Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Models are needed to consistently needed to mimic cancer metastasis through the interaction with lymph nodes. In patients with both colorectal cancer and urothelial cell cancer of the bladder. Our orthotopic xenographic models described in this paper can help answer these important biologic questions and test new therapies efficiently and in a clinically relevant manner.

Our orthotopic xenograft tumors produced in mice resemble the patients'tumor. In the presence of lymph node stromal cells both models have a higher tumor implantation and distant organ metastasis rates and locations that mimic human metastatic patterns. Also, both animal models have zero procedural-related mortality.

After removing patient derived tumors from euthanized mice, use steril surgical scissors to mince luciferase positive tumor pieces as small as possible in a petri dish under a laminar flow hood. Transfer the tumor pieces into a sterile 50 mL conical tube. To prepare digest solution, add 10 mL of collagenase type 4, 80 microliters of hyaluronidase and 160 microliters of deoxyribonuclease type 1 to 40 mL of HBSS and mix by inverting.

Add 35-40 mL of the digest solution to minced tumor and incubate at 37 degrees Celsius with continuous rotation for 2 hours with periodic vigorous shaking. To remove debris after digestion, filter the digested tumor through sterile 100 micrometer cell strainer followed by filtration through a 40 micrometer cell strainer saving the flow through in a 50 mL tube each time and discarding the debris. To wash free cells, add 20 mL of HBSS to the flow through and centrifuge at 329 times gravity for 5 minutes.

Aspirate the supernatant and resuspend the pellet in 30 mL of HBSS. Transfer 100, 000 tumor cells to a sterile 15 mL conical tube and add 300, 000 previously prepared fresh HK cells. Centrifuge the cells at 329 times gravity for 5 minutes and discard the supernatant by aspiration.

Resuspend the cells in complete RPMI medium and keep on ice until ready for use. To prepare the animal for UCC cell injection use hair removal cream to shave the lower back of a 6-8 week old female NOD-SCID mouse. To administer UCC cells into a bladder, first set up a monopolar electrocautery machine to a power of 4 watts.

After anesthetizing the mouse, place it in supine position with its snout in a isoflurane nose cone and bare back firmly grounded on a dispersed electrode. Lubricate a 24 gauge angiocatheter with lubricating jelly and then insert the angiocatheter gently but firmly through the mouse urethra. If catheter bends on entry, insert guide wire halfway into catheter to provide stability.

Then, fully insert the 0.64 millimeter fixed core straight guide wire 1 millimeter past the end of the angiocatheter. Hold the monopolar pin to the guide wire for one second allowing for an electrical irritation of the bladder mucosa. Attach a fresh, sterile angiocatheter to 1 mL leur lock syringe and draw up to 200 microliters of UCC cells prepared in previous steps.

Remove guide wire and angiocatheter from the urethra and then insert angiocatheter with syringe of cells attached to it. Inject 50 microliters of the cells to the mouse bladder and to allow the cells to adhere to the bladder wall, wait a few seconds before removing the angiocatheter. Finally, remove the mouse from isoflurane nose cone and grounding pad and observe it for 1 hour following procedure for signs of distress.

To create CRC mouse model, place an anesthetized 6-8 week old male NOD-SCID mouse in supine position under a dissecting microscope securing the snout to and isoflurane nose cone and the front limbs with tape for stability. Place a small object such as a small gauze section under the base of the tail elevating the anus to improve visability and angle. Use curved lubricated blunt tipped forceps to dilate the anal canal and expose the distal anal and rectal mucosa and remove feces.

Connect a sterile 30 gauge removable needle to a 50 microliter glass syringe. Draw 10 microliters of tumor and HK cells cell suspension prepared in previous steps into the syringe. Inject the 10 microliters into the distal posterior rectal submucosa 1-2 millimeters above the anal canal making sure not to pass into the pelvic cavity.

Finally, remove the mouse from isoflurane nose cone and observe it for one hour following procedure for signs of distress. To weekly monitor the primary tumor, liver and lung metastatic burden in either mouse model, first, weigh the mouse. Inject 150 mg/kg luciferin intra-peritoneally and wait 5 minutes for the substrate to circulate in the body.

Place the anesthetized mouse in a bioluminescent imaging system with nose fixed in nose cone making sure that the area of interest, which is the ventral side of the animal is facing the camera for each image. Image mouse in supine position weekly for luciferase activity. After slicing and staining paraffin-embedded isolated tissues, observe them using a high-powered microscope.

In the UCC mouse model, 83.3 percent of animals generated primary tumors and displayed time-dependent primary tumor growth based on weekly bioluminescence measurements. In the CRC mouse model, 96.9%of mice grew primary tumors. Depending on the patient tumor, mouse tumor growth had a different latency period, which reflects the difference in the patients'clinical characteristics.

In both intra-vesicle and intra-rectal models, tumor cell injection generated orthotopic primary tumors. Furthermore, 33.3%and 53.1%of mice instilled with UCC cells and CRC cells with HK cells, respectively, developed distant organ metastasis. Histopathology of xenografts in the UCC model was compared to primary patient bladder carcinoma.

The staining results from xenografts were similar to those in the original surgical biopsies, confirming similar tissue morphology. Antibody to Ki67, a human cell proliferation marker, shows positive nuclear staining indicating highly proliferating, fast-growing human tumor cells. Similarly, in the CRC model, H&E staining indicates the similarity of architecture between xenografts and patient tumors.

Antibody staining against cytokeratin 20 also showed similar tumor growth pattern in both CRC models. The key to these procedures is to treat the animals gently, but firmly. Practice with a steady hand is the best way to perform these techniques safely and achieve the best outcomes.

Once these models are made, multiple different forms of cancer therapies can be tested, including combinations of current drugs as well as newly developed therapies prior to clinical trials in patients. It is important to remember that the handling of both cancer cells and the needles should be performed by experienced personnel for their safety as well as to achieve optimal results.