Kang-Da Liu

Biological characteristics of fluorescent protein-expressing human hepatocellular carcinoma xenograft model in nude mice.

Objectives To study biological characteristics of stable red fluorescent protein (RFP)-expressing or green fluorescent protein (GFP)-expressing HCCLM3 cell lines and those of their relevant xenograft models in nude mice. Methods HCCLM3, a human hepatocellular carcinoma cell line with high metastatic potential was infected with RFP or GFP full-length cDNA via lentivirus. Stable RFP-expressing or GFP-expressing HCCLM3 cells, namely HCCLM3-R and HCCLM3-G, were subcutaneously injected and two patient-like metastatic models of HCCLM3-R and HCCLM3-G in nude mice were established using surgical orthotopic implantation from subcutaneous tumor tissues. Cell proliferation, karyotype, biomarker expression, tumor growth, and metastasis of HCCLM3-R and HCCLM3-G were analyzed in vitro and in vivo. Results RFP and GFP genes were integrated in genomic DNA of HCCLM3. HCCLM3-R and HCCLM3-G expressed red and green fluorescence, stable and intense, 300 days after 60 consecutive passages, and also positively expressed CK8+, P16+, AFP+ and negatively expressed HBsAg−. Their biomarker expression and karyotype were found to be similar to those of the parental HCCLM3, and their tumorigenesis occurred in 10 nude mice without exception after a subcutaneous injection and did the same in 20 nude mice after an orthotopic implantation. The results showed that the rate of spontaneous metastasis to the liver and lung and peritoneal seeding was 100, 100, and 90%, respectively. Conclusion Stable fluorescent protein-expressing HCCLM3-R and HCCLM3-G xenografts in nude mice could be of two useful models for studying mechanisms of hepatocellular carcinoma growth and metastasis in real time.

Improved long-term survival for unresectable hepatocellular carcinoma (HCC) with a combination of surgery and intrahepatic arterial infusion of 131I-anti-HCC mAb. Phase I/II clinical trials

Abstract Resectional therapy has been accepted as the only curative therapy for hepatocellular carcinoma (HCC). Unfortunately, it is estimated that only 10% of HCC are resectable at the time of diagnosis. Cytoreduction and sequential resection offer a new hope for patients with unresectable HCC. Radioimmunotherapy (RIT) is an attractive approach for cytoreduction. We have previously shown that intrahepatic arterial 131I-labelled anti-HCC monoclonal antibody (131I-Hepama-1 mAb) could be used safely in combination with hepatic artery ligation for treatment of unresectable HCC, and encouraging results have been achieved. In this paper, the long-term survival and the prognostic factors in HCC patients treated with radioimmunotherapy will be analysed. Sixty-five patients with surgically verified unresectable HCC were treated with hepatic artery ligation plus hepatic artery cannulation and infusion from 1990 to 1992. Thirty-two patients were enrolled in a phase I–II clinical trial with infusion of 131I-radiolabelled anti-HCC monoclonal antibody (Hepama-1 mAb) via the hepatic artery (the RIT group). Another 33 patients formed the group treated with intrahepatic-arterial chemotherapy (the non-RIT group). T cell subsets were measured in 24 patients and human anti-(murine Ig) antibody (HAMA) were monitored in the RIT group. The 5-year survival rate was significantly higher in the RIT group than in the chemotherapy group, being 28.1% compared to 9.1% (P < 0.05); this was mainly a result of better cytoreduction and a higher sequential resection rate (53.1% compared to 9.1%). Significant prognostic factors in the RIT group included tumour capsule status and the number of tumour nodules. HAMA incidence and CD4+ T lymphocytes influenced short-term, but not long-term survival. It is suggested that intrahepatic-arterial RIT, using 131I-Hepama-1 mAb, combined with hepatic artery ligation might be an effective approach to improve long-term survival in some patients with unresectable HCC, which may successfully be made resectable by intra-arterial infusion of 131I-Hepama-1 mAb.

Metastatic models of human liver cancer in nude mice orthotopically constructed by using histologically intact patient specimens.

In this study of orthotopic implantation of histologically intact surgical specimens, the authors constructed metastatic models of human hepatocellular carcinoma (HCC) in nude mice. Histologically intact human liver cancer specimens, derived from patients, were implanted directly into the liver of nude mice, and their orthotopic growth and metastases were observed. The transplantability and metastatic rate of two specimen groups (primary and metastatic lesions) were analysed. α-Fetoprotein (AFP) was also determined in transplanted tumours by an immunohistochemical method. Orthotopic growth was observed in 14 of 30 transplanted specimens and formation of metastases in 7 cases, which exhibited the variety of clinical behaviours seen in patients with HCC. These behaviours included local growth, regional invasion, spontaneous intrahepatic, lymph node and lung metastasis and peritoneal seeding. In two groups the growth rate of metastatic lesions following implantation was clearly higher than that of primary tumours. Chromosome analysis from locally growing tumours confirmed their morphologically human origin. An immunohistochemical study showed that implanted tumours originating from AFP-positive specimens maintained AFP expression. These results indicated that the animal models should prove valuable for developing new treatment modalities and studying the mechanism of metastasis of human HCC.

Radioimmunotherapy for unresectable hepatocellular carcinoma using131I-Hepama-1 mAb: preliminary results

Twenty-three patients with surgically verified unresectable hepatocellular carcinoma (HCC) have been treated by intrahepatic arterial administration of131I-labeled anti-HCC monoclonal antibody (Hepama-1) combined with hepatic artery ligation. Radioimmunoimaging demonstrated that the median tumor/liver ratio was 2.1 (1.1–3.6) at day 5. A decline in α-fetoprotein level and shrinkage of tumor were observed in 75% (12/16) and 78% (18/23) of patients respectively. Sequential resection was done in 11 patients (48%) after treatment. The surgical specimens revealed massive necrosis of tumor, but residual cancer cells were found at the edge of the specimens. Anti-antibody was determined in 43% (10/23) of patients 2–4 weeks after the administration of131I-Hepama-1 mAb. No marked toxic effects were noted. It is suggested that131I-Hepama-1 mAb might be of value as one of the multimodality treatments for unresectable HCC.

Apoptosis of human BEL-7402 hepatocellular carcinoma cells released by antisense H-ras DNA-in vitro and in vivo studies

Recent findings suggest that over-expression of activated H-ras inhibited apoptotic cell death by blocking the activity of apoptotic endonuclease(s). This study was designed using antisense H-ras oligodeoxynucleotides (ODN) to evaluate whether alterations of H-ras expression in BEL-7402 human hepatocellular carcinoma cells could influence the induction of apoptosis in vitro and in vivo. We found that, in vitro, continuous suppression of H-ras expression could decrease the proliferation of BEL-7402 cells and inhibit H-ras-induced entry into S phase. In situ end labeling showed that a large number of cells underwent apoptotic cell death after treatment with antisense H-ras ODN (P<0.01), and gel electrophoresis of DNA extracted from these cells demonstrated a typical DNA ladder, characteristic of apoptosis. In vivo study indicated that pretreatment with antisense H-ras significantly retarded tumor growth in comparison with the untreated controls or tumors treated with non-specific ODN (P<0.01,P<0.01). In situ end-labeling revealed that pronounced apoptotic nuclei were also present in the tissue treated with antisense H-ras ODN (P<0.01). Immunocyto-histochemical study showed that expression of p21H-ras was significantly decreased after treatment with antisense H-ras. These results indicate that suppression of H-ras over-expression by antisense ODN could effectively inhibit tumor growth and revive the apoptotic pathway by releasing the activity of apoptotic endonuclease(s). The data also suggest the need for further studies to elucidate molecular events involved in antisense H-ras-released apoptosis and evaluate its therapeutic implications.

Radioiodinated anti-hepatocellular carcinoma (HCC) ferritin Targeting therapy, tumor imaging and anti-antibody response in HCC patients with hepatic arterial infusion

SummaryRadioimmunoimaging and radioimmunotherapy with radioiodinated anti-(hepatocellular carcinoma ferritin) antibody (131I-or125I-FtAb) have been applied in patients with primary liver cancer. A total of 41 patients with surgically unresectable hepatocellular carcinoma (HCC) and receiving hepatic artery ligation and cannulation during exploratory laparotomy were treated with this regimen by intrahepatic arterial infusion. Compared with the control group, a decline of serum α-fetoprotein (65.7% versus 42.9%) and shrinkage of tumor (68.3% versus 33.9%) were observed in the treated group, and a higher second-look resection rate (31.7% versus 5.1%) and longer survival (1-year: 61.0% versus 37.3%, 3-year: 25.0% versus 6.9%) resulted. The administration of antibody through a hepatic arterial catheter (n=16) was compared with intravenous injection (n-17) in terms of the tumor-imaging sensitivity in 33 patients with liver cancer. The results indicated that hepatic arterial infusion was superior to intravenous injection. The sensitivity 7 days after the administration was 100% in the i.a. group and 76.5% in the i.v. group, the uptake ratio of tumor to liver being 1.74±0.57 in the former and 1.34±0.29 in the latter. Furthermore, intrahepatic arterial infusion revealed a lower anti-antibody detection rate than intravenous injection (0/14 versus 4/11).

Analysis of the cellular origin of hepatocellular carcinoma by p53 genotype

The polymerase chain reaction andHaeIII enzymatic digestion were used to study the seventh exon of the p53 gene in 29 primary and recurrent hepatomas in paraffin-embedded samples from 11 patients. The mutation rate of the p53 gene and its geno-types in samples of primary and recurrent tumours and multiple nodules were investigated. The cellular origins of hepatocellular carcinoma were analysed by p53 genotype. p53 mutation rates were found to be 69.0% (20/29) in the primary and recurrent tumours, 58.8% (10/17) in tumours with a single nodule and 83.3% (10/12) in tumours with multiple nodules. The p53 genotypes were found to be different in 6 pairs of primary and recurrent tumours, and another 5 pairs had the same p53 genotypes. The samples with multiple nodules in the same patients had the same p53 geno-types. Seven recurrences were of multicentric origin and four were of unicentric origin. It is suggested that the recurrent lesions developed from both unicentric and multicentric origins.

Human anti-(murine Ig) antibody responses in patients with hepatocellular carcinoma receiving intrahepatic arterial 131I-labeled Hepama-1 mAb. Preliminary results and discussion

Human anti-(murine Ig) antibody (HAMA) responses were monitored in 32 patients with unresectable hepatocellular carcinoma (HCC) undergoing radioimmunotherapy using131I-labeled anti-HCC monoclonal antibody (Hepama-1 mAb) intrahepatic arterial infusion. Dosages of Hepama-1 mAb ranged from 5 mg to 20 mg and the mAb was radiolabeled with 0.74–4.00 GBq (20–108 mCi)131I (4–6 mCi/mg). T lymphocyte subsets were examined before and after radioimmunotherapy in 24 patients. In this series, 34.4% (11/32) of patients developed HAMA within 2–4 weeks after the infusion. All patients with a negative HAMA response (n=14). had CD4+ T lymphocyte subsets (T helper/inducer) much lower than those of the HAMA-positive (n=10) patients and the control group (n=40) (P<0.01) prior to infusion. The sequential resection and survival rates in the HAMA-negative group were also lower than that of the HAMA-positive group. Thus, the determination of T lymphocyte subsets might help to predict the HAMA response in HCC patients during radioimmunotherapy.

Reconstruction and expression of chimeric anti-HBx antibody in Escherichia coli.

The variable regions of murine monoclonal anti-HBx immunoglobulin and the constant region of human antibody were cloned by reverse transcriptpolymerase chain reaction (RT-PCR). The heavy-chain and light-chain variable regions were connected and coexpressed with human constant region C-r3 and C-k3 in the reconstructed vector ofE. coli. The products showed high specificity and binding ability with HBx, which is closely associated with hepatocarcinogenesis. This makes it possible to humanize the mouse monoclonal antibodies and express the fusion protein inE. coli for potential radioimmunotherapy in patients with hepatocellular carcinoma.

mRNA levels of nm23 in murine ascites hepatoma (H22) clones with different lymphatic metastatic potential

Levels of expression of the nm23 gene inversely correlated with metastatic potential in several rodent tumor model systems and human breast carcinoma. In the present study, we examined nm23 mRNA levels in two murine ascites hepatoma models (H22-16A3-F and H22-A2-P) with different metastatic potentials. Metastatic H22-16A3-F (80% metastatic rate) and non-metastatic H22-A2-P clones were both derived from murine ascites hepatoma (H22). We found that a 0.8-kb nm23 transcript was expressed in both cell clones. The nm23 gene was expressed at a higher level in non-metastatic H22-A2-P: approximately 8.6-fold higher than in metastatic H22-16A3-F. The present data suggest that the expression of nm23 mRNA might be associated with metastasis of murine ascites hepatoma (H22), though heterogeneity of nm23 steady-state expression levels among the H22 clones remains to be investigated.