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The response of standard-risk hepatoblastoma (HB) to neoadjuvant cisplatin (CDDP) chemotherapy is excellent; however, in high-risk HB, drug resistance remains a major challenge. Alternative therapeutic strategies may consider combining cytotoxic drugs with apoptosis sensitizers as this has shown additive effects in various types of malignancies. Analysis of published expression databases have revealed an anti-apoptosis state in HB samples. Herein, we evaluated the synergistic effects of ABT-737 as a modulator of apoptosis in combination with CDDP in HB. To this end, clonogenic assays were performed with HepT1 and HUH6 HB cells to evaluate the synergistic effects of CDDP and ABT-737. Combination treatment with CDDP and ABT-737 reduced the clonogenicity of HB cells more than 5-fold compared to treatment with CDDP alone. Furthermore, the HUH6 mixed-type HB cells showed higher sensitivity to CDDP and combination treatment compared to the HepT1 embryonal-type cells. Subcutaneous HUH6 tumors in NOD/LtSz-scid IL2Rγnull mice were treated with CDDP (1.25 and 3 mg/kg body weight, n=6), ABT-737 (100 mg/kg, n=5) and the combination of both agents (n=5). Combined treatment led to a significantly reduced tumor growth compared to CDDP treatment alone (p<0.02). When using higher doses of CDDP (3 mg/kg) alone or in combination with ABT-737, dose-dependent toxicity was observed in this mouse strain. In conclusion, our results demonstrated the enhancement of chemotherapy efficacy by using modulators of apoptosis together with cytotoxic agents. Additive effects of ABT-737 may allow reduction in CDDP dosages with maintenance of antitumor activity. Sensitizing HB to apoptosis may also render resistant HB susceptible to established chemotherapy regimens.

Expression profiling of tumor tissue allows a systematic search for targeted therapies and offers relevant prognostic information. Molecular studies on rhabdomyosarcoma (RMS) revealed a more differentiated classification than the histological subgrouping into embryonal (RME) and alveolar (RMA) rhabdomyosarcoma, and reflected the chromosomal aberrations found in RMS. We addressed biological processes like cell migration and emerging drug resistance by expression profiling to identify mechanisms of metastasic invasion and differential response to chemotherapy in RMS. Gene expression analysis was performed in 19 RMS samples using the Affymetrix U133 Plus2 array. Validation of target genes was performed by qRT-PCR. Data were analyzed using Pathway analysis software. Involvement of these genes in invasion processes was evaluated in knock-down experiments using specific interference RNA and Matrigel(TM) invasion assay. In RMA tissues 211 of 534 genes were overexpressed, in RME tissues 323 genes were overexpressed. Pathway analysis software identified a group of genes involved in cell growth, morphology and motility. In patients with distant metastases especially transcription factors such as FOXF1 and LMO4 showed a high expression, which were described as determinants of tumor cell migration. Down-regulation of these factors inhibited the invasion of RMS cells >10-fold. Microarray technology is a powerful method not only to classify RMS samples, but also to identify major regulatory processes. Functional evaluation of LMO4 and FOXF1 identified targets of a molecular network for preventing metastatic invasion in RMS.

Multidrug resistance (MDR) is a major reason for poor treatment results in hepatoblastoma (HB). The objective of this study was to establish a drug resistance model for HB to analyse alternative treatment options in vitro. Both HB cell lines HUH6 and HepT1 were xenotransplanted in NMRI mice (nu/nu) and 2 cycles of cisplatin (CDDP) treatment were administered. Thereafter, xenotransplants were excised and viable tumour cells were re-cultured. 3D cultures of HUH6 and HepT1 cells were generated on a low binding culture surface. Cell viability in response to CDDP/DOXO (doxorubicin) and apoptosis was assessed by MTT-assay and caspase 3 activity, respectively. Efflux of doxorubicin was measured by flow cytometry. Cellular levels of ABC-transporters (MDR1, MRP1, cMOAT and BRCP) were determined by real time rt-PCR. Only HepT1 cells isolated from HB xenografts showed resistance to CDDP, but did not survive repeated passages. Culturing HUH6 and HepT1 cells as spheroids was successful and 3D cultures showed an IC50-drift to higher drug concentrations for CDDP and DOXO compared to 2D cultures. Treatment with CDDP and DOXO led to homogeneous apoptosis in spheroids. Increased doxorubicin efflux in HUH6 spheroids was not influenced by the P-glycoprotein inhibitor tariquidar. Expression levels of MDR1, MRP1, cMOAT and BRCP in 3D cultures were similar to those in 2D cultures and were higher in HepT1 than in HUH6 cells. In conclusion, a 3D cell culture model for multidrug resistance was established for hepatoblastoma. The underlying mechanism involves altered accessibility of the cells for drugs rather than up-regulation of ABC-transporters.

The outcome of patients with advanced stage rhabdomyosarcoma (RMS) is still sobering. This outcome has not improved through conservative treatments. Therefore, novel treatment approaches such as immunotherapy need to be evaluated in human-adapted animal models. The aim of this study was to develop a humanized mouse model of childhood RMS as a basis for the study of immunotherapeutic approaches. Therefore, NOD/LtSz-scid IL2rgammanull-mice were used for all the experiments (n=19). The animals underwent sublethal irradiation on days 1 and 2 (1 x 300 cGy). After irradiation, the transplantation of human CD34+-cells (1,000,000 cells per animal i.v.) was carried out. Five animals served as the control and did not undergo stem cell transplantation. The engraftment of human cells was assessed in peripheral blood on days 21 and 55 by FACS analysis. Eight weeks after transplantation, the subcutaneous xenotransplantation of human alveolar and embryonal RMS cell lines was carried out. Tumor growth was monitored and tumors were resected 93 days after CD34+-transplantation. The tumor specimens were evaluated histologically. The successful engraftment of human cells with the establishment of a human immune system was observed in 12 out of 14 animals. B and T cells were mostly detected in the peripheral blood. There were only a few monocytes and almost no natural killer cells. The xenotransplantation of alveolar RMS resulting in subcutaneous tumor growth was feasible in 7 animals. The xenotransplantation of embryonal RMS was performed in 5 animals and led to tumor growth in 1 animal. A histological work up showed either alveolar or embryonal RMS cells with central necrosis. This is the first time a xenotransplantation model of human RMS has been developed in a humanized mouse model. The establishment of subcutaneous tumor xenografts was more effective in the alveolar subtype. This model offers a basic tool for further analyzing novel immunotherapeutic approaches in RMS, and could possibly be used in other solid pediatric tumors.

Multidrug resistance (MDR) is a common problem in the treatment of childhood rhabdomyosarcoma (RMS). A complete reversal of MDR is currently not possible. The aim of this study was to investigate the role of glutathione-S-transferase (GST) as mechanism of MDR in childhood RMS and to analyze possible reversal strategies. Female athymic mice underwent xenotransplantation with embryonal or alveolar RMS cells and were treated with vincristine. Gene expression analysis using Affymetrix HU-Gene 1.0 arrays revealed 2314 differentially expressed genes between the groups in alveolar RMS and 1387 in embryonal RMS. Ingenuity pathway analysis revealed a cluster of 5 overexpressed genes of the GST family in animals treated with vincristine, putative mediating the development of MDR. In order to analyze possible GST activity after chemotherapy with other commonly used drugs (doxorubicin, topotecan), cell culture experiments with alveolar and embryonal RMS cells were carried out. Specific GST activity was quantified using the clorodinitrobenzol conjugation with glutathione. Increased GST activity was found after incubation with cytotoxic agents in all cell lines. Highest induction of GST activity was found in embryonal RMS (up to 12-fold). After incubation with the GST inhibitors, tumor cell viability was decreased depending on the type of tumor cell and inhibitor used. We detected a novel mechanism for MDR in childhood RMS mediated via genes and proteins of the GST family. Reversal of these effects may be achieved by GST inhibitors in part. The GST family represents a promising target for further treatment strategies in childhood RMS.