Generating PDOX Mouse Model of Colorectal Cancer: Studying Progression of Colorectal Cancer in a Murine Model
Transkript
– Lymph nodes, or LNs, provide a microenvironment for tumor growth due to the stromal cells present in LNs. Follicular dendritic cells, or FDCs, are the most abundant types of stromal cells that release chemokines, cytokines, and growth factors, which interact with colorectal cancer stem cells and increase tumor growth.
FDCs also promote extranodal metastasis of colorectal cancer to organs such as the lungs and liver. To study the colorectal cancer progression, we implant patient tumor-derived cancer cells into the organ, which matches the tumor histotype in an immunocompromised mouse model known as patient-derived orthotopic xenograft, or PDOX.
Begin by securing an anesthetized mouse in a supine position on a dissection board using lab tape. Now, mix a required amount of luciferase-tagged colorectal cancer cells and FDCs in a tube. Take the resulting suspension in a syringe. Next, locate the anal opening of the mouse and inject the prepared colorectal cancer mix into the submucosal layer of the rectum.
To observe metastatic burden, inject luciferin intraperitoneally every week and image the mouse using bioluminescent imaging. In the presence of luciferase enzyme and ATP, luciferin in cancer cells converts to oxyluciferin, emitting blue-green light. In the following protocol, we will generate an orthotopic xenograft mouse model for studying colorectal cancer progression.
– To create CRC mouse model, place an anaesthetized six to eight-week-old male NOD SCID mouse in supine position under a dissecting microscope, securing the snout to an 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 visibility 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 cell suspension prepared in previous steps into the syringe. Inject the 10 microliters into the distal posterior rectal submucosa, 1 to 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.
