Subcutaneous Injection of Human Colorectal Cancer Cells in Athymic Nude Mice to Evaluate Antitumor Efficacy

This research presents a reproducible preclinical protocol using a subcutaneous colorectal cancer mouse model to assess anti-tumor effects of bioactive compounds. Recent developments include refined xenograft techniques, improved animal handling, and advanced imaging methods that enhance tumor monitoring and reproducibility in preclinical cancer studies. To begin, transfer a working solution of D-Luciferin Potassium Salt Bioluminescent Substrate through a 0.2-micrometer syringe filter for sterilization.

Place the extracellular matrix on ice and using a pre-chilled P1000 micropipette, aliquot 200 microliters of extracellular matrix into sterile tubes. Aspirate the culture medium carefully to avoid disturbing adherent HCT 116 bioluminescent cells. Next, wash the cells with eight milliliters of sterile PBS, pre-warned at 30 degrees Celsius to remove residual serum.

Add two milliliters per flask of warm trips in EDTA at 0.05%and incubate at 37 degrees Celsius for four to five minutes. Monitor cell detachment under an inverted microscope. Next, add 10 milliliters of complete growth medium containing fetal bovine serum to neutralize trypsin.

Then, transfer the cells into a 15-milliliter conical tube using a 10-milliliter serological pipette. Now, centrifuge the cell suspension at 200g for five minutes at room temperature. Carefully aspirate the supernatant without disturbing the cell pellet.

Gently resuspend the cell pellet in three milliliters of seeding medium to achieve a homogeneous suspension. Determine cell viability by mixing 10 microliters of the cell suspension with 10 microliters of trypan blue and counting viable cells under an inverted microscope. After resuspending the cells in sterile PBS, prepare 200-microliter aliquots.

Mix 200 microliters of the prepared bioluminescent colorectal cancer cell aliquots with 200 microliters of extracellular matrix in a cryo vial inside a biosafety cabinet. Gently pipette with a P1000 pipette to avoid air bubble formation and dissociation. Transfer the mixture to a one-milliliter syringe, remove air bubbles, and proceed immediately to injection.

Remove the mouse from the chamber once it is anesthetized and place the animal snout in an outlet supplying the same anesthetic. Place the mouse on its right side on a flat surface. Using the tips of the fingers of the non-dominant hand, gently pinch and lift the animal skin in the lower left quadrant.

Insert the needle bevel up 2/3 into the lifted skin parallel to the body. Inject the contents of the syringe into the lifted skin right between the two fingers. Hold briefly before withdrawing the needle slowly.

Manually restrain the mouse in dorsal recumbents, abdomen side up, with the cranial end of the animal pointed down to allow the intestinal contents to move downward. Keeping the needle bevel side up and angled at 15 to 20 degrees, push the needle into the abdominal cavity, so the tip just penetrates the abdominal wall of the left lower abdominal quadrant. Slowly inject the D-Luciferin into the intraperitoneal cavity, then remove the needle.

After placing the anesthetized animal in the imaging system chamber and ensuring proper alignment to capture the tumor area, open the linked software. Choose the visual or photographic imaging mode to confirm the animal's position. Save the data and change the folder's name.

Set up a region of interest around the tumor for analysis and copy it for further analysis. Measure the light of the region of interest and export the results, then paste previously created and copied regions of interest of the same size for subsequent tumors. Use a calibrated digital caliber to measure tumor length, width, and depth.

Then, calculate the volume using the formula for spherical or ellipsoid tumors. Bioluminescence imaging enables sensitive real-time imaging of small tumor lesions with homogeneous signals. A linear correlation was observed between tumor volume and total light flux, confirming that bioluminescence accurately reflects tumor size.

Tumor volume estimates calculated with Formula 1 significantly overestimated actual volume compared to Formula 2. This protocol enables non-invasive IVUS tumor monitoring, offering a controlled reproducible platform that reduces animal use and minimizes experimental variability. Future research will develop human-relevant preclinical models, refine protocols for reproducibility, prioritize animal welfare, and optimize metastasis monitoring.