Hi,

Since MCF-7 cells require the estrogen hormone for their growth, Beta-Estradiol pellets are used for the artificial release of this hormone.

You can follow the following protocol for preparation of your xenograft model.

One week prior to cell implant:

1. Anesthetize the mouse and make a small incision on its back.

2. Using a blunt end forcep, raise the skin and make a pouch like structure in the incision site using another blunt end forcep or a trochar.

3. Push the Beta-estradiol pellet in using the trochar and slowly pull the trochar out making sure the pellet doesn't roll out along with it.

4. Seal the incised region with Vetbond (or whatever adhesive you use routinely).

The reason it is preferred to do this procedure a week in advance is to allow sufficient time for the mouse to recover from the estrogen depletion-induced changes in hemodynamics and avoid the effect of endogenous estrogen, which may vary between the animals.

Now, on the day of cell implantation:

1. Grow sufficient amount of cells in a 75mm T-flask.

NOTE: Make sure the flask is not more than 60% confluent. Cells should not be under any form of stress.

2. Trypsinize the cells, collect them in a 15ml tube, count them, and centrifuge them at 1500rpm at 4 deg C for 3 mins.

3. Give two 1X PBS washes at 1500rpm at 4 deg C for 3 mins.

4. Say you wish to implant 1 million cells (generally 1-5 million cells are used for implantation; varies with standardization); collect 1 million cells in 100ul of PBS.

NOTE: Don't exceed volumes greater than 100ul for subcutaneous implantations. As there is a higher chance of cells being spread out and this may cause problems in tumor formation. Although it is better to take additional volume as the syringe may accumulate some dead volume too if you are using a tuberculin syringe. If you are comfortable with injecting lower volumes then you can go as low as 50ul too. I personally prefer injecting 50ul so that the cells are in as close proximity as possible and this allows for better tumor formation.

5. Keep the cells on ice until implantation. Prefer to have the time significantly lower in between the cell collection and cell implantation.

6. Tap the cells properly to have a homogenous mixture.

7. Preferably use a 26G syringe for cell implantation to avoid any damage to the cells when being passed through the gauge of needle.

8. Take about 150-200ul of cells in the syringe and remove any air bubbles.

9. Raise the skin on the flank of the mouse and inject 100ul of the total volume taken in the syringe.

NOTE: Make sure you release the volume slowly so that the cells stay in close proximity.

Monitor the tumor growth with time.

Hope that will help.

If you have any more questions in the given protocol feel free to ask.

Good luck!

Amir

The publication title" Estrogen receptor beta inhibits human breast cancer cell proliferation and tumor formation by causing a G2 cell cycle arrest" published in 2004 on Cancer Research Journal used xenograft studies in nude mice with MCF-7 cells in response to estradiol. I hope this paper serves to you.

They grafted tumor cells under the kidney capsule of nude mouse but I was looking for s.c. model in the flank.

Hi,

Since MCF-7 cells require the estrogen hormone for their growth, Beta-Estradiol pellets are used for the artificial release of this hormone.

You can follow the following protocol for preparation of your xenograft model.

One week prior to cell implant:

1. Anesthetize the mouse and make a small incision on its back.

2. Using a blunt end forcep, raise the skin and make a pouch like structure in the incision site using another blunt end forcep or a trochar.

3. Push the Beta-estradiol pellet in using the trochar and slowly pull the trochar out making sure the pellet doesn't roll out along with it.

4. Seal the incised region with Vetbond (or whatever adhesive you use routinely).

The reason it is preferred to do this procedure a week in advance is to allow sufficient time for the mouse to recover from the estrogen depletion-induced changes in hemodynamics and avoid the effect of endogenous estrogen, which may vary between the animals.

Now, on the day of cell implantation:

1. Grow sufficient amount of cells in a 75mm T-flask.

NOTE: Make sure the flask is not more than 60% confluent. Cells should not be under any form of stress.

2. Trypsinize the cells, collect them in a 15ml tube, count them, and centrifuge them at 1500rpm at 4 deg C for 3 mins.

3. Give two 1X PBS washes at 1500rpm at 4 deg C for 3 mins.

4. Say you wish to implant 1 million cells (generally 1-5 million cells are used for implantation; varies with standardization); collect 1 million cells in 100ul of PBS.

NOTE: Don't exceed volumes greater than 100ul for subcutaneous implantations. As there is a higher chance of cells being spread out and this may cause problems in tumor formation. Although it is better to take additional volume as the syringe may accumulate some dead volume too if you are using a tuberculin syringe. If you are comfortable with injecting lower volumes then you can go as low as 50ul too. I personally prefer injecting 50ul so that the cells are in as close proximity as possible and this allows for better tumor formation.

5. Keep the cells on ice until implantation. Prefer to have the time significantly lower in between the cell collection and cell implantation.

6. Tap the cells properly to have a homogenous mixture.

7. Preferably use a 26G syringe for cell implantation to avoid any damage to the cells when being passed through the gauge of needle.

8. Take about 150-200ul of cells in the syringe and remove any air bubbles.

9. Raise the skin on the flank of the mouse and inject 100ul of the total volume taken in the syringe.

NOTE: Make sure you release the volume slowly so that the cells stay in close proximity.

Monitor the tumor growth with time.

Hope that will help.

If you have any more questions in the given protocol feel free to ask.

Good luck!

Amir

Thank you so much Amirali Bukhari

It all depends on the type of experiment you are carrying out, John. Generally 1.7mg/pellet is used as an optimal dose so I believe it should be ideal for tumor growth and should not cause any side effects or affect the efficacy of your chemotherapeutic drug.

We have been using the same dose in the form of 60 and 90 day release pellets and in combination with chemotherapeutic drugs and we have not faced any issues so far.

I hope this helps.

Hello John,

Apologies for the delayed response. 1.7mg/pellet would facilitate better formations of tumors. Of course it would all ultimately come down to the quality of immunocompromised mice you are using and how susceptible they are to tumor formations. While 0.72mg supplication of estrogen should suffice tumor formation, the growth dynamics may be a little slow given the estrogen dependent nature of the MCF-7 cells.

As far as your second question is concerned, again, depending on the quality of the mice you are using, and the level of estrogen being artificially supplied, it may take somewhere in between 5-7 weeks for tumors to reach a diameter of 200mm3.

Hope this will help.

Amir

Hi John,

I have not tried growing MCF-7 cells in matrigel. Well, in my experience, MCF-7 cells grow in very close proximity (with lots of cell-cell contact) and matrigel is not required for their growth. However, matrigel is essential for the growth of MDA-MB-231 cells due to their migratory nature.

Injecting MCF-7 cells after resuspending them in PBS should be just fine.

As far as your other question is concerned regarding a published article (sorry for the late reply), I will have to look for articles wherein they have followed the procedure and the article from my previous group wherein I did that has not been published. But rest assured of the protocol that I am providing as I have myself optimized these conditions in the best possible manner.

Hope this helps.

Amir

Hello Amir Sir,

I am also working on MCF-7 and Xenograft in nu/nu athymic nude mice,but I  am very new for this all process.I really appreciate your effort for posting the protocol herein,its very helpful for me.

Regarding implantation of estrogen pellet, is it okay to  implant the mice with estradiol pellets subcutaneously.What are the other key points to concern to minimize the estrogen side-effects while maximizing sustained tumor growth.

Thank You

Sincerely

Kamal

Hi Kamal,

You are required to implant the estradiol pellet subcutaneously. The pellets supplicate the mouse with artificial release of the hormone thereby facilitating optimal growth of cell lines that are estrogen dependent, for example, in this case MCF-7. The other breast cancer cell line that is estrogen dependent and tumorigenic in nature is Zr-75-1.

The only thing you need to be careful during the procedure is to keep everything sterilized. Beta-estradiol pellets are supplied in packets by Innovative Research of America so I always prefer opening those packets in a biosafety cabinet and always used sterile forceps to take out individual pellets and a sterile trochar to implant them.

Be sure to clean the trochar with disinfectant and drying it well before placing the pellet on it and inserting it in the mice to avoid any chances of infection from one animal to the other.

Be sure to seal the incision site properly with the tissue adhesive and applying pressure with the forcep to ensure stronger attachment after pellet implantation.

It is a fairly easy to carry out procedure and you shouldn't face any issues ideally.

Good luck. Hope this helps.

- Amir

Hello Amirali,

you seem to be very experienced with these Xenografts, so I would have another question:

Do you know whther there is a difference in E2 release if th epellet is put in the area of the neck or lower dorsal part , close to the limb?

I use NOD SCID mice and put the pellet s.c. on the lower dorsal part, close to the limb at the back. I inject usually 3d later 5 million cells on the other site s.c. (in PBS). And noticed that 90% of the masses reached 50-120 mm3...(10% stopped at 30mm3 and didnt grow further)

but in my CTR group than 3 out of 5 masses grew not anymore or VERY slow , while the others grew much faster. A collaborator uses nude mice and puts the pellet on the neck and they had also the 10% of not growing masses but the rest all grew quite similar.....

I know there were no technical problems, but can't understand why the masses grow so differnetly...

Thanks so much much in advance to whoever can help!

Hello Vanessa,

Apologies for the late response. There should essentially be no difference if the pellets are being injected near the neck region or in the lower dorsal region of the mouse. The reason I prefer (and suggest) to inject them on the shoulder flanks is because it is difficult for the mouse to reach that region with either it's teeth or paws and scratch off the tissue adhesive.

It is very weird that you are getting this uneven growth of tumors especially if you are confident there were no technical issues. Could you please elaborate more on how you did and what you did exactly and I will try to help out to the best of my knowledge and expertise. Could you also please tell me which cell line did you try injecting?

Thanks and sorry once again for the delayed response.

- Amir

Hey,

still thanks a lot for your considerations.

I guess, rereading all posts I had the cells grown too confluent when harvesting the fist time. ..This would explain the slow growth ...but not the unequal as I mixed cells every time before injecting ....but the only weak oint I can immagine.

I started a 2nd experiment now and cells have not been more than 60% I'd say . I can let you know.

The pellet again put at the dorsal flank and I can tell that it's not an issue in terms of mice reaching the wound or the site.

Thanks for still answering to this chat.

@ John Tat , some curiousities (I am using NOD SCID, but still interested )

- how old are your nude mice when putting the pellet and when starting the exp AND how much do they weigh?

- after how many days did your mice devolpe masses ? As I am supposed to use 5x10^6 and was wondering whther to go down with the quantity....

- did you us eAMtrigel ?

Just to let u know, my mass development is much faster now, so most pobably my cells have been to confluent the fist time. But the variation I have is still big.

I also had physiological problems with the mice, almost 20% died within the 2-6 weeks after cell injection and I think it might be the pellet, so going down now with the concentration....

Hmmm, ok, thanks,

I inject 5x10^6 but tried also once 3x10^6 and after 10 days in both cases, I started getting palpable masses.I started my experiment after 14/15 days, as then all mice developed palpable masses, some were already 120/130mm3.

But I observe then, that maybe 20% grow until approx 70m3 and then stopp......

Confluency at harvesting time point is crucial. They should not be over 60/70% ...and try to be quick, lets say 1-2h from trypsinising til injection.

I grow them usually in DMEM 4.5g/L Glucose incl. Glgutamax , 10% FCS S.A, 1% NEAA,

To John Tat: 

what you've heard about Matrigel is not exactly right. It is easy to use, just have to follow the instructions which come with the bottle. Matrigel is a perfect matrix , basically keeping cells in 3D, while providing them also with growth factors.

MCF7 cells should not have 100% penetrance. If you want all of the mice to bear tumors in this case there are different, more malignant cell lines.

Mortality comes from inadequately high estrogenic background, which promotes urinary tract inflammation and sometimes even uterus prolapse.

Hello John and Venessa,

Late again on this post but I will try to address most of the concerns raised here:

John, 1 x 106 MCF7 cells is still a very small number given the fact you are using nude mice for your experiments. It is important to remember that nude mice lack thymus and therefore they greatly reduced T cell count. Having said that, it is crucial to remember that these mice have B cells, NK cells, and normally functioning macrophages. In addition to this, they may also develop extrathymic T cell function with age. Therefore you would expect that not all your mice injected with 1 x 106 MCF7 cells would form tumors (mouse to mouse variation is a very common problem). If you do plan on continuing to use nude mice, it would indeed be wise to inject at least 5 x 106 MCF7 cells.

Regarding Matrigel, in addition to what Veronika already mentioned, matrigel is relatively easy to work with as long as you keep it on ice until you have to inject cells. When injecting cells, what I find most helpful is injecting lower volumes (50ul total), that is mixed with matrigel in a 1:1 ratio (25ul matrigel : 25ul cell suspension in PBS).

Vanessa, the time it takes for tumors to form in your mice would vary drastically to that of the time it takes John's mice to develop tumors given the fact that you are using NOD/SCID mice for your experiments. The 20% mice that died in your case, did you carry out their postmortem to determine the cause of death? I was told by a veterinarian once in our facility that estrogen pellets may sometime cause adverse side effect in NOD/SCID mice eventually leading them to die. I am not sure how that works though so I can't comment further on that.

Hope this helps.