Establishing Experimental Metastases Mouse Model: Implanting GFP Expressing CRC Organoid Cells in PDX Model to Detect Micrometastases

- Micrometastases are a small group of cancer cells that shed from a primary tumor and spread to different parts of the body via blood or lymph nodes. To detect micrometastases, suspend colorectal cancer organoid cells in PBS to maintain pH and load the cell suspension into a syringe. Next, place an anesthetized mouse in a prone position on a surgical bed. Make a small incision using scissors on the lower part of the left flank and expose the spleen. Gently pull the spleen out using tweezers and slowly inject the cell suspension into the spleen. Close the incision using sutures and return the animal to its cage. Monitor it until it recovers.

After the desired period of injection, sacrifice the mouse to dissect primary tumors and different tissues. Observe dissected tissues under a fluorescence microscope. GFP-expressing cancer cells develop a primary tumor at the injected site in the spleen. A few cancer cells from the primary tumor move to a different organ, usually the liver, to form micrometastatic colonies. In the following protocol, we will establish an experimental metastases mouse model of GFP-expressing colorectal cancer organoid cells.

- To establish an experimental metastasis model of CRC patient derived tumor xenografts, dilute the single tumor cell suspensions at a 4 times 10 to the fourth cells per 50 microliters concentration and load 50 microliters of cells per mouse into a syringe equipped with a 22 gauge needle. Place the first anaesthetized NOG mouse in a prone position on the bench and make a small incision in the lower region of the left flank.

Use sterile scissors and tweezers to carefully cut the peritoneum and expose the spleen, and use the tweezers to grasp the fat attached to the spleen. Then, slowly inject 50 microliters of the tumor cell suspension into the spleen, taking care that no leakage is observed.

After all of the mice have been injected and sutured as necessary, return the animals to their home cages and check for suture leakage and viability one day after the tumor cell delivery. Use calipers to measure the tumors weekly, harvesting the primary tumors and relevant tissues up to the appropriate experimental end points. Transfer the dissected tumors and organs into 5 milliliters of ice-cold PBS in a 10 centimeter Petri dish on ice and observe the dissected tissues under a stereo-fluorescence microscope to assess their GFP expression.