Orthotopic Hepatocellular Carcinoma Model Research Articles

Abstract 4710: Sunitinib prevents tumor-induced immunotolerance in novel murine model of hepatocellular carcinoma (HCC) through modulating CD4+CD25+Foxp3+ regulatory T cells via TGF-β signaling.

Abstract The high rate of mortality, frequent incidence of recurrence, and limited benefit of current therapies for hepatocellular carcinoma underline the urgent need for new therapeutic approaches. Immunotherapy has been demonstrated to be a promising strategy for this lethal disease. However, tumor progression induced immunotolerance abrogates effective immune responses and prevents the development of successful immunotherapeutic strategies. In this study, we evaluated the role of sunitinib, a FDA approved chemotherapeutic agent, in preventing tumor-induced immunotolerance and investigated the underlying cellular and molecular mechanisms. Recently, we've established a novel fibrotic and orthotopic murine model of HCC through seeding of tumorigenic hepatocytes from SV40 T antigen (Tag) transgenic MTD2 mice into the livers of syngeneic C57BL/6 mice followed by carbon tetrachloride treatment. This model mimics human HCC progression and recapitulates typical histologic, biologic, and immunologic features of this disease. Using this model, we've demonstrated that the treatment of sunitinib, a small molecule receptor tyrosine kinase inhibitor (RTKI), restrains the tumor growth in advanced HCC. Interestingly, sunitinib treatment also induces the proliferation and differentiation of tumor antigen-specific CD8 T cells in tumor bearing mice, evidenced by significantly higher portion of CD8 T cells that produced IFN-γ and TNF-α in response to tumor antigen stimulation. Subsequent findings revealed that sunitinib treatment led to the reduction in the frequency of CD4+CD25+Foxp3+ Treg and decrease in TGF-β expression. In vitro experiments indicated that Tregs in tumor-bearing mice express higher level of TGF-β and strongly suppress the function of CD4 and CD8 T cells in a dose-dependent manner compared to that in tumor-free mice. Tregs in tumor-bearing mice treated with sunitinib exhibited reduced suppressive function compared to that in tumor-bearing mice treated with vehicle. Further, the suppressive function of Tregs from tumor-bearing mice can be partly inhibited by blocking TGF-β with anti-TGF-β antibody. In addition, we've demonstrated that tumor lysate drives CD4+CD25- naïve T cells differentiation into CD4+CD25+Foxp3+ Tregs and this differentiation could be partially neutralized with anti-TGF-β antibody. In conclusion, our findings indicated that sunitinib treatment prevents tumor-induced immunotolerance through reducing the frequency and suppressive function of Tregs via TGF-β signaling pathway. Citation Format: Dai Liu, Guangfu Li, Eric T. Kimchi, Qing X. Yang, Todd Schell, Kevin F. Staveley-O'Carroll. Sunitinib prevents tumor-induced immunotolerance in novel murine model of hepatocellular carcinoma (HCC) through modulating CD4+CD25+Foxp3+ regulatory T cells via TGF-β signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4710. doi:10.1158/1538-7445.AM2013-4710

Insulin-like growth factor-binding protein-7 (IGFBP7): a promising gene therapeutic for hepatocellular carcinoma (HCC).

Hepatocellular carcinoma (HCC) is a highly fatal disease mandating development of novel, targeted therapies to elicit prolonged survival benefit to the patients. Insulin-like growth factor-binding protein-7 (IGFBP7), a secreted protein belonging to the IGFBP family, functions as a potential tumor suppressor for HCC. In the present study, we evaluated the therapeutic efficacy of a replication-incompetent adenovirus expressing IGFBP7 (Ad.IGFBP7) in human HCC. Ad.IGFBP7 profoundly inhibited viability and induced apoptosis in multiple human HCC cell lines by inducing reactive oxygen species (ROS) and activating a DNA damage response (DDR) and p38 MAPK. In orthotopic xenograft models of human HCC in athymic nude mice, intravenous administration of Ad.IGFBP7 profoundly inhibited primary tumor growth and intrahepatic metastasis. In a nude mice subcutaneous model, xenografts from human HCC cells were established in both flanks and only left-sided tumors received intratumoral injection of Ad.IGFBP7. Growth of both left-sided injected tumors and right-sided uninjected tumors were markedly inhibited by Ad.IGFBP7 with profound suppression of angiogenesis. These findings indicate that Ad.IGFBP7 might be a potent therapeutic eradicating both primary HCC and distant metastasis and might be an effective treatment option for terminal HCC patients.

Dovitinib preferentially targets endothelial cells rather than cancer cells for the inhibition of hepatocellular carcinoma growth and metastasis

BackgroundDovitinib is a receptor tyrosine kinase (RTK) inhibitor targeting vascular endothelial growth factor receptors, fibroblast growth factor receptors and platelet-derived growth factor receptor β. Dovitinib is currently in clinical trials for the treatment of hepatocellular carcinoma (HCC).MethodIn this study, we used five HCC cell lines and five endothelial cell lines to validate molecular and cellular targets of dovitinib.ResultsTumor growth and pulmonary metastasis were significantly suppressed in an orthotopic HCC model. Immunoblotting revealed that among known dovitinib targets, only PDGFR-β was expressed in two HCC cell lines, while four of five endothelial lines expressed PDGFR-β, FGFR-1, and VEGFR-2. Dovitinib inhibited endothelial cell proliferation and motility at 0.04 μmol/L, a pharmacologically relevant concentration; it was unable to inhibit the proliferation or motility of HCC cells at the same concentration. Immunohistochemical analyses showed that dovitinib significantly decreased the microvessel density of xenograft tumors, inhibiting proliferation and inducing apoptosis in HCC cells.ConclusionOur findings indicate that dovitinib inhibits HCC growth and metastasis preferentially through an antiangiogenic mechanism, not through direct targeting of HCC cells.

Telomerase‐specific oncolytic adenoviral therapy for orthotopic hepatocellular carcinoma in HBx transgenic mice

The telomerase-specific replication-competent oncolytic adenovirus, Telomelysin, was developed for virus-mediated preferential lysis of tumor cells. Its selectivity is derived from a human telomerase reverse transcriptase (hTERT) promoter-driven active viral replication, which occurs in cancer cells with high telomerase activity but not in normal cells lacking such activity. Because the TERT activity is elevated in most cases of hepatocellular carcinoma (HCC), the current study aims to investigate whether Telomelysin can be used for treatment of HCC. The oncolytic effect of Telomelysin has been investigated both in vitro using cell culture and in vivo using an immunocompetent in situ orthotopic HCC model. In this model, HCC developed spontaneously in the liver of HBx transgenic mice, which is pathologically and genetically similar to human HCC. In cell culture assay, Telomelysin lyses HCC cell lines at a low multiplicity of infection (MOI), ranging 0.77-6.35 (MOI [PFU/cell]). In the orthotopic HCC model, Telomelysin showed a potent oncolytic effect on HCC but spared normal liver tissue. Dose escalation analysis identified a safety dose of 1.25 × 10(8) PFU for this model. The effect of multiple injections of Telomelysin was also evaluated in this immunocompetent HCC model. We found that the virus replicates in HCC after a second intratumoral injection despite an immune response induced by the previous injection. This preclinical study shows that Telomelysin can be used for treatment of human HCC at an appropriate dosage and that its tumor-killing activity persists after multiple injections.

Estrogen Represses Hepatocellular Carcinoma (HCC) Growth via Inhibiting Alternative Activation of Tumor-associated Macrophages (TAMs)

Hepatocarcinoma cancer (HCC) occurs more often in men than in women, and little is known about its underlying molecular mechanisms. We identify that 17β-estradiol (E2) could suppress tumor growth via regulating the polarization of macrophages. Estrogen functions as a suppressor for macrophage alternative activation. These studies introduce a novel mechanism for suppressing male-predominant HCC. Hepatocarcinoma cancer (HCC), one of the most malignant cancers, occurs significantly more often in men than in women; however, little is known about its underlying molecular mechanisms. Here we identified that 17β-estradiol (E2) could suppress tumor growth via regulating the polarization of macrophages. We showed that E2 re-administration reduced tumor growth in orthotopic and ectopic mice HCC models. E2 functioned as a suppressor for macrophage alternative activation and tumor progression by keeping estrogen receptor β (ERβ) away from interacting with ATP5J (also known as ATPase-coupling factor 6), a part of ATPase, thus inhibiting the JAK1-STAT6 signaling pathway. These studies introduce a novel mechanism for suppressing male-predominant HCC.

In Vivo and In Vitro Suppression of Hepatocellular Carcinoma by EF24, a Curcumin Analog

The synthetic compound 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24) is a potent analog of curcumin that exhibits enhanced biological activity and bioavailability without increasing toxicity. EF24 exerts antitumor activity by arresting the cell cycle and inducing apoptosis, suppressing many types of cancer cells in vitro. The antiproliferative and antiangiogenic properties of EF24 provide theoretical support for its development and application to liver cancers. We investigated the in vitro and in vivo activities of EF24 on liver cancer to better understand its therapeutic effects and mechanisms. EF24 induced significant apoptosis and G2/M-phase cell cycle arrest in mouse liver cancer cell lines, Hepa1-6 and H22. The expression levels of G2/M cell cycle regulating factors, cyclin B1 and Cdc2, were significantly decreased, pp53, p53, and p21 were significantly increased in EF24-treated cells. In addition, EF24 treatment significantly reduced Bcl-2 concomitant with an increase in Bax, enhanced the release of cytochrome c from the mitochondria into the cytosol, resulting in an upregulation of cleaved-caspase-3, which promoted poly (ADP-ribose) polymerase cleavage. EF24-treated cells also displayed decreases in phosphorylated Akt, phosphorylated extracellular signal-regulated kinase and vascular endothelial growth factor. Our in vitro protein expression data were confirmed in vivo using a subcutaneous hepatocellular carcinoma (HCC) tumor model. This mouse HCC model confirmed that total body weight was unchanged following EF24 treatment, although tumor weight was significantly decreased. Using an orthotopic HCC model, EF24 significantly reduced the liver/body weight ratio and relative tumor areas compared to the control group. In situ detection of apoptotic cells and quantification of Ki-67, a biomarker of cell proliferation, all indicated significant tumor suppression with EF24 treatment. These results suggest that EF24 exhibits anti-tumor activity on liver cancer cells via mitochondria-dependent apoptosis and inducing cell cycle arrest coupled with antiangiogenesis. The demonstrated activities of EF24 support its further evaluation as a treatment for human liver cancers.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Antitumor effect of vascular endothelial growth factor inhibitor sunitinib in preclinical models of hepatocellular carcinoma

Tumor recurrence and metastasis is the most common cause of mortality in hepatocellular carcinoma (HCC) patients. Despite positive results with vascular endothelial growth factor (VEGF) inhibitors in preclinical studies using HCC xenograft models, the clinical outcome in HCC patients has been disappointing. So far, only the multitargeted tyrosine kinase inhibitor sorafenib has been shown to significantly improve survival in HCC patients, suggesting that this class of agents could be effective against HCC. Recently, another VEGF inhibitor, sunitinib, showed survival benefits in HCC hepatitis B-positive patients, but failed to improve survival in HCC hepatitis C-positive patients. Obviously, concomitant liver disease, liver function in general, and the local liver environment have a huge impact on treatment outcomes. In this study, we aimed to examine the antiproliferative effect of sunitinib in different HCC cell lines in vitro, and then in xenograft and orthotopic models of HCC in order to assess the effect of the local liver vasculature on drug efficacy. Human cancer cell lines Huh7.5, Hep3B, and SK-Hep-1 were used for in-vitro studies. In in-vivo studies, each mouse carried Huh7.5 cells in both the subcutaneous and the intrahepatic compartment; therefore, drug exposure and treatment regimen were identical in both tumors. Sunitinib has the potential to moderately inhibit proliferation in the Huh7.5 cell line, induce p53 in the p53-wild-type cell line SK-hep-1, and to increase the S-phase and the sub-G1 component of the cell cycle in the Hep3B cell line. Diverse responses to sunitinib in HCC cell lines emphasize the heterogeneity of HCC tumors and may further explain the discrepancy between preclinical and clinical results. The in-vivo results show that sunitinib treatment was far less effective against intrahepatic tumors compared with xenografts. Histological data indicate that large solid intrahepatic tumors are severely affected by sunitinib as shown by large areas of necrosis and diminished number of viable tumor cells. The real problem when treating intrahepatic tumors with sunitanib and/or other VEGF inhibitors seems to arise from unopposed local growth of the small tumors and perhaps the development of distant micrometastases. Even though both xenograft and orthotopic models have limitations, these models add value to our understanding of tumor biology and help to better design treatment paradigms for patients with HCC. In comparison with xenograft models, the orthotopic HCC model allows for a more realistic assessment of drug efficacy in patients, in particular by enhancing our knowledge of the role that organ vasculature plays in the development of local metastasis and tumor resistance to antiangiogenic treatments.

Antitumor effects of Stat3-siRNA and endostatin combined therapies, delivered by attenuated Salmonella, on orthotopically implanted hepatocarcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive carcinomas. Limited therapeutic options, mainly due to a fragmented genetic understanding of HCC, and major HCC resistance to conventional chemotherapy are the key reasons for a poor prognosis. Thus, new effective treatments are urgent and gene therapy may be a novel option. Signal transducer and activator of transcription 3 (Stat3) is a highly studied member of the STAT family. Inhibition of Stat3 signaling has been found to suppress tumor growth and improve survival, providing a molecular target for cancer therapy. Furthermore, HCC is a hypervascular tumor and angiogenesis plays a crucial role in tumor growth and metastasis. Thus, anti-angiogenic therapy, combined with inhibition of Stat3, may be an effective approach to combat HCC. We tested the effect that the combination therapy consisting of endostatin (a powerful angiogenesis inhibitor) and Stat3-specific small interfering RNA, using a DNA vector delivered by attenuated S. typhimurium, on an orthotopic HCC model in C57BL/6 mice. Although antitumor effects were observed with either single therapeutic treatment, the combination therapy provided superior antitumor effects. Correlated with this finding, the combination treatment resulted in significant alteration of Stat3 and endostatin levels and that of the downstream gene VEGF, decreased cell proliferation, induced cell apoptosis and inhibited angiogenesis. Importantly, combined treatment also elicited immune system regulation of various immune cells and cytokines. This study has provided a novel cancer gene therapeutic approach.

Abstract 3936: A hemoglobin-based oxygen carrier enhances chemotherapy in a rat orthotopic HCC model

Abstract Background and aims: Chemotherapy, as a regular treatment for non-resectable and recurrent hepatocellular carcinoma (HCC), its efficiency is largely limited by the high incidence of chemo-resistance. In the past several years, extensive evidence has demonstrated that tumor cells are exposed to diverse levels of hypoxia (oxygen deficiency) in vivo and this hypoxic stress acts as one of the major forces inducing chemo-resistance in tumors. Additionally, some recent studies reported that improving tumor oxygenation could enhance the therapeutic efficacy, thereby serving as a novel strategy for cancer management. In this study we aimed to examine whether a hemoglobin-based oxygen carrier could increase tumor oxygenation and thus enhance the efficiency of TACE in a rat orthotopic HCC model. Materials and methods: An orthotopic HCC model was established and TACE performed as reported previously. Real-time partial pressure of oxygen (pO2) inside tumor tissue was monitored with OxyLab pO2TM. Efficacy of O2 carrier in delivering O2 to tumor was tested by comparing pO2 measurements before or after systemic delivery of the carrier. Sensitization of chemotherapy (cisplatin) in TACE by O2 carrier was examined with 4 different therapeutic regimens, which included cisplatin (1mg/kg) + O2 carrier (0.2g/kg), cisplatin (1mg/kg) + O2 carrier (0.4g/kg), cisplatin (3mg/kg) + O2 carrier (0.2g /kg), cisplatin (3mg /kg) + O2 carrier (0.4g/kg). In each regimen, a separate group of rats receiving mock buffer instead of O2 carrier was set as control. For all the therapeutic regimens, single administration of O2 carrier or mock buffer via tail vein was performed 1 hr before TACE. Tumor response was evaluated at macroscopic and microscopic levels at day 21 after therapy. Results: Both significant augmentation in pO2 readings inside tumor tissues and a right-shift of pO2 frequency distribution were observed after O2 carrier administration (0.4g/kg). Tumor growth was most strikingly inhibited in rats treated with regimen combining 3mg/kg of cisplatin and 0.4g/kg of O2 carrier. Alteration of average estimated tumor volume in this regimen was 570 mm3 (pre-trx) vs 410 mm3 (post-trx) in O2 carrier -treated rats in contrast to 620 mm3 (pre-trx) vs 1980 mm3 (post-trx) in comparable control rats (p<0.05). Microscopic examination revealed that complete or nearly complete necrosis occurred more frequently in rats received this regimen (21%) compared with all the other regimens (0∼10%). Conclusion: Hypoxic levels inside tumor tissue could be alleviated by systemic delivery of a hemoglobin-based oxygen carrier in the rat orthotopic HCC model. Pre-administration of a certain amount of O2 carrier (0.4g/kg) could greatly improve the therapeutic efficacy of cisplatin (3mg/kg) in TACE. Whether delivery of O2 carrier in other ways (multiple dosages or continuous perfusion) is more efficient in sensitizing cisplatin is worth further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3936. doi:1538-7445.AM2012-3936

Abstract 5387: Exploring effects of nanosecond and microsecond pulsed electric field ablation in a rat orthotopic hepatocellular carcinoma model

Abstract Hepatocellular carcinoma (HCC) is the third most common cancer in the world. A diagnosis of HCC carries a guarded prognosis, especially when metastasis is documented. Reliable, curative treatment for very early, early, and intermediate stages is still an inconsistent ideal. Nanosecond (ns), or microsecond (μs) pulsed electric fields (PEFs) that produce irreversible electroporation (IRE) could be used to ablate local tumor masses through minimal invasive or open surgery delivery. Both nsPEFs and μsPEFs induce permanent permeabilization of cells resulting in cell death without any thermal effect. NsPEFs can also impact intracellular organelles and induce apoptosis. Previous studies indicated under certain condition, there was a protective tumor response following nsPEFs treatment in mouse subcutaneous HCC model. In these studies, we specifically test the efficacy of nsPEFs and μsPEFs in an orthotopic HCC rat tumor model. N1S1 cells stably transfected with luciferase were used to generate tumors. Orthotropic insertion of transfected N1S1 HCC cells in Sprague Dawley rat livers was used as the model. Different pulses numbers, rise times and pulse durations were applied. The effect on treated tumor tissue suggests that a single treatment of 500 or 1000 100ns pulses with either slow (50ns) or fast (10ns) rise times and 50 kV/cm can eliminate HCC tumors. Continuous delivery of eighty 100μs microsecond pulses at 2.5 kV/cm dramatically reverses lesion enlargement. 1000 pulses of 100 ns PEFs with fast rise times provoke an innate immune response. Immune responses in this orthotopic HCC model are still under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5387. doi:1538-7445.AM2012-5387

GHGKHKNK Octapeptide (P-5m) Inhibits Metastasis of HCCLM3 Cell Lines via Regulation of MMP-2 Expression in in Vitro and in Vivo Studies

P-5m, an octapeptide derived from domain 5 of HKa, was initially found to inhibit the invasion and migration of melanoma cells. The high metastatic potential of melanoma cells was prevented by the HGK motif in the P-5m peptide in vitro and in an experimental lung metastasis model, suggesting that P-5m may play an important role in the regulation of tumor metastasis. The aim of this study was to measure the effect of P-5m on tumor metastasis of human hepatocarcinoma cell line (HCCLM3) in vitro and in vivo in a nude mouse model of hepatocellular carcinoma (HCC), and detect the mechanisms involved in P-5m-induced anti-metastasis. By gelatin zymography, matrix metallo-proteinases 2 (MMP-2) activity in HCCLM3 was dramatically diminished by P-5m peptide. In addition, the migration and metastasis of HCCLM3 cells was also inhibited by the peptide in vitro. In an orthotopic model of HCC in nude mice, P-5m treatment effectively reduced the lung metastasis as well as the expression of MMP-2 in the tumor tissues. Overall, these observations indicate an important role for P-5m peptide in HCC invasion and metastasis, at least partially through modulation MMP-2 expression. These data suggests that P-5m may have therapeutic potential in metastatic human hepatocarcinoma.

Arresting the Culprit: Targeted Antagomir Delivery to Sequester Oncogenic miR-221 in HCC

Various treatment options are available for hepatocellular carcinoma (HCC); however, the overwhelming majority of them fail to extend patient survival, especially in cases of advanced disease. Although a number of different genetic lesions have been linked to HCC, the expression of oncogenic microRNAs (miRNAs), such as miR-221 and miR-222, are consistently found elevated in HCC tumor specimens,1,2 suggesting that silencing of these miRNAs might slow or stop tumor growth. In a recent study, Park et al. show that systemic administration of a chemically modified oligonucleotide that binds to and sequesters miR-221 is efficacious in HCC.3 Their approach identified a preferred chemical modification on the oligonucleotide that allowed its accumulation in hepatocytes, which led to therapeutic silencing of miR-221, reduced cellular proliferation, and most importantly increased survival of an orthotopic mouse model of HCC. Although this approach has yet to be tried in human patients, it appears likely that treatments involving miRNA overexpression or silencing, such as these, will translate efficiently.

Foretinib demonstrates anti-tumor activity and improves overall survival in preclinical models of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death. Although sorafenib has been shown to improve survival of patients with advanced HCC, this improvement is modest and patients eventually have refractory disease. The purpose of this study is to assess the anti-tumor and anti-angiogenic activities of foretinib, a vascular endothelial growth factor receptor 2 (VEGFR-2) and c-Met inhibitor using mouse models of human HCC. SK-HEP1 and 21-0208 HCC cells as well as patient-derived HCC models were employed to study the anti-tumor and antiangiogenic activities of foretinib. Changes of biomarkers relevant to hepatocyte growth factor (HGF) signaling pathways were determined by Western blotting. Microvessel density, apoptosis and cell proliferation were analyzed by immunohistochemistry. Treatment of SK-HEP1 cells with foretinib resulted in growth inhibition, G2/M cell cycle arrest, reduced colony formation and blockade of HGF-induced cell migration. In both orthotopic and ectopic models of HCC, foretinib potently inhibited tumor growth in a dose-dependent manner. Inhibition of angiogenesis correlated with inactivation of VEGFR-2/c-Met signaling pathways. Foretinib also caused elevation of p27 and Bim but reduced cyclin B1 expression and p-c-Myc, which resulted in a reduction in cellular proliferation and the induction of tumor cell apoptosis. In an orthotopic model, foretinib potently inhibited primary tumor growth and significantly prolonged mouse survival. Foretinib demonstrated significant antitumor activities in patient-derived HCC xenograft models. This study provides a compelling rationale for clinical investigation in patients with advanced HCC.

Regression of Established Hepatocellular Carcinoma Is Induced by Chemoimmunotherapy in An Orthotopic Murine Model

The high rate of mortality and frequent incidence of recurrence associated with hepatocellular carcinoma (HCC) reveal the need for new therapeutic approaches. In this study we evaluated the efficacy of a novel chemoimmunotherapeutic strategy to control HCC and investigated the underlying mechanism that increased the antitumor immune response. We developed a novel orthotopic mouse model of HCC through seeding of tumorigenic hepatocytes from SV40 T antigen (Tag) transgenic MTD2 mice into the livers of syngeneic C57BL/6 mice. These MTD2-derived hepatocytes form Tag-expressing HCC tumors specifically within the liver. This approach provides a platform to test therapeutic strategies and antigen-specific immune-directed therapy in an immunocompetent murine model. Using this model we tested the efficacy of a combination of oral sunitinib, a small molecule multitargeted receptor tyrosine kinase (RTK) inhibitor, and adoptive transfer of tumor antigen-specific CD8(+) T cells to eliminate HCC. Sunitinib treatment alone promoted a transient reduction in tumor size. Sunitinib treatment combined with adoptive transfer of tumor antigen-specific CD8(+) T cells led to elimination of established tumors without recurrence. In vitro studies revealed that HCC growth was inhibited through suppression of STAT3 signaling. In addition, sunitinib treatment of tumor-bearing mice was associated with suppression of STAT3 and a block in T-cell tolerance. These findings indicate that sunitinib inhibits HCC tumor growth directly through the STAT3 pathway and prevents tumor antigen-specific CD8(+) T-cell tolerance, thus defining a synergistic chemoimmunotherapeutic approach for HCC.

MiR-221 Silencing Blocks Hepatocellular Carcinoma and Promotes Survival

Patients with advanced hepatocellular carcinoma (HCC) face a dismal prognosis because of a lack of any effective therapies. To address this situation, we conducted a preclinical investigation of the therapeutic efficacy of oligonucleotides directed against the oncogenic microRNA miR-221, which has been implicated in HCC. Of 9 chemistries evaluated, we determined that a 2'-O-methyl phosphorothioate-modified anti-miR-221 oligonucleotide was most effective at reducing proliferation in vitro. A cholesterol-modified isoform of anti-miR-221 (chol-anti-miR-221) exhibited improved pharmacokinetics and liver tissue distribution compared with unmodified oligonucleotide. Chol-anti-miR-221 significantly reduced miR-221 levels in liver within a week of intravenous administration and in situ hybridization studies confirmed accumulation of the oligonucleotide in tumor cells in vivo. Within the same period, chol-anti-miR-221 reduced tumor cell proliferation and increased markers of apoptosis and cell-cycle arrest, elevating the tumor doubling time and increasing mouse survival. Taken together, our findings offer a preclinical proof of efficacy for chol-anti-miR-221 in a valid orthotopic mouse model of HCC, suggesting that this targeted agent could benefit treatment for patients with advanced HCC.

Abstract B247: The MEK inhibitor BAY 869766 inhibits tumor growth and metastatic spread, prolongs the survival and acts synergistically with standard of care drugs in models of hepatocellular carcinoma and pancreatic cancer.

Abstract Introduction: BAY 869766 (RDEA119) is an orally available, allosteric MEK1/2 inhibitor, targeting the central switch in the MAP kinase pathway. BAY 869766 demonstrates potent in vitro and in vivo activity in different human cancer cell lines and preclinical models. BAY 869766 is currently in clinical phase I/II studies in the indications HCC (hepatocellular carcinoma) and pancreatic cancer (PaCa). In the described preclinical studies BAY 869766 was studied in two orthotopic, syngeneic HCC and PaCa models and five subcutaneous PaCa xenografts as a single agent and combination therapy. In contrast to subcutaneous xenografts, orthotopic syngeneic models provide additional important information about microenvironment-dependent tumor growth, metastatic spread, immune response and survival. Results: To establish a syngeneic, orthotopic HCC model, the rat HCC cell line MH3924a was first evaluated in vitro. BAY 869766 induced antiproliferative effects in MH3924a HCC cells in monotherapy and showed synergistic effects in combination with sorafenib. In the corresponding rat allograft model efficacy of BAY 869766 in mono- and combination therapy with sorafenib was confirmed. Primary tumor growth and metastatic spread were strongly reduced. These effects were accompanied by a reduction of pERK levels, proliferation index and microvessel density. Finally, the median overall survival in combination treatment was increased by 100% in comparison with vehicle treatment. To characterize BAY 869766 in an orthotopic syngenic PaCa model, the Panc02 mouse allograft model was established. BAY 869766 was tested in mono- and combination therapy with Gemcitabine. Especially, combination treatment led to significant lower tumor weight, reduction of metastatic spread and increase of median survival by 135%. To test BAY 869766 in further clinical relevant pancreatic cancer models, five patient-derived pancreatic cancer tumor samples were subcutaneously implanted in mice and subsequently treated with BAY 869766. Significant tumor growth inhibition in these patient-derived pancreatic cancer xenograft models was observed (Treatment/Control ratios from 23,5% − 44%). Conclusion: These preclinical results support the clinical development of the MEK inhibitor BAY 869766 in HCC and pancreatic cancer. Inhibition of Ras-Raf-MEK-Erk-signaling, direct antiproliferative effects on tumor cells, reduction of metastatic spread and anti-angiogenic properties were demonstrated in these studies. In combination with the respective SoC drugs a significant benefit in survival could be achieved in the orthotopic models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B247.

Antitumor effects of hyaluronic acid inhibitor 4-methylumbelliferone in an orthotopic hepatocellular carcinoma model in mice

Liver cirrhosis is characterized by an excessive accumulation of extracellular matrix components, including hyaluronan (HA). In addition, cirrhosis is considered a pre-neoplastic disease for hepatocellular carcinoma (HCC). Altered HA biosynthesis is associated with cancer progression but its role in HCC is unknown. 4-Methylumbelliferone (4-MU), an orally available agent, is an HA synthesis inhibitor with anticancer properties. In this work, we used an orthotopic Hepa129 HCC model established in fibrotic livers induced by thioacetamide. We evaluated 4-MU effects on HCC cells and hepatic stellate cells (HSCs) in vitro by proliferation, apoptosis and cytotoxicity assays; tumor growth and fibrogenesis were also analyzed in vivo. Our results showed that treatment of HCC cells with 4-MU significantly reduced tumor cell proliferation and induced apoptosis, while primary cultured hepatocytes remained unaffected. 4-MU therapy reduced hepatic and systemic levels of HA. Tumors systemically treated with 4-MU showed the extensive areas of necrosis, inflammatory infiltrate and 2-3-fold reduced number of tumor satellites. No signs of toxicity were observed after 4-MU therapy. Animals treated with 4-MU developed a reduced fibrosis degree compared with controls (F1-2 vs F2-3, respectively). Importantly, 4-MU induced the apoptosis of HSCs in vitro and decreased the amount of activated HSCs in vivo. In conclusion, our results suggest a role for 4-MU as an anticancer agent for HCC associated with advanced fibrosis.

Comparing the Efficacy of Sunitinib with Sorafenib in Xenograft Models of Human Hepatocellular Carcinoma: Mechanistic Explanation

Hepatocellular carcinoma (HCC) is the fifth most common and third deadliest malignancy. Sorafenib has demonstrated 44% survival advantage over placebo and has emerged as a standard of care in advanced HCC. The therapeutic effects of sorafenib are however transient and hence additional treatment options are warranted. In this study, we aimed to compare the efficacy of sunitinib relative to sorafenib, two potent inhibitors of protein tyrosine kinases involved in tumor growth, metastasis, or angiogenesis. We reported that sorafenib and sunitinib suppressed tumor growth, angiogenesis, cell proliferation, and induced apoptosis in both orthotopic and ectopic models of HCC. However, the antitumor effect of 50 mg/kg sorafenib was greater than that of 40 mg/kg sunitinib. Sorafenib inhibited p-eIF4E Ser209, p-p38 Thr180/Tyr182 and reduced survivin expression. This was not seen with sunitinib. In addition, the antitumor and apoptotic effects of sorafenib, which are associated with upregulation of fast migrating Bim and ASK1 and downregulation of survivin, were greater than that of sunitinib. These observations explained in part the apparent superior anti-tumor activity of sorafenib compared to sunitinib. In conclusion, sunitinib demonstrated an inferior anti-tumor activity compared to sorafenib in ectopic and orthotopic models of human HCC. It remains to be seen whether such observations would be recapitulated in humans.

EGFR-specific PEGylated immunoliposomes for active siRNA delivery in hepatocellular carcinoma

The development of immunoliposomes for systemic siRNA (small interfering RNA) delivery is highly desired. We reported previously the development of targeted LPD (liposome–polycation–DNA complex) conjugated with anti-EGFR (epidermal growth factor receptor) Fab′ (TLPD-FCC) for siRNA delivery, which showed superior gene silencing activity in EGFR-overexpressing breast cancers. However, TLPD-FCC did not achieve satisfactory gene silencing activity in EGFR-overexpressing hepatocellular carcinoma (HCC). In this study, some modifications including increased antibody conjugation efficiency and reduced PEGylation degree were made to TLPD-FCC to increase gene silencing activity in HCC. The resultant optimized liposomes denoted as TLPD-FP75 efficiently bound and delivered to EGFR-overexpressing HCC, resulting in enhanced gene silencing activity compared to untargeted LPD (NTLPD-FP75). Tissue distribution in vivo revealed that the accumulation of TLPD-FP75 was higher than NTLPD-FP75 in orthotopic HCC model of mice. The promoted uptake of TLPD-FP75 in HCC cells was confirmed by confocal microscopy. To investigate the in vivo gene silencing activity, we administered TLPD-FP75 by intravenous injections into mice bearing orthotopic HCC. The results showed TLPD-FP75 potently suppressed luciferase expression, while little silencing was observed in NTLPD-FP75. TLPD-FP75 was demonstrated to possess potent gene silencing activity in HCC and will potentially increase the feasibility of HCC gene therapy.