Spontaneous Metastasis Mouse Models: A Platform to Study the Metastatic Potential of Orthotopically Injected Colorectal Cancer Cells

- Begin with a culture of green fluorescent protein, or GFP, expressing colorectal cancer organoids. Harvest the organoids. Treat with a cell detachment enzyme solution to dissociate the organoids into a single-cell suspension. Centrifuge to separate the enzyme-containing supernatant. Remove the supernatant. Resuspend the GFP-expressing colorectal cancer cells using an artificial extracellular matrix-containing media. Load the cell suspension into a syringe and maintain it on ice to prevent matrix solidification at ambient temperatures.

Next, prep an anesthetized mouse in the supine position. Gently pull out the mucosal layer of the rectum through the anal opening. Now, inject the prepared GFP-expressing cancer cell suspension into the rectal submucosal layer containing the network of blood vessels. Allow the mouse to recover.

Within a few days, a GFP-expressing primary tumor develops at the site of injection. With spontaneous metastasis, these tumor cells detach and enter the circulation. Eventually, the cells extravasate into distant organs and proliferate to form GFP-positive metastatic tumors. In the following protocol, we will show the generation of a spontaneous metastasis mouse model of colorectal cancer by orthotopically injecting GFP-expressing colorectal organoid cells.

- To establish a spontaneous metastasis mouse model of CRC patient-derived tumor xenografts, dissociate the GFP-labeled organoids as demonstrated and dilute the single tumor cell suspensions at a 5 times 10 to the five tumor cells per 50 microliters of PBS, supplemented with 50% artificial extracellular matrix concentration. Load 50 microliters of cells per mouse into a syringe equipped with a 22 gauge needle and place the tumor cells on ice.

Next, confirm a lack of response to toe pinch and place the first anesthetized NOG mouse in the supine position on the laboratory bench. Using sterile tweezers, gently pull the rectal mucosa out of the anus and immediately inject the tumor cells into the rectal submucosa.