there are many reasons.

1) 100 mm3 is a small tumor of about 3x3 mm and it could as well be local reaction to the injected cells/liquid, but no tumor take. so, no surprise if it lasts for a while in the nude mouse and then disappears and never grows out.

2) Also, it is common in xenograft (even those of very agressive lines) to see tumors that grow very slowly or dont grow at all. it might be because few cells survived the inection and found a "hostile" microenvironment and never produced enough vegf (to mention one). For example, if you inject right under the epidermis it is different than injecting deeper.

2a) some ovarian cancer cell lines for example, grow poorly when injected under the skin, but rapidly metastasize all over if injected intraperitoneal.

3) hormone dependency.I think pc3 are know to be hormone independent (although you never know how YOUR cells have become after passaging in you lab for years...) but the sex of the recipient mouse might affect tumor take and growth.

4) number of injected cells. keep in mind many of the cells you inject die, so if your injection falls below a critical number you might not get anything. see point (1)

in this case, you might want to try inject a lot (in the range of millions): you will get tumors that are more uniform in size and grow faster; it could be ok if you want to do a drug treatment.

Hi, I have used this prostate cell line for xenograft experiments. I used always male Nude/scid mice and I have always a good rate of tumour growth, probably because these cells need to growth that are specific hormons. What kind of injections do you practice?

Nude mice retain a fair amount of immune system function outside of B and T cells.

Give scid/beige or nod/scid/il2r-KO mice a shot

We havealso in some case regression of tumors, wich another kind of cell lines (glioma cell), but it was not always (subcutaneous injection ) I agree with ML about a residual immune system

As your nudes get older the possibility of tumor regression increased just because the inmune system is getting stronger. Start the experiment with 5-6 week-old mice.

Nude mice have NK [natural killer] cells that kill tumor cells; they use a completely different recognition system known as KIR as opposed to cytotoxic T lymphocytes, which recognize cells by MHC [major histocompatibility complex] proteins.

The beige mutation inhibits NK function, so scid/beige or bnx [beige-nude-xid] mice should permit your tumor to grow, but as noted above, you probably should use male mice, not female mice, because the tumor likely needs testosterone for growth. The NOD/scid/IL2R KO mice also lack NK function, because NK cells use IL-2 as a growth factor.

there are many reasons.

1) 100 mm3 is a small tumor of about 3x3 mm and it could as well be local reaction to the injected cells/liquid, but no tumor take. so, no surprise if it lasts for a while in the nude mouse and then disappears and never grows out.

2) Also, it is common in xenograft (even those of very agressive lines) to see tumors that grow very slowly or dont grow at all. it might be because few cells survived the inection and found a "hostile" microenvironment and never produced enough vegf (to mention one). For example, if you inject right under the epidermis it is different than injecting deeper.

2a) some ovarian cancer cell lines for example, grow poorly when injected under the skin, but rapidly metastasize all over if injected intraperitoneal.

3) hormone dependency.I think pc3 are know to be hormone independent (although you never know how YOUR cells have become after passaging in you lab for years...) but the sex of the recipient mouse might affect tumor take and growth.

4) number of injected cells. keep in mind many of the cells you inject die, so if your injection falls below a critical number you might not get anything. see point (1)

in this case, you might want to try inject a lot (in the range of millions): you will get tumors that are more uniform in size and grow faster; it could be ok if you want to do a drug treatment.

Was the bolus of cells inoculated with matrigel? If not that could be an issue. Try inoculating cells in matrigel.

I agree with Lorenzo, and as assure ur techniques of injection or etc etc is correct please also if pssible to have kind of positive control ..... So at least u hv a better idea why the inoculation is not working or so on .....

For the female nude mice, im not sure wheather it work or not ... Although there is many theories been mention here, but why not try on it and if it work then we hv to find a novel answer ....

I agree tieh Lorenzo, too. Inject more cells or cells and Matrigel mixture should be inproved.

hello,i think firstly you should check the PC-3cell line being pure.

Thank u very much to all of you for giving such a nice suggestions...Special thanks to LB,GG,GC.i would like to add about technique i m using is subcutaneous injection of PC-3 into serum free medium in 6-8 weeks old CD1 Male Nude mice.

In CD1 nude mice, Does Tumorigenicity decrease after few generations of inbreeding i mean wheather they can regain their immunity after regular breeding as they have their Precursor T cells, B cells,functional NKs and macrophages?

I'm not sure what you mean by interbreeding - are you crossing the CD1 nudes, or are you breeding them to non-nudes? As I recall, breeding nudes is not trivial, but the specifics elude me at the moment.

So long as the mice are nudes, they still will lack T cells because they are athymic. That being said, as nudes age - over a year old - they do develop limited T cell immunity - the so-called peripheral T cell pathway. Those T cells tend to be of limited diversity - possibly clonal - so they react to a limited number of antigens.

To GG, thank u GG...i mean to say inbreeding of Male Nude homozygous CD1 with female Nude heterozygous CD1.

That would be the proper method.

Since the mothers are heterozygous nudes, they are immune competent, and will pass antibodies [mostly IgG] and a few lymphocytes to the pups via the placenta, and some antibodies [mostly IgA] via milk. Most of the antibodies are antibacterial, and I doubt they would interfere with your tumor implants.

I suspect NK is the relevant issue for you. There are some anti-NK monoclonals available, as well as anti-asialoGM1 antiserum that abrogate NK activity. Alternately, scid-beige or NOD/scid/IL2R KO would get you there.

Good luck!

For what it's worth, I had the same problem in these mice when I was trying to grow my breast cancer xenografts. These mice are known to have extra-thymic T cell development, which is pronounced when they age. I found this to be true in mice around the same age as yours since splenocytes were activated in response to anti-CD3. I got the best growth in NOD SCID mice, which are also lacking NK cells. Sometimes it just takes a long time for tumours to grow - my breast cancer line took 21 days for palpable tumours to grow (30 mm^3) in NOD SCIDs. I also used sf medium. You'll get better tumour take if you use early passage cells (this makes a big difference) and only inject a few mice at a time (viability does not mean tumourigenicity, and the latter is lost quickly when the cells are left in a syringe). Hope this helps! Good luck!

We have a lot of experience in growing PC-3 cells subcutaneously in nu/nu males. It is very reproducible. The grow is certainly dose dependant. We find that injection of 2-4 million cells SC will induce palpable tumors after about 21 days which then grow linearly for about another month by which time tumors tumors can reach 2.5cm^3. (This is as long as we are allowed to keep them!) In the beginning of this project we did inject female mice as well, but no tumor grew.

With regards the regression, I agree with the previous comments that 100mm3 reactions may well not be tumor at all, but rather local inflammation. It should also be remembered that in all these so-called "immunodeficient" mice there is some small and variable amount of "immune leakage" so there may be an immune reaction against the graft. We have not worked with CD1 mice but this may explain at least some of the regression you are seeing.

Hi,

I have been inoculating 1-5 million cells of HK1-rfp line in NSG mice and have encountered similar probs of shrinkage of tumor, tumor disappears after a while or some tumors simply have an odd growth curve.

Some are human errors but sometimes biological factors plays a part influences the outcomes of these experiments. The common causes of tumor shrinkage in my lab is due to infections that originates from primary xenografts of patient samples.

PC-3 xenografts do not recruit new blood vessels (neovasculature) very well. If you compare PC-3 to C4-2 or LNCaP xenografts, you'll notice the C4-2 and LNCaP are dark, red, and vascularized. PC-3 xenografts don't look this way. Rather, they form small, confined, white/clear tumors in mice, lacking new vasculature. I am unsure of the molecular reasons for this, but I have extensive experience producing human prostate cancer xenografts in nude mice. My thoughts are that it is a lack of ability to bring in a blood supply and nutrients. If you are not already doing so, you should inject the PC-3 cells with Matrigel (extracellular matrix) that helps to produce more stable xenografts.

As Robert mentioned Matrigel will facilitate tumor take in nude mice. For our LNCaP xenograft study, we suspended 1X10^6 cells in 50ul of Reduced Growth Factor-Matrigel +50ul serum-free media. LNCaP are slow growing compared to some of the more aggressive prostate cancer cells lines so we did not detect palpable tumors until a month into the study. Also, we injected RFP or GRP labeled LNCaP cells so that we could image tumor cells using IVIS imaging. This helped us determine when cells were present.

There are a few issues with PC-3 xenografts, but the general procedure is sufficiently simple (see an example here: http://altogenlabs.com/xenograft-models/prostate-cancer-xenograft/pc-3-xenograft-model/). Matrigel is necessary, but the main issue is likely with the mouse strain. As others have mentioned, CD-1 is not the best one to use, and other nude mice strains are going to be better suited for tumor growth (such as nu/nu mice).