Impact of metronomic UFT/cyclophosphamide chemotherapy and antiangiogenic drug assessed in a new preclinical model of locally advanced orthotopic hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is an intrinsically chemotherapy refractory malignancy. Development of effective therapeutic regimens would be facilitated by improved preclinical HCC models. Currently, most models consist of subcutaneous human tumor transplants in immunodeficient mice; however, these do not reproduce the extensive liver disease associated with HCC or metastasize. To address this deficiency, we developed an orthotopic model. Human HCC cells were transfected with the gene encoding secretable beta-subunit human choriogonadotropin (beta-hCG), which was used as a surrogate marker of tumor burden. The HCC cells were implanted into the left liver lobe of severe combined immunodeficient (SCID) mice, after which the efficacy of different therapies was evaluated on established, but liver-confined human Hep3B cell line HCC. Treatments included sorafenib or metronomic chemotherapy using cyclophosphamide (CTX), UFT, an oral 5-fluorouracil prodrug, or doxorubicin either alone or in various combinations, with or without an antiangiogenic agent, DC101, an anti-vascular endothelial growth factor receptor-2 antibody. Sorafenib inhibited tumor growth in a dose-dependent manner but caused severe weight loss in SCID mice, thus necessitating use of DC101 in subsequent experiments. Although less toxicity was observed using either single or doublet metronomic chemotherapy without any added antiangiogenic agent, none, provided survival benefit. In contrast, significantly improved overall survival was observed using various combinations of metronomic chemotherapy regimens such as UFT + CTX with DC101. In conclusion, using this model of liver-confined but advanced HCC suggests that the efficacy of a targeted antiangiogenic drug or metronomic chemotherapy can be mutually enhanced by concurrent combination treatment.

Figure 1

Summary of the development of an orthotopic HCC model. (A) One million human HCC cells expressing β-hCG were implanted into the left lobe of the liver of SCID mouse, as described in Materials and Methods. (B) At week 6 after PLC-hCG cell implantation, multiple tumor nodules developed in liver, indicated by the yellow arrows. (C) Hep3B-hCG cells developed into a single tumor mass in the liver 6 weeks after cell implantation. Normal liver is indicated by blue arrowheads.

Figure 2

Human β-hCG levels in urine samples from HCC-bearing SCID mice. (A) The relative HCC tumor burden is reflected by the level of β-hCG detected in urine samples (n = 12). Note that the latency period for growth of the HCC tumors is approximately 2 to 3 weeks. (B) Correlation of tumor mass and urine β-hCG levels from the pooled data obtained from the subcutaneous (n = 4) and orthotopic transplant models (n = 10).

Figure 3

Dose-response of sorafenib treatment in the HCC orthotopic and ectopic (subcutaneous) tumor therapy models (n = 5). (A) Tumor volumes in subcutaneous transplant model; sorafenib induced a dose-dependent inhibition of tumor volumes. (B) Relationship of β-hCG levels and relative tumor volume in the subcutaneous transplant model shown in Fig. 3A (n = 5). β-HCG levels correlated with tumor volumes. (C) Relationship of β-hCG levels and relative tumor volumes in HCC transplant models; β-hCG levels parallel relative tumor volumes in a dose-dependent manner. (D) Relative mouse weights in the subcutaneous HCC transplant model. Toxicity was detected after 7 days treatment with both the 30- and 60-mg/kg per day doses of sorafenib. (E) Relative weights detected in one orthotopic transplant model; toxicity became apparent after 7 days treatment at the 60 mg/kg/day dose. (F) Comparisons of tumor mass in the orthotopic and subcutaneous transplant models. The tumor mass in the orthotopic model control group is significantly greater compared with the subcutaneous transplant model group (P = .0029). The vertical arrows in panels A to E represent start of sorafenib treatment.

Figure 4

Evidence of b-hCG expression in vivo and of metastatic and recurrent HCC after surgical resection of primary tumor mass by immunohistochemical staining. Hepatectomy was performed in two mice 7 weeks after implantation of Hep3B cells in the liver. Immunohistochemical staining was done with anti-β-hCG antibody on formalin-fixed paraffin sections. The liver (A) and lung (B) specimens were collected 8 weeks after hepatectomy. Reddish brown staining indicates positive β-hCG staining of HCC cells. Red arrows indicate HCC metastasis.

Figure 5

Combination of various low-dose metronomic (LDM) chemotherapy regimens/drugs with or without DC101 in the orthotopic Hep3B HCC model (n = 5 per group). (A) Survival curves observed using single and combination LDM chemotherapy treatments, without DC101. (B) Survival curves using combination LDMchemotherapy treatments with or without DC101. The smaller lower five graphs shown in panels A and B are taken from the larger figure at the top of panels A and B. Together, these smaller graphs comprise the larger composite graph and are shown to more clearly illustrate the differences or similarities between each treatment group and the control untreated group. Note the prolongation of survival when metronomic UFT + CTX or UFT + DOX is combined with DC101. UFT = tegafur + uracil, a 5-FU prodrug. See Table 1 for median survival times and statistical significance.

Figure 5

Combination of various low-dose metronomic (LDM) chemotherapy regimens/drugs with or without DC101 in the orthotopic Hep3B HCC model (n = 5 per group). (A) Survival curves observed using single and combination LDM chemotherapy treatments, without DC101. (B) Survival curves using combination LDMchemotherapy treatments with or without DC101. The smaller lower five graphs shown in panels A and B are taken from the larger figure at the top of panels A and B. Together, these smaller graphs comprise the larger composite graph and are shown to more clearly illustrate the differences or similarities between each treatment group and the control untreated group. Note the prolongation of survival when metronomic UFT + CTX or UFT + DOX is combined with DC101. UFT = tegafur + uracil, a 5-FU prodrug. See Table 1 for median survival times and statistical significance.