Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

The overall goal of this procedure is to generate subcutaneous or int hepatic human hepatocellular carcinoma or HCC xenografts in immunodeficient mice. This is accomplished by first preparing tumor fragments or a single cell suspension from a fresh human HCC specimen for implantation into the mice for subcutaneous xenografts. The next step is to inject tumor cells or to implant tumor fragments into a subcutaneous dorsal pocket for intra hepatic xenografts.

The next step is to inject tumor cells into the spleen or liver, or to implant tumor fragments directly into the liver in the final step. The animals are observed for several weeks until tumors develop. Ultimately, the human HCC Xenografts can be used to study many aspects of tumor biology, including the mechanisms of tumorgenesis and responses to novel therapies.

The main advantage of this technique over existing methods like the use of cell lines or subcutaneous xenografts only, is that with this technique, the primary human liver cancer specimens and patient derived intrahepatic xenografts provide a more realistic biological model of human hepatocellular carcinoma. Demonstrating the procedure will be Sharif Ahmed, a postdoctoral research scientist from my laboratory For subcutaneous implantation of the tumor fragments. First place the anesthetized mouse in the prone position, maintaining the inhalational isof fluorine anesthesia with a mouthpiece.

Apply tear gel to protect the animal's eyes from traumatic injury, and then sterilize the dorsal shaved area with Betadine surgical scrub, followed by 70%ethanol, and then lastly by povidone iodine solution. Next, use sharp scissors to make a five millimeter incision in the skin. Insert the tip of a pair of closed blunt scissors into the subcutaneous space, and then gently open the scissors to develop a pocket large enough to accommodate a tumor fragment.

Then use a pair of fine forceps to insert the tumor fragment into the subcutaneous pocket using clips to close the incision after the fragment has been placed just prior to injection lotus suspension of tumor cells in matrigel into an insulin syringe capped with a 29 gauge by half inch needle. Then insert the needle into the subcutaneous space of an anesthetized and shaved animal advancing the needle several millimeters along the subcutaneous plane away from the puncture site and discharge the contents of the syringe To prepare the animal for engraftment with human tumor tissue. Begin by subcutaneously administering 350 microliters of sterile normal saline solution with a 27 gauge by half inch needle on a one milliliter syringe into the dorsum of an anesthetized animal's neck to preemptively compensate for any intraoperative fluid loss.

Then after anesthetizing the supine mouse on a preheated pad, extend the animal's limbs and secure them with tape to the operating surface to optimize the exposure of the ventral abdomen and thorax. Position a magnifying lamp above the animal and then sterilize the shaved skin on the ventral abdomen and thorax as just demonstrated. Using sharp scissors, make a transverse bilateral subcostal skin incision, and then divide the muscle layers to expose the entire liver.

Place a stitch in the skin above the xiphoid process and secure it to the mouthpiece with tape in order to allow better exposure of the liver and the surrounding structures. Next, stabilize the liver with two cotton tipped applicators adjacent and posterior to the tissue. Then use a pair of sharp scissors to divide the falciform ligament attached to the median lobe of the liver.

Mobilize the left lobe of the liver with a cotton tip applicator and position a loose prettied knot of five oh silk suture around the lobe. Advance the knot as close as possible towards the bi pedicle of the left lobe, and then tighten it. Then use a pair of scissors to excise the lobe distal to the ligature, leaving a small stump to prevent slippage of the knot and subsequent hemorrhage.

Using a similar technique, ligate and resect the majority of the median lobe of the liver, and then use surgicel and gentle pressure with a cotton tipped applicator along the cut surfaces of the liver parenchyma to achieve complete hemostasis. Finally, perform intra hepatic xenograft of a tumor fragment or tumor cells, or intra hepatic xenograft of tumor cells via splenic injection as demonstrated in the following sections. For intra hepatic implantation of tumor fragments, use a number 10 scalpel blade to first make an incision three millimeters in length and depth on the surface of the liver.

Then immediately apply surgicel and gentle pressure to the incision site for 60 to 90 seconds to achieve hemostasis When complete hemostasis has been achieved, use a fine forcep to place a tumor fragment into the liver incision. Then apply a small piece of surgicel over the incision for hepatic implantation of tumor cells via direct injection into the liver. Insert an insulin syringe needle loaded with tumor cell suspension into the exposed liver and advance the tip a few millimeters beyond the puncture site along the subcapsular plane.

Gently discharge the syringe contents and remove the needle from the liver. Then place surgicel over the puncture site and apply gentle pressure with a cotton tipped applicator to prevent leakage of the tumor cell suspension and to achieve complete hemostasis for intra hepatic xenograft of tumor cells via injection into the spleen. Position the mice in the right lateral decubitus position and use sharp scissors to make a one centimeter left subcostal incision to enter the peritoneal cavity.

Then use a cotton tipped applicator to reflect the stomach cran into the animal's right side. In order to expose the spleen by handling the surrounding adipose tissues with fine a traumatic forceps, deliver the spleen into the incision and place a cotton tip duplicator behind the spleen to stabilize the tissue. Now use a five oh silk suture to place a loose pretti knot around the spleen above the lower pole.

Insert a tumor cell suspension loaded insulin syringe needle into the lower pole of the spleen and advance it past the level of the loose prettied knot. Then slowly discharge the contents of the syringe, remove the needle from the spleen and tighten the knot to prevent any leakage of the injected cell suspension. Finally, replace the spleen into the peritoneal cavity.

In this image, a liver resection specimen from a patient with HCC who had not received any adjuvant therapy to the tumor is shown. The white box indicates a viable portion of the tumor near the periphery that was obtained for xenograft. The development and growth of subcutaneous xenografts can be readily monitored by daily examination of the recipient mice.

For example, the typical appearance of a subcutaneous human HCC xenograft, which is visually distinct from the surrounding tissues and the corresponding histopathological appearance of the tumor are shown. The histology of the tumor demonstrates the typical features of HCC, including hepatocyte like cells with nuclear AIA and high nuclear dec cytoplasmic ratio, absence of portal tracks and distorted trabeculae with increased thickness of hepatocellular plates. In these images, the typical appearances of an intra hepatic human HCC xenograft achieved from direct implantation of tumor tissue into the liver, as well as that achieved from injection of tumor cells into the spleen are shown with the intraparenchymal tumors indicated by the black arrow in each image.

This histology section through the margin of an intra hepatic xog graft demonstrates the typical features of human HCC and the adjacent to normal mouse liver. The table summarizes the tumor engraftment rates achieved after implantation of primary human hepatocellular carcinoma tissues in immunodeficient mice. Using the different techniques just demonstrated for each implantation method, the denominator of the tumor take rate reflects a unique human HCC sample.

These data demonstrate that for primary human HCC tissues, subcutaneous engraftment rates are inferior to the rates of intra hepatic engraftment achieved by intra splenic tumor cell injection or intra hepatic tumor fragment implantation, and that partial hepatectomy appears to enhance intra hepatic engraftment. While attempting this procedure, it's important to remember to use sterile technique and achieve complete hemostasis as small six to eight week old immunocompromised mice are susceptible to infections and may not survive even a small amount of postoperative bleeding.