I have worked exclusively with triple negative MDA-MB-231. We are thinking about redoing projects using a different TNBC cell line or ER+ line such as BT20 or MCF7, respectively. The xenograft publications I have read are a bit inconsistent, but I gather that BT20 does not grow well. MCF7 appears to establish tumors, but they do not grow as fast/large as MDA-MB-231. What are your experiences? We do scapular/flank injections, not MFP.
Hi Beth
I've grown both MCF7 and 231's in immunodeficient mice. We use the NSGs that Mauro talks about, and although they are purportedly more receptive to xenotransplant, I don't think they differ in the spectrum of cell lines that grow. Thus if you're growing in nude mice the kinetics will likely be slower, but that's all.
For MCF7 I transplant 100K cells in what I colloquially call the side-boob - it's at the fatty extremity of the inguinal fat pad, very close to the flank, but a further up towards the head. The aim is to provide a semi-appropriate niche. (There is evidence that the fat pads that end at the scapula provide a more tumourigenic environment and so I wouldn't worry so much about site of injection). The cells I inject are in 50% growth factor reduced matrigel (BD) in a final volume of 100 uL. I also implant a silastic estrogen pellet (17B E2, 0.72mg/90d slow release pellet, Innovative Research of America) - this is essential as without this the cells won't grow appreciably. (I should also comment that there is mixed opinion on whether you should oviarectomise or not to reduce endogenous E2 levels..I tend not to as the affinity of murine E2 for human ER is very low, coupled with the comparatively much lower circulating E2 levels in mouse compared to human, and the fact that MCF7's in the absence of E2 supplementation do not grow). I measure them over 4-5 weeks using a combination of calipers and bioluminescence. The trick I have found is not to let the tumours grow too big as the necrosis that ensues tends to lead to regions of hypoxia, reduces ER/PR expression and to increased CC3 staining. This will detrimentally affect any downstream analyses. The bioluminescent analyses are particularly helpful in judging this endpoint as the signal will flatten out as necrosis becomes evident (usually at 5 wks in my model).
For MDA-MB-231 I follow a similar protocol, however I omit the E2 pellet. And, to be honest, the kinetics are not that dissimilar. Perhaps I would subtract a week from what I'd expect from MCF7s. However, with 231's, hypoxia is an issue (unless this is your endpoint of course).
Hope this is of some help. Feel free to contact me if you need any further info.
Best of luck with your experiments.
Al
For MDA-MB-231 cells, we use male NUNU mice and for MCF-7 cells we use female NUNU mice. In male mice, MCF-7 cells do form xenografts but the growth rate is much slower compared to MDA-MB-231 cells. I recommend female mice for MCF-7 cells.
Attached below are two well cited publications regarding use of human breast cancer cell lines that grow in nude mice. Luminal cell lines (MCF7, T47D, BT474) don’t grow as well as triple negative highly metastatic cell lines (MDA-MB-231, Sum159PT). In our experience, MDA-MB-231 grows extremely well however when understanding the mechanism with this particular cell line its a little difficult. Many researchers are using Sum159PT, mesenchymal stem-like cell line, grows extremely well in nude mice. The duration of tumors formed will be within 3-4 weeks. These are quick growing tumors. Find attached article that describes these cell lines in more detail. (http://www.jci.org/articles/view/45014)
Best human breast cancer cell lines as model of tumorigenicity in nude mice:
http://www.ncbi.nlm.nih.gov/pubmed/2297709
http://www.ncbi.nlm.nih.gov/pubmed/8738607
About tumor cell: i would recommend a beautiful review ofMina Bissell, from Berkeley, where phenotypes, growth characteristics, tumorigenicity etc. of breast cancer cell lines are clearly reported (Molecular Oncology 2007;1 :84-96).
About immunodeficient mice: new strains have been developed to facilitate tumor cell growth. As an example, NOD scid gamma (NSG) mouse (2007). This mutant mouse combines the features of the NOD/ShiLtJ background (conferring a number of deficiencies in innate immunity), the severe combined immune deficiency mutation (scid) and an IL2 receptor gamma chain deficiency which disables cytokine signalling. As a result, the NSG mice lack mature T cells, B cells, functional NK cells, and are also deficient in cytokine signaling. NSG are then the most immunodeficient mice, engrafting the widest range of human cells and tissues, making it the model of choice for tumor xenografts, stem cell biology and humanized mice, for a lot of investigations, including the activity of human immune cells against cancer.
Male or female: usually male mouse is more aggressive, but the choice male/female depends on what is the rationale of the study. Hormone-dependent cells grow better in an hormone rich environment and female mice environment can be manipulated by hormonal (estrogen, androgen, glucocorticoid.....) or by anti-hormonal implants, both commercially available.
Hi Beth
I've grown both MCF7 and 231's in immunodeficient mice. We use the NSGs that Mauro talks about, and although they are purportedly more receptive to xenotransplant, I don't think they differ in the spectrum of cell lines that grow. Thus if you're growing in nude mice the kinetics will likely be slower, but that's all.
For MCF7 I transplant 100K cells in what I colloquially call the side-boob - it's at the fatty extremity of the inguinal fat pad, very close to the flank, but a further up towards the head. The aim is to provide a semi-appropriate niche. (There is evidence that the fat pads that end at the scapula provide a more tumourigenic environment and so I wouldn't worry so much about site of injection). The cells I inject are in 50% growth factor reduced matrigel (BD) in a final volume of 100 uL. I also implant a silastic estrogen pellet (17B E2, 0.72mg/90d slow release pellet, Innovative Research of America) - this is essential as without this the cells won't grow appreciably. (I should also comment that there is mixed opinion on whether you should oviarectomise or not to reduce endogenous E2 levels..I tend not to as the affinity of murine E2 for human ER is very low, coupled with the comparatively much lower circulating E2 levels in mouse compared to human, and the fact that MCF7's in the absence of E2 supplementation do not grow). I measure them over 4-5 weeks using a combination of calipers and bioluminescence. The trick I have found is not to let the tumours grow too big as the necrosis that ensues tends to lead to regions of hypoxia, reduces ER/PR expression and to increased CC3 staining. This will detrimentally affect any downstream analyses. The bioluminescent analyses are particularly helpful in judging this endpoint as the signal will flatten out as necrosis becomes evident (usually at 5 wks in my model).
For MDA-MB-231 I follow a similar protocol, however I omit the E2 pellet. And, to be honest, the kinetics are not that dissimilar. Perhaps I would subtract a week from what I'd expect from MCF7s. However, with 231's, hypoxia is an issue (unless this is your endpoint of course).
Hope this is of some help. Feel free to contact me if you need any further info.
Best of luck with your experiments.
Al
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