand the mechanisms of mammary tumor progression. The outlined procedures demonstrate a reliable method to: isolate and culture mammary epithelial and mesenchymal cells and orthotopically transplant these cells in immunocompetent mice to form mammary tumors. Using the outlined procedures, we have grafted over 40 mice using different experimental groups and different endpoints with over a 97 % successful transplant rate, demonstrating the reliability of this system. The effects of mammary fibroblasts on tumor growth in this system are consistent with previously published studies involving subrenal capsule grafting of fibroblasts with mammary carcinoma cells 9. We have found few concerns with the system in this report. If the concentration of the resulting stock is too low for embedding cells, one may further concentrate the collagen to a lower volume or isolate more collagen from additional tails. To ensure consistent tumor formation, it is important to disperse the cells evenly in the collagen plug throughout the collagen by pipetting and avoid the presence of bubbles in the collagen plug.
The low background of tumor formation in C57BL/6J mice 10, 11 allows us to investigate the functional contribution of specific oncogenes or inactivation of tumor suppressors in stromal: epithelial interactions during mammary tumor progression. Expression of specific oncogenes and tumor suppressors can be modified in stromal cells or epithelial cells through stable expression of siRNAs for example, prior to transplantation. Because primary cells senesce with continual passaging, genetic manipulation of cultured cells is easier if the cells are immortalized, for example through spontaneous immortalization or expression of an oncogene 12, 13, 14, 15. Immortalization of different stromal cell types has been achieved with macrophages and endothelial cells 16, 17, 18. Immunofluorescence staining for specific stromal and epithelial markers revealed highly purified populations of fibroblasts and epithelial cells isolated in these studies. However, the identification of contaminating cell populations may necessitate the use of flow sorting to further purify the desired cell populations. As an alternative to immortalization and genetic manipulation in culture, investigators have also isolated specific cell types from transgenic mice through flow sorting and directly transplanted these cells into mice for the study of different tissues, including the mammary gland 19, 20. This approach This approach yields physiologically relevant primary cells, but requires isolation of cells from mice each time. However, the supply of cells and purity of cell populations are dependent on the abundance of the cell type and on the numbers of mice available. These challenges may be overcome with transplantation of tumor cells in mice carrying fluorescent reporter such as actin-GFP mice 21. In this system, one would be able to investigate the effects of the overall stroma on tumor progression, but not distinguish the contribution of specific stromal cells. Each of the approaches comes with specific advantages and disadvantages, and may be combined with the system described in this report, according to the investigators’ needs. In addition, the described system can be adapted or modified for transplantation in different genetic backgrounds, transplantation of different stromal cell types and mammary epithelial cells at varying states of malignancy to determine how these different variables affect stromal: epithelial interactions and subsequent tumor progression.
The authors have nothing to disclose.
This project was funded through by NIH/NCI grant number R00 CA127357 and University of Kansas Cancer Center Endowment.
Name of the reagent | Company | Catalogue number | Comments (optional) |
---|---|---|---|
C57BL/6N mice | Harlan | N/A | |
MMTV-PyVmT transgenic mice | Jackson laboratories | 002374 | |
Fetal Bovine Serum | Fisher | SH3039603PR | |
DMEM | VWR | 10000113873 | |
Penicillin/streptomycin | Fisher | MT-30-001 | |
amphotericin | fisher | BP2645-20 | |
Amicon filtration columns ultracel 50k | Millipore | UFC905008 | |
Tubes for Beckman TI rotor | Beckman | 355618 | |
Rat tail collagen | Fisher | CB 40236 | |
10x EBSS | Sigma Aldrich | E7510-100ML | |
Trypsin 1X, 0.25% in HBSS w/o Calcium and Magnesium | Fisher | MT-25-050-CI | |
Glacial acetic acid | Fisher | A491-212 | |
Coomasie blue | Fisher | BP101 25 | |
Trypsin | Sigma Aldrich | T3924-100ml | |
Collagenase A | Sigma AldrichC0130-50 | ||
hyalronidase | Sigma Aldrich | H3884 | |
DNase | Sigma Aldrich | D5025 | |
Kaleidoscope Protein standard | Biorad | 1610375 | |
Glass slides | Fisher | 12545-78 | |
Glass coverslips | VWR | 101400-042 | |
Vimentin antibody S-20 | Santa Cruz Biotechnology | SC-7558 | |
α-smooth muscle actin antibody | Abcam | ab5694 | |
CK14 antibody | Santa Cruz Biotechnology | 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 | NC9326254 | |
Glass Pasteur pipet | Fisher | 22-042-815 | |
Ethanol | Fisher | A406P 4 | |
betadine | fisher | NC9386574 | |
Wound clips | Fisher | 12032-07 | |
Wound staple | Fisher | 12031-07 |