Establishment and serial quantification of intrahepatic xenografts of human hepatocellular carcinoma in severe combined immunodeficiency mice, and development of therapeutic strategies to overcome multidrug resistance.

A murine model in which to study multiple drug resistance in human hepatocellular carcinoma was developed. PRF/PLC/5 hepatoma cells (Alex 0) and an induced multidrug resistant clone (Alex 0.5) were injected intrasplenically into severe combined immunodeficiency mice. In 70% of injected mice, hepatoma cells engrafted in the liver and grew as intrahepatic metastasis. Since Alex cells contain an integrated hepatitis B virus genome and secrete hepatitis B surface antigen (HBsAg), the serum HBsAg concentration in tumor-bearing mice was used to quantitate tumor burden. Tumor wet weight determined at necropsy was directly proportional to the serum HBsAg concentration. In Alex 0 cells, IC50s for doxorubicin, vinblastine, and cis-platinum were 0.35 microM, 0.029 microM, and 3.70 microM, respectively. Alex 0.5 cells were 25-, 14-, and 1.4-fold more resistant to doxorubicin, vinblastine, and cis-platinum, respectively. Immunoblotting of Alex 0 cell membranes with an anti-P-glycoprotein antibody (C219) revealed small amounts of P-glycoprotein, whereas Alex 0.5 membranes overexpressed the protein. Concurrent exposure to verapamil (10 microM) sensitized both cell lines to the cytotoxic action of vinblastine and doxorubicin but had no effect on the cytotoxicity of cis-platinum. Mice bearing intrahepatic xenografts derived from Alex 0 and 0.5 cells had no response to treatment with i.v. vinblastine or doxorubicin, as was anticipated from in vitro drug testing. Addition of verapamil to vinblastine treatment did not improve the success of in vivo chemotherapy. Immunotherapy with a human anti-P-glycoprotein antibody (MRK16) suppressed the in vivo growth of tumors derived from both cell lines. The effect was most pronounced in mice bearing Alex 0.5 tumors. Immunoblotting of tumors which initially responded to MRK16 therapy, but subsequently relapsed, revealed a marked decrease in P-glycoprotein expression when compared to results in tumors that were untreated or treated with vinblastine or control antibody. In summary, we have developed an intrahepatic tumor xenograft model of human hepatocellular carcinoma in mice that permits noninvasive serial quantification of tumor burden by determination of serum HBsAg levels and demonstrated a positive response to immunotherapy with anti-P-glycoprotein antibodies.