Adjuvant treatment of pancreatic carcinoma in a clinically adapted mouse resection model.

BACKGROUND: The high rate of local recurrence after radical resection of pancreatic adenocarcinoma fosters intensive efforts to develop new approaches for adjuvant treatment. The established animal models show significant limitations in simulating an adjuvant therapeutic setting. For optimal approximation to the clinical situation we therefore improved a murine orthotopic human xenotransplantation model. METHODS: Subtotal pancreatectomy in mice was performed after orthotopic inoculation of human pancreatic cancer cells and manifestation of solid tumours. The natural course of disease, tumour growth and metastases were analysed. Gemcitabine as a cytotoxic drug was tested in vitro on the cell line used in this model and the effect of adjuvant treatment with gemcitabine in vivo was investigated. RESULTS: All tumour-resected animals showed local recurrence. Organ metastases occurred in 67% in resected compared to 25% of non-resected animals. Gemcitabine in vitro was ineffective, but as adjuvant monotherapy resulted in a highly significant reduction of tumour weight and metastatic events. CONCLUSION: Subtotal pancreatectomy for xenotransplanted pancreatic cancer in SCID beige mice is feasible. Due to high rates of local recurrence and increased organ metastases, this model offers a relevant option for preclinical adjuvant testing, especially as in vitro and in vivo effects of cytotoxic drugs differ enormously.

Resistance developing after long-term ganciclovir prodrug treatment in a preclinical model of NSCLC.

BACKGROUND: We recently demonstrated a 100% increase in the survival period with ganciclovir (GCV) therapy in mice hearing orthotopic HSV-TK-positive non-small cell lung cancer (NSCLC) tumors. However, long-term survival was not achieved. The aim of the present study was to evaluate tumor growth during extended GCV therapy and to monitor the herpes simplex virus thymidine kinase (HSV-TK) gene and protein in tumors at different time points. MATERIALS AND METHODS: The human NSCLC cell line KNS 62 was retrovirally transduced with the HSV-TK30 gene. Cell suspensions in which 100% or 25% of the cells were TK30-positive were inoculated subcutaneously in SCID bg mice. Tumor growth was evaluated during GCV therapy and HSV-TK DNA, RNA and protein were analyzed at different time points using PCR, RT-PCR and immunoblotting. RESULTS: HSV-TK DNA, RNA and TK30 protein were demonstrated in the tumors 21 days after subcutaneous tumor inoculation. TK-positive tumors regressed during GCV therapy and tumors in which 25% of the cells were TK-positive grew significantly more slowly than control tumors did. After 4 weeks of GCV therapy, HSV-TK DNA, RNA and TK protein were not detectable in the remaining tumors, which were therefore resistant to further GCV therapy. CONCLUSION: Prodrug therapy of the NSCLC cell line KNS 62, including bystander effects, is sufficient. Nevertheless, GCV-resistant tumors develop after functional loss of the TK gene. In the clinical context, further studies will need to evaluate immunological bystander effects or combinations with other drugs.

Resistance of pancreatic cancer to gemcitabine treatment is dependent on mitochondria-mediated apoptosis.

Palliative chemotherapy with gemcitabine, a common mode of treatment of pancreatic cancer, has little influence on patients' survival. We investigated the impact of anti-apoptotic Bcl-xL protein and its antagonist Bax on gemcitabine-induced apoptosis in human pancreatic carcinoma cells in vitro and in vivo. The level of Bcl-xL and Bax expression was determined in 3 established pancreatic cancer cell lines that differ in their sensitivity to gemcitabine-mediated apoptosis. Bcl-xL and Bax genes were transduced into Colo357 cells by retroviral infection. In addition, cells were transfected with c-FLIP to assess involvement of CD95 and caspase-8. The impact of Bax/Bcl-xL expression on gemcitabine-sensitivity in vivo was evaluated in orthotopic Colo357 tumors in SCID mice. The apoptotic index revealed a strong inverse correlation between Bcl-xL expression and gemcitabine-induced apoptosis in the pancreatic carcinoma cell lines tested. Caspase-8 and Bid were cleaved in Colo357 cells exposed to gemcitabine, and there was no correlation with either Bcl-xL or with Bax expression. In contrast, the lack of mitochondrial transmembrane potential transition, release of cytochrome-c and absence of caspase-9- and PARP-cleavage showed a strong correlation with Bcl-xL expression. Expression of c-FLIP significantly increased the resistance towards gemcitabine. Orthotopically growing Colo357-bcl-xl tumors in SCID mice were refractory to gemcitabine treatment, and in contrast to the in vitro data, Colo357-bax tumors exhibited a 12-fold greater tumor regression than Colo357-wild-type tumors in the control group. Gemcitabine-induced apoptosis involves the mitochondria-mediated signaling pathway. A functional restoration of this pathway appears to be essential to overcome the resistance mechanisms of pancreatic tumor cells and to improve the response to therapy as demonstrated by Bax overexpression in a clinically relevant tumor model.

Influence of tumor-associated E-cadherin mutations on tumorigenicity and metastasis.

In this study, we investigated whether tumor-associated E-cadherin mutations impair the tumor-suppressive function of the cell adhesion molecule and influence metastasis formation in a severe combined immunodeficiency mouse model. The investigated E-cadherin mutations were in frame deletions of exons 8 (del 8) or 9 (del 9) and a point mutation in exon 8 (p8). Transfected human MDA-MB-435S carcinoma cells stably expressing wild-type (wt) or mutant E-cadherin were injected into the mouse mammary fat pad. Mice transplanted with wt E-cadherin transfectants developed significantly smaller tumors than animals transplanted with the E-cadherin-negative parental cell line. Animals transplanted with del 9 or p8 E-cadherin transfectants produced medium size tumors, indicating that these mutations impair the tumor-suppressive function of E-cadherin. In contrast, mice transplanted with del 8 E-cadherin transfectants developed tumors of approximately the same sizes as animals transplanted with wt E-cadherin expressing cells. Lung metastases were induced by all cell lines without significant differences. Immunohistochemical analysis of E-cadherin expression in the tumors revealed a heterogeneous staining pattern, indicating loss or down-regulation of E-cadherin in some tumor cells. Metastases were completely negative for E-cadherin. Our data suggest that the type of mutation determines whether the tumor-suppressive function of E-cadherin is impaired.

Usage of the NF-kappaB inhibitor sulfasalazine as sensitizing agent in combined chemotherapy of pancreatic cancer.

Sulfasalazine is commonly used as an anti inflammatory agent and is known as a potent inhibitor of NF-kappaB. Some pancreatic carcinomas are characterized by a constitutively elevated NF-kappaB activity accounting for chemoresistance. To elucidate whether blockade of NF-kappaB activity with sulfasalazine is suitable for overcoming this chemoresistance in vivo, we employed a mouse model with subcutaneously xenotransplanted human Capan-1 pancreatic carcinoma cells. Fourteen days upon tumor inoculation, animals were randomized in 6 groups, receiving no treatment, treatment with gemcitabine or with etoposide, either alone or in combination with sulfasalazine, or with sulfasalazine alone. Two therapy regimens were given with a 7-day interval in between. Upon treatment with etoposide or gemcitabine alone, tumor sizes were moderately reduced to 65-68% and 50-65%, respectively, as compared to untreated tumors. Sulfasalazine alone only decreased temporarily the tumor sizes. Sulfasalazine in combination with gemcitabine showed only partially higher reduction in tumor sizes than gemcitabine alone, whereas the combination with etoposide reduced significantly the tumor sizes in all experiments (down to 20%). TUNEL-staining showed higher numbers of apoptotic cells in tumors from the combination groups, in particular with etoposide, and proliferation as indicated by Ki67 staining was strongly reduced. Furthermore, combined treatment of sulfasalazine with the cytostatic drugs led to a decreased blood vessel density. Immunohistochemical staining of the activated p65 subunit showed that sulfasalazine treatment abolished the basal NF-kappaB activity in tumor xenografts. These data imply that the well established anti-inflammatory drug sulfasalazine sensitizes pancreatic carcinoma cells to anti cancer drugs, in particular to etoposide in vivo by inhibition of NF-kappaB. This combined chemotherapy offers great potential for improved anti-tumor responses in pancreatic carcinomas.

Ganciclovir prodrug therapy is effective in a murine xenotransplant model of human lung cancer.

BACKGROUND: Therapy failures have been reported for retroviral gene transfer of herpes simplex virus thymidine kinase (HSV-TK) gene followed by systemic ganciclovir application in human lung cancer. Use of the HSV-TK mutant TK30 in combination with a VSV-G pseudotyped retroviral vector was found to enhance the efficacy of prodrug therapy. The present study evaluated this therapeutic strategy in human non-small cell lung cancer cell lines in a preclinical murine xenotransplant model. METHODS: Intrathoracally induced by HSV-TK30 transduced non-small cell lung cancer cell lines Colo 699 (adenocarcinoma) and KNS 62 (squamous cell carcinoma) were monitored for local tumor growth, survival, and metastases. So-called bystander effects were investigated in tumors consisting of as little as 25% TK30 transfected cells and by analysis of gap junctional protein connexin-43 expression. RESULTS: Survival was significantly prolonged, and tumor growth and pleural metastases were reduced in HSV-TK30-positive tumors of both cell lines. A significant therapeutic effect in bystander experiments was observed in squamous cell carcinoma. This was correlated with higher expression of connexin-43. CONCLUSIONS: Delivery of HSV-TK30 in a VSV-G pseudotyped retroviral vector and subsequent ganciclovir application provided therapeutic efficacy. Despite of low transduction rates achievable in gene transfer in situ, prodrug therapy appears to be feasible in tumor cells with efficient bystander effects.