View source for Tumour cell implant into mammary fat pad

In order to carry out studies involving mammary tumours, cells or a fragment of a tumour cell line may be implanted in the mammary fat pad of female mice. The traditionally-used technique involves anaesthetising the mouse and injecting or implanting the tumour cells via a surgical approach. This method describes injection of the tumour cells percutaneously, therefore not requiring surgery. [Tumour fragments still require a surgical procedure to implant.] 

The mammary glands of the mouse appear as thick 'fat pads' and lie just underneath the skin of the abdomen and thorax. They're easily visible in a nude (hairless) mouse but can also be seen easily if the mouse is gently clipped to remove the hair in the relevant area. Some groups implant tumours into the thoracic mammary gland, as the resulting mass appears to cause less hinderance to the mouse as it develops but the technique is equally applicable to implant into either inguinal or thoracic gland.

The mouse is health-checked, then anaesthetised using gaseous anaesthesia, such as isofluorane in oxygen, and placed on a thermostatic heated pad to prevent chilling during the procedure. The hair over the relevant gland is gently removed using small clippers, taking care to avoid the nipples. Cells are prepared as usual (e.g. in media or Matrigel) and kept cool until injection. A suitably sized syringe (e.g. 500mcL) and needle (25g or smaller) is used for the injection. Injection volumes are usually less than 100mcL.

For cell injection into the "3rd" (thoracic) gland, the tip of the needle is inserted just caudal and lateral to the nipple and the needle directed laterally to follow the contour of the mammary gland around the chest wall. Cells are gently injected and the needle withdrawn.

For injection into one of the inguinal glands (eg "4th" gland) the mammary gland is identified and gently held between the fingers, or with forceps, then the needle is inserted through the skin caudal to the nipple and the injection given into the gland. 

The procedure takes only a few seconds and the mouse is allowed to recover in a warmed cage and returned to its home cage and cage-mates.

For some mice it may be possible to inject cells into the inguinal mammary gland using only manual restraint (particularly in nude mice, where the glands are very easy to see). This requires a more proficient and confident operator.

Training hint: when starting out, it is helpful to inject a dye, such as Vital Blue into the mammary glands of cadaver (dead) mice, so that the placement of the injection is evident and gives instant feedback to the trainee. Both thoracic and inguinal glands can be injected on the same cadaver, to maximise learning opportunities.