Indeed 4T1 is an aggressively growing tumor. Mice generally succumb to metastasis or asphyxiation arising from the increased numbers of circulating white blood cells. That said, we have observed that primary 4T1 tumors grow faster and tend to be less metastatic, than clonal derivatives. Our bone metastasizing variant (4T1.2) does not reach 1500mm3 (our ethical cull point) as a primary tumor within 30 days, yet has extensive metastatic disease. At a molecular level, we observe necrosis in the centre of the tumor, largely due to an incomplete vasculature. It is a region that is poorly perfused, and as Clemens stated, induces necrosis due to hypoxia/anoxia.

Things I would consider for your experiments:

1) Never let your tumors grow beyond 2cm; it is ethically irresponsible, causing much distress to the mice, including skin rupture like you describe.

2) Inject tumors in the 4th inguinal mammary fat pad. This region, not only being orthotopic and more biologically relevant, does not encumber the mouse. Especially compared to tumors growing around the neck.

3) Inject fewer cells. We routinely inject 50000 cells in 20ul of PBS/Matrigel solution into the mammary gland, and get 100% tumor take. Less cells, will allow you to grow tumors for longer prior to reaching an ethical cull point. Time is also a factor for the development of spontaneous metastatic disease in this model.

4) Try deriving single cell clones from the 4T1 parental cell line. It is a heterogenous cell line, you will discover variants within this population that will grow slow and metastasize better.

Dear Sajid

4T1 is a very agressively growing tumor that we never grow beyond 6 weeks since at that time the tumor has metastasized to lungs and other sites. The mice will get in a bad shape and according to our guidlines we have to euthenize them. After 2-3 weeks we indeed see the first signs of necrosis due to the rapid growth and hyoxia/anoxia in the center.

Indeed 4T1 is an aggressively growing tumor. Mice generally succumb to metastasis or asphyxiation arising from the increased numbers of circulating white blood cells. That said, we have observed that primary 4T1 tumors grow faster and tend to be less metastatic, than clonal derivatives. Our bone metastasizing variant (4T1.2) does not reach 1500mm3 (our ethical cull point) as a primary tumor within 30 days, yet has extensive metastatic disease. At a molecular level, we observe necrosis in the centre of the tumor, largely due to an incomplete vasculature. It is a region that is poorly perfused, and as Clemens stated, induces necrosis due to hypoxia/anoxia.

Things I would consider for your experiments:

1) Never let your tumors grow beyond 2cm; it is ethically irresponsible, causing much distress to the mice, including skin rupture like you describe.

2) Inject tumors in the 4th inguinal mammary fat pad. This region, not only being orthotopic and more biologically relevant, does not encumber the mouse. Especially compared to tumors growing around the neck.

3) Inject fewer cells. We routinely inject 50000 cells in 20ul of PBS/Matrigel solution into the mammary gland, and get 100% tumor take. Less cells, will allow you to grow tumors for longer prior to reaching an ethical cull point. Time is also a factor for the development of spontaneous metastatic disease in this model.

4) Try deriving single cell clones from the 4T1 parental cell line. It is a heterogenous cell line, you will discover variants within this population that will grow slow and metastasize better.

Sajid,

I think that there might be no problems with your 4T1 cells. Its is unethical to let grow such an aggressive tumor far from the cull point. Mice with a 4t1 tumor load beyond 1.5 cc become very stressed or moribund.

Please find attached a copy of the chapter about 4t1 cells model in Current Protocols in Immunology.

Many syngeneic mouse models including 4T1 grow faster than the mouse can supply blood vasculature. What you are observing is most likely the tumor collapsing upon itself due to very necrotic centers from insufficient nutrients and oxygen. (The center of tumor is most likely fluid, blood or mushy tissue). The black you observed is an ulceration due to the aggressive and inflammatory nature of the tumor. These are very painful for the mouse and should be euthanized immediately after an opening is observed.

I have worked with 4T1 cells and did with Xenograft. the best way to induce tumour growth and metastasis is to inject orthotopically, into the mammary fat pad, of SCID mice. The tumours develop quickly and do metastasise. If you follow harvesting with nested PCR to determine metastasis. I found metases in both lung and liver tissues, of some mice, not them all.

Re your ulceration status with sub cutaneous injection. With in UK Home office standards, the mice must be sacrificed if ulceration develops. Tumour size and ulceration status must be monitored daily. Due to rapid growth of these sub cutaneous tumours, it is very common for the tumours to ulcerate.

Re cells: Always ensure the cells that are being used, are tested and free from Mycoplasma, prior to injection.

Initial Cell number is also crucial for the size of the tumor and mortality of the mice

It seems 10^6 cells is high number and large tumors will develop with in short period of time. if you try 10000 to 20000 cells may be you may get tumors with our necrosis within 2-3 weeks period.

If I inject 0.25 million cells in fourth mammary fat pad, in how many days I would be able to see the tumor?

Hi Ana,

Thank you for the response.