We are attempting to establish long-term xenografts in the mammary fat pads of SCID mice using the T-47D breast cancer cell line. The cell line is estrogen dependent; therefore, mice must be supplemented with exogenous estrogen to allow sufficient primary tumor formation.
In particular, we are having trouble with high mortality rates due to calcium oxalate bladder stones forming after implanting the mice with estradiol pellets (subcutaneous). Ovariectomized SCID mice were pelleted with silastic pellets containing 0.36 mg of 17-beta-estradiol. The pellets were produced in-house as described in the attached paper. We did not utilize commercially available pellets as we have found them to be unreliable. Starting at ~5 weeks, mice began to exhibit weight loss, urine scalding, hunching, and palpable bladders. Eventually this affected the majority of implanted mice. Mice presenting with these symptoms were sac'd and necropsies revealed enlarged bladders containing calcium salt deposits.
We would like to minimize the estrogen side-effects while maximizing sustained tumor growth; however, we would prefer not to venture into detailed dosing studies. Any suggestions from someone experienced in this model would be greatly appreciated. I look forward to your replies.
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Thought I'd given an update on the issue. There are now some low-cost alternatives that have been researched (Article Low Dose, Low Cost Estradiol Pellets Can Support MCF-7 Tumou...
) and these are worth investigating. When we do T-47D xenografts, we vary the estrogen administration based on the goals of the study and possible interactions between estrogen and target therapeutics (http://altogenlabs.com/xenograft-models/breast-cancer-xenograft/t-47d-xenograft-model/). It's worked so far, and so have estrogen pellets.
You could try using estradiol in water, just like is described in this paper.
I couldn't say that the levels you reach will be reliable (it is very variable!) but it may work.
It seems others have seen this as well: Kang, Biol. Pharm. Bull.
32(1) 150—152 (2009). Lowering the dose by half seemed to work for them. Another possibility could be reducing calcium in diet and adding sodium bicarbonate to drinking water.
We occasionally had this issue with our NOD/SCID mice implanted with a pellet containing estrogen. Any mouse unable to urinate as a result of estrogen pellet supplementation had their distended bladders expressed manually. We monitored the mice closely, looking for signs of pain or distress (such as abdominal enlargement of significant change in gait) in animals with the pellets. The bladders were checked and expressed once or twice daily as necessary for mice receiving pellets for longer than 1 month.
An alternative, as mentioned by others, was to reduce the pellet dose significantly.
I would suggest you to increase the number of T47D cells up to 107/mouse, tumor take rate will be -100%. Even though T47D cells are ER+, cell proliferation is not estrogen-dependent. In cell culture, cells grow very well without E supplementation. It's true that E addition can promote cell proliferation and tumor growth in the immune deficient mice. My point is that you don't have to use E pellet but increase cell number only.
I agree with Licun that increasing cell number can help plus the age of the mice, I faced similar problem with our mammary carcinoma model where we faced problems with tumor take rate sometimes and to solve the problem with less efforts we found doing transplantation in 3-4 weeks old mice is better than 6-8 weeks, may be you can try that as well.
Our group has used 1 µg 17β-estradiol valerate in peanut oil (100 ul volume subcutaneous) every fourth day for many years in many xenograft models using NOD/SCID mice. It is longer lasting and no side effects on mice. For tumor establishment I strongly suggest you start at 2-5 million cells per injection and you may want to consider a matrix support like matrigel to encourage establishment
Jacqueline, I would like to know you mean 10 µg/ml 17β-estradiol valerate in peanut oil (100µl per mouse) ?
Hi Jacqueline, Ca you share any of the publications done with the mentor you mentioned above. I searched your profile and couldn't find a papers that follow this method.
If it is a tumor cell line injecting cells in the abdominal cavity would form tumors along the peritoneum to be studied... How long are you waiting for tumors to form? Maybe you are impatient...You certainly need more than 4 weeks to obtain small tumors.
Why to use oophorectomized mice?
How about instead or also using male castrated...then using estrogen you should be able to grow tumors there too...
You seems to want to boost the estrogen...the natural stimulus could be better...as in people, they are not necessarily boosted unless they have hyperestrogenism.
Hi Henry
I'm sure you've found the solution you were after by now, but I thought I'd share our (and others) experience anyway.
We use NSG mice which have slightly higher take rates, however they have the same urinary issue with prolonged E2 supplementation. We originally made our silastic pellets in house at 2 mg 17-beta-estradiol/pellet. To combat the bladder issue we tried halving and quartering the dose (which had limited benefits to the urinary complications) and ended up having to stop our assays at 5 weeks post-implantation.
After discussion with various colleagues (in Manchester, London and abroad) we decided to go down the significantly more expensive route of using commercial pellets (Innovative Research of America) 0.72 mg/pellet (90 day release). Since the switch we haven't had any urinary complications. Be warned, these pellets are extortionate - however we really didn't have a viable alternative.
I have to say, I like Jacqueline's approach as a possible alternative. It's a lot more hands-on, however if it lets you go out beyond 3 months then that's an excellent benefit.
Best of luck.
Al
Thought I'd given an update on the issue. There are now some low-cost alternatives that have been researched (Article Low Dose, Low Cost Estradiol Pellets Can Support MCF-7 Tumou...
) and these are worth investigating. When we do T-47D xenografts, we vary the estrogen administration based on the goals of the study and possible interactions between estrogen and target therapeutics (http://altogenlabs.com/xenograft-models/breast-cancer-xenograft/t-47d-xenograft-model/). It's worked so far, and so have estrogen pellets.- Badges
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