Using just a model to develop a hepatocellular cancer mass model could help people elucidate their tumor-induced immunotolerance and specific treatment-induced, any tumor immunoresponses. The orthotopic tumor that develops with the liver fibrosis technique mimics the clinical features of human liver cancer. This method could provide a useful tool in hepatocellular cancer immunological study.
Visual demonstration of this method can illustrate the more technically challenging steps, such as the intrasplenic injection, that are essential for a successful execution of this protocol. Next, manually restrain a male, six to eight-week old C57 black 6 mouse dorsal-side up. Use alternate 70%ethanol and Betadine scrubs to clean the injection site three times.
Then deliver 160 microliters of carbon tetrachloride to the mouse by intraperitoneal injection and return the animal to its home cage. After confirming a lack of response to toe pinch, in a five-month old simian virus 40 T antigen transgenic mouse, place the mouse in the supine position and secure the limbs with tape. Make a midline laparotomy incision along the length of the linea alba, large enough to provide an adequate exposure of the liver, and displace the intestines to the left.
Dissect above the liver to exposure the inferior vena cava. Induce an artery clamp to ligate the vessel. Use a butterfly needle to gain intravenous access to the portal vein and manually secure the catheter.
Carefully switching syringes for each solution, successfully deliver 15 milliliters of solution one, 15 milliliters of 0.75%collagenase solution two, and 15 milliliters of solution two without collagenase via catheter, at an approximate 8.9 milliliters of solution per minute flow rate. At the end of the perfusion, harvest the tumor mass into a 50-milliliter conical tube containing 10 to 15 milliliters of PBS. Excise the tumor from the next MTD2 mouse as just demonstrated.
Use scissors to cut the livers into smaller pieces. Transfer the tissues into a new container of fresh PBS to remove the rest of the blood from the tissue before transferring the tissue pieces into a 50-milliliter conical tube containing five milliliters of complete RPMI medium. Mince the washed liver samples until the pieces can fit easily through the tip of a five-milliliter pipette.
Add enough medium to the tube to reach a final volume of 30 milliliters. Use a five-milliliter pipette to triturate the tissue fragments, before filtering the solution through a 70-micrometer strainer into a new 50-milliliter tube. Wash the strainer several times with complete medium.
Adjust the final volume to 50 milliliters with additional complete medium. Quickly spin the suspension by centrifugation to a maximum of 50 times g before stopping the centrifuge and decanting the supernatant. Then resuspend the pellet in 20 milliliters of PBS for counting.
For intrasplenic injection of the MTD2 hepatocytes, load a one-milliliter syringe equipped with a 27-gauge needle per each recipient animal with 220 microliters of hepatocytes per mouse. Confirm a lack of response to toe pinch in an anesthetized, carbon tetrachloride-treated animal. Beginning just below the spine muscle, next, make a one-centimeter incision on the left flank parallel to the thirteenth rib from the dorsal extreme and use blunt-pointed forceps to exteriorize the spleen.
Clip the spleen with two medium-sized titanium clips between the splenic artery and vein, and deliver 200 microliters of cells into the inferior pole of the spleen. Clip the inferior branch of the pedicle with one medium-sized clip and cut the spleen between the two initially placed clips. Remove the inferior pole of the spleen that was directly injected with tumor cells and use 3-0 polyglactin 910 interrupted suturing to close the inner muscle layer.
Then use sterilized steel wound clips to close the outer skin layer and subcutaneously administer five milligrams per kilogram of carprofen. After isolation from T antigen transgenic mice as demonstrated, both hepatocytes and tumor infiltrating cells can be observed. The cells typically demonstrate an almost 100%viability after processing.
After intrasplenic inoculation into wild-type carbon tetrachloride-treated animals, the transplanted hepatocytes successfully and reliably grow orthotopic hepatocellular carcinoma tumors that express the tumor-specific simian virus 40 T antigen. The preparation of our viable and pure population of hepatocytes from MTD2 mass and accurate administration of the intrasplenic injection are important to the success of the procedure. Although our team has tested hepatocyte inoculation into recipient mice via intravascular and intraperitoneal injection, these other methods weren't able to induce orthotopic hepatocellular carcinoma.
This model provides a unique platform enabling the investigation for immune surveillance and the tolerance in its early and the later stages of tumor progression. Please be careful when using the carbon tetrachloride, as it is toxic.
