Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

The overall goal of this procedure is to investigate breast cell progression after tumor cell engraftment in the mouse memory fat pad. This is accomplished by injecting breast cancer cells into the memory fat pad of an adult mouse, and then measuring the tumor volume with a caliper at predetermined time levels. At the end of the experiment, the tumor mass and volume were determined and the blood and lungs of the animal are harvested for further analysis.

Ultimately, macro and micro metastases can be quantified by visual and immunohistochemical analysis respectively. The main advantage of this technique over existing methods like subcutaneous tumor cell injection, is that the mammary fat pad injection site mimics the original site of breast tumor formation, yielding more dependable results. This method can help answer key questions in the breast cancer field, such as which tumor, suppressors, oncogenes, or stromal compartment components are involved in cancer progression.

A day before the procedure shave the N-O-D-S-C-I-D gamma mice from the fourth nipple to the midline and weigh them to verify that all the animals have roughly comparable weights. The next day, wash a human breast adenocarcinoma cell culture once with PBS and then tryps anize the cells when most of the cells have detached. Quench the reaction with 10 milliliters of serum containing DMEM media and then spin down the cells for seven minutes at 1, 200 RPMs at room temperature, wash the pellet to remove any trace of serum, and then resuspend the second pellet in PBS.

After counting, aliquot the cells in 500, 000 per 150 microliter concentrations into sterile micro centrifuge tubes on ice. Then fill one 50 milliliter conical tube with 35 milliliters of PBS and another with 70%ethanol and soak cotton swabs in each. When all of the tools are ready for each animal, apply ophthalmic ointment to the animal's eyes.

Then use tape to gently restrain an anesthetize mouse on a heating pad and confirm sedation by toe pinch. Use an ethanol soaked cotton swab to clean the shaved area and then use a scalpel blade to make a small incision between the fourth nipple and the midline. Insert the PBS soaked cotton swab into the incision to make a pocket and then use tweezers to expose a memory fat pad, which can be detected by its white color.

Use another pair of tweezers to squeeze the fat pad at its base and fully expose the tissue. Then pipette the adenocarcinoma cells up and down a few times to make a homogenous cell solution and gently aspirate 150 microliters of cells into an insulin syringe. Taking care to hold a syringe horizontally with the needle hole facing up.

Inject the cells into the mammary fat pad confirming a successful injection by swelling of the tissue. Then release the fat pad gently and use mosquito forceps to suture the incision, turning the suture around the mosquito forceps in a clockwise, anti-clockwise and clockwise direction to make three knots. After the surgery, inject an analgesic and place each animal in a sternal recumbent position in an individual cage, monitoring the mice until they're fully recovered.

Eight weeks after the cell implantation, mice are prepared for organ harvest. Fix the animal to a foam base and use calipers to measure the tumor volume. Next, make a long vertical midline incision and then make two horizontal incisions right below the front leg and above the rear leg.

Pin the skin to the foam to expose the tumor, and then use scissors to dissociate the tumor from the skin. Then gently remove the lungs, placing the left lung into Bowen's solution for three days after which this superficial metastatic folky will become clearly visible to the naked eye snap freeze part of the tissue in liquid nitrogen for later RNA isolation and place the remaining part of the tumor into one of the conical tubes filled with formin. Next, after sacrifice of the animal by cardiac puncture, dispensed the 450 microliter blood sample into a micro centrifuge tube containing 50 microliters of 3.2%sodium citrate, then spin down the red blood cells and store the plasma samples at negative 20 degrees Celsius until further use experimental errors such as an imprecise inoculation of the cells or leakage may lead to variations in tumor size or even the absence of a tumor leading to the formation of a structure that appears similar to memory.

Fat pad injected with control buffer. The growth rate of the tumor is also dependent on the nature of the injected cell line, and in general can be observed through the skin of the mouse. In addition, the area around the tumor may display large vessels that arise from vascular remodeling and that may play crucial roles in the removal of waste products and supplying the tumor tissue with nutrients and oxygen.

Unlike in vitro experiments, the growth rate of the tumor cells may not be constant in time in vivo due to hypoxia and a lack of nutrients. Necrotic areas may form within the surrounding tissue, and these areas will affect the growth rate of the tumor. In addition, injected cells expressing specific genes of interest may impact cancer progression as the genes may induce tumor growth that begins quickly, but slows down at the end or vice versa.

Collection of the lungs in Bowen's solution helps to visualize the superficial metastatic foci, which appears as raised pale formations on the surface of the lung tissue. The formation of foci is cell line dependent. Non-aggressive cells may give rise to micro metastases only, which can be easily identified with h and e staining on paraffin embedded lung tissue.

Following this procedure, other methods like Eliza can be performed to answer additional questions like, how does the growth of the experimental tumor affect the levels of cytokines or pro angiogenic molecules in the plasma.