Susanne Serba

New doxorubicin-loaded phospholipid microbubbles for targeted tumor therapy: in-vivo characterization.

Doxorubicin(DOX) is a potent chemotherapy drug that is often limited by severe adverse effects such as cardiac toxicity and myelosupression. Drug targeting with non invasive techniques would be desirable, aiming at increased local drug concentration and reduced systemic side effects. Ultrasound(US) targeted destruction of drug loaded microbubbles(MBs) has evolved as a promising strategy for non invasive local gene and drug delivery. A recently developed novel DOX-loaded microbubble (DOX-MB) formulation was previously tested in-vitro, with optimal DOX loading capacity, ideal physical characteristics and preserved antiproliferative efficacy. The aim of this study was to evaluate applicability and efficacy of DOX-loaded MBs in a pancreas carcinoma model of the rat. First, immediate toxicity was tested in rats ruling out in-vivo MB agglomeration/capillary adhesion with subsequent embolisation/occlusion of the pulmonary vasculature. In a second set of experiments, tumors derived from pancreas carcinomas were implanted in both flanks of Lewis rats. After establishing the tumors, DOX-MBs were administered intravenously while one of the two tumors was exposed to US (1.3 MHz; mechanical index 1.6). DOX tissue concentration was measured in tumors and control organs after the experiment. Finally, efficacy of US targeted destruction of DOX-MBs in tumors was studied, looking at tumor growth after two therapeutic applications. All rats survived the DOX-MB administration without any sign of embolisation/occlusion of the pulmonary vasculature. US targeted destruction of DOX-MBs leads to a 12-fold higher tissue concentration of DOX and a significantly lower tumor growth in the target tumor compared to the contralateral control tumor. In conclusion, novel DOX-loaded MBs can be safely administered to rats, leading to a relevant increase in local drug concentration and reduction in tumor growth.

5-Fluorouracil and interferon-α immunochemotherapy enhances immunogenicity of murine pancreatic cancer through upregulation of NKG2D ligands and MHC class I.

Pancreatic cancer has the poorest prognosis of all gastrointestinal cancers, driving the need for new therapeutic approaches. Adjuvant 5-fluorouacil (5-FU) chemotherapy proved effective in increasing the survival of patients with resected tumors. Furthermore, the addition of interferon alpha (IFN-&agr;) immunotherapy to 5-FU has shown encouraging clinical results. The aim of this study was to determine the relevance of different immune cell populations, namely natural killer (NK) cells, CD8 T cells, and dendritic cells, in the anticancer immune response mediated by the combination therapy using an orthotopic mouse model of pancreatic carcinoma and to get more insight into the underlying mechanisms of action. Depleting CD8 T cells, NK cells, or dendritic cells significantly reduced the anticancer effects mediated by the combination therapy. Tumors of mice treated with 5-FU+IFN-&agr; harbored higher numbers of infiltrating NK cells in comparison with control mice. In addition, NK cells isolated from these mice showed enhanced cytotoxicity against Panc02 pancreatic cancer cells. Furthermore, 5-FU+IFN-&agr; treatment increased the expression of major histocompatibility complex class I and NKG2D ligands on Panc02 cells that could be a potential key for enhancing the immunogenicity of tumors. Understanding how this combination therapy enhances the immunogenicity of pancreatic tumors in our model may provide potential predictive biomarkers. This will allow to evaluate the efficacy of this immunochemotherapy more effectively in future clinical trials and to identify patients who will benefit most from it.

Transfection with CD40L induces tumour suppression by dendritic cell activation in an orthotopic mouse model of pancreatic adenocarcinoma

Objective: Patients with adenocarcinoma of the pancreas have only limited promising therapy options. Therefore, immunotherapeutic approaches might be considered promising and have gained importance over the last few years. In this study, CD40L gene transfer was tested as potent immunotherapy. Methods: The efficacy of CD40L gene transfer in initiating anti-tumour immune response was investigated in a pancreatic ductal adenocarcinoma orthotopic syngeneic mouse model. In addition, the role of dendritic cells was determined. Results: A significantly slower tumour growth rate and less metastasis were observed following administration of the CD40L plasmid. Such an effect of the plasmid was not observed in immunodeficient mice. Tumours of treated mice were found to be infiltrated with T cells and dendritic cells. The latter were mature and of myeloid origin. Tumour-infiltrating lymphocytes were tumour-specific as shown in IFN-γ ELISPot assays. Using intravital microscopy it was possible to show a significant induction of leukocytes sticking to the tumour endothelium after CD40L treatment. Adoptive cell transfer experiments have revealed that tumour-derived dendritic cells and CD8 cells from CD40L-treated donor mice either harbour anti-tumour activity or induce it in the recipients. Distinctly, CD8 cells from donor spleens were found to migrate directly into the recipient’s tumour. Conclusions: The induction of anti-tumour activity initiated after treating mice with the CD40L plasmid was achieved. Further investigations showed that this is mediated by mature myeloid dendritic cells which activate CD8 cells. Clinical trials investigating CD40L-based therapies should be extended.

Involvement of the epidermal growth factor receptor in the modulation of multidrug resistance in human hepatocellular carcinoma cells in vitro

BackgroundHepatocellular carcinoma (HCC) is a molecular complex tumor with high intrinsic drug resistance. Recent evidence suggests an involvement of the tyrosine kinase pathway in the regulation of ATP-binding cassette protein (ABC-transport protein) mediated multidrug resistance in cancer cells. The aim of this study was to examine whether EGFR inhibition sensitizes HCCs to chemotherapy and to elucidate its mechanism.ResultsChemotherapeutic treatment induces multidrug resistance and significantly increases ABC-transport protein expression and function in a time- and dose-dependent manner in HCC cells. Furthermore, cytostatic treatment increases the mRNA expression of tyrosine kinases and induces the phosphorylation of ERK. EGF activation of the tyrosine kinase pathway up-regulated the ABC-transport protein mRNA expression and enhanced the survival of resistant HCC cells. Consistent with these effects, inhibition of the EGFR using siRNA decreased the ABC-transport protein mRNA expression and inhibited the proliferation of resistant cells. Additional treatment with Gefitinib, a clinically approved EGFR inhibitor, caused a dose-dependent reversal of resistance to conventional chemotherapy.ConclusionThe present study demonstrates that the multidrug resistance of HCC is modulated through the EGF-activated tyrosine kinase cascade. Consequentially, the restoration of chemosensitivity by EGFR inhibition may lead towards new tailored therapies in patients with highly resistant tumors.

Ex vivo expanded telomerase‐specific T cells are effective in an orthotopic mouse model for pancreatic adenocarcinoma

Telomerase activity is over‐expressed in nearly all pancreatic carcinomas, but not in chronic pancreatitis. Here, we investigated various protocols for expansion of telomerase‐specific T cells for adoptive cell transfer and their use in a syngeneic pancreatic carcinoma mouse model. Telomerase‐specific T cells were generated by stimulation of splenocytes from peptide‐immunized donor mice with either interleukin (IL)‐2, IL‐15, artificial antigen‐presenting cells, anti‐signalling lymphocyte activation molecule (SLAM) microbeads or allogeneic dendritic cells in combination with a limited dilution assay. T cells were tested for antigen specificity in vitro and for anti‐tumour activity in syngeneic mice with orthotopically implanted tumours pretreated with cyclophosphamide. The immune cells from recipients were immunophenotyped. During a period of 2 weeks, the expansion approach using IL‐2 was very successful in generating a high number of telomerase‐specific CD8+ T cells without losing their function after adoptive cell transfer. Significantly slower tumour growth rate and less metastasis were observed after adoptively transferring telomerase specific CD8+ T cells, expanded using IL‐2. Further investigations showed that anti‐tumour efficacy was associated with a significant shift from naive CD8+ T cells to CD8+ central memory T cells, as well as recruitment of a high number of dendritic cells. Remarkable amounts of telomerase‐specific T cells were detectable in the tumour. Generation of telomerase‐specific T cells is feasible, whereat IL‐2‐based protocols seemed to be most effective and efficient. Antigen‐specific T cells showed significant cytotoxic activity in a syngeneic, orthotopic mouse model, whereas central memory T cells but not effector memory T cells appear to be of high importance.

Immunological in vivo effects of B7-H1 deficiency.

B7-H1 regulatory protein, a member of the B7-H family, plays a crucial role in the modulation of immune response in healthy steady-state conditions as well as in different pathologies. B7-H1 knockout mice represent an important model to elucidate further molecular and cellular mechanisms involved, among others, in autoimmunity development and cancer progression. However, a deep immunologic characterization of this model is not complete yet. This study examined the role of B7-H1 in vivo further by direct comparison of specifically phenotyped spleen immune-cell subpopulations and their activation and naïve/memory state as well as cytokine profile in wild-type and B7-H1 knockout mice. Our results demonstrated that B7-H1 deficiency in vivo modulates several immunological parameters, including the amount and composition of Gr1(+)CD11b(+) myeloid population, the composition and activation state of the DC compartment, the frequency and status of NK and NKT cells, B-cells, naïve/memory state of CD8 T-cells and production of IL-2 and IL-10 cytokines. Moreover, we observed an increase in the PD-1 expression in the immune cells in B7-H1 knockout mice compared to the wild-type animals. Valuing the importance of B7-H1 knockout mice for their use in disease models, these data underline the role of B7-H1 in vivo also in healthy state and should be taken into account in future studies on this immunosuppressive molecule.

Anti-tumor properties of the cGMP/protein kinase G inhibitor DT3 in pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in the world. Therefore, new therapeutic options are urgently needed to improve the survival of PDAC patients. Protein kinase G (PKG) conducts the interlude of cGMP signaling which is important for healthy as well as for cancer cells. DT3 is a specific inhibitor of PKG, and it has been shown to possess an anti-tumor cytotoxic activity in vitro. The main aim of this work was to investigate anti-tumor effects of DT3 upon PDAC in vivo.Expression of PKG was assessed with real-time PCR analysis in the normal and tumor pancreatic cells. In vitro cell viability, proliferation, apoptosis, necrosis, migration, and invasion of the murine PDAC cell line Panc02 were assessed after DT3 treatment. In vivo anti-tumor effects of DT3 were investigated in the murine Panc02 orthotopic model of PDAC. Western blot analysis was used to determine the phosphorylation state of the proteins of interest.Functional PKGI is preferentially expressed in PDAC cells. DT3 was capable to reduce viability, proliferation, and migration of murine PDAC cells in vitro. At the same time, DT3 treatment did not change the viability of normal epithelial cells of murine liver. In vivo, DT3 treatment reduced the tumor volume and metastases in PDAC-bearing mice, but it was ineffective to prolong the survival of the tumor-bearing animals. In addition, DT3 treatment decreased phosphorylation of GSK-3, P38, and CREB in murine PDAC.Inhibition of PKG could be a potential therapeutic strategy for PDAC treatment which should be carefully validated in future pre-clinical studies.

Abstract 1791: Immunochemotherapy with 5-Fluorouracil and Interferon-α enhances the recognition of pancreatic cancer cells by NK and CD8+ T cells via increasing the expression of NKG2D ligands and MHC Class-I

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Pancreatic cancer has the poorest prognosis of all cancers urging the necessity of new therapeutic approaches. Recent clinical data show encouraging results for combining Interferon-α (IFN-α) immunotherapy with 5-Fluorouacil (5-FU) chemotherapy. However, the role of IFN-α in this immunochemotherapy scheme is still elusive. Here we investigate in vivo the relevance of different immune cells in the anti-cancer immune response mediated by IFN-α in combination with 5-FU using an orthotopic mouse model of pancreatic carcinoma. Luciferase-transfected Panc02 cells were implanted in the pancreas of C57BL/6 mice. Five days later, mice were treated with 5-FU alone, 5-FU + IFN-α or with a vehicle control. In parallel, CD4+ T cells, CD8+ T cells, and NK cells were depleted by neutralizing antibodies. Depletion of CD11c+ dendritic cells (CD11c+ DCs) and regulatory T cells (T regs) was performed using transgenic mice based on the diphtheria toxin receptor-mediated conditional and targeted cell ablation. On day 21, tumor volume was measured and NK cells were isolated and enriched from spleens of mice then used as effectors against Panc02 cells in a standard chromium release assay. Tumors were frozen for further immunohistochemistry analysis to evaluate the immune cells infiltration. In parallel, we performed flow cytometry analysis to evaluate the effects of different treatments on the expression of MHC Class-I and NKG2D ligands (MULT-1 and Rae-1) in Panc02 cells in vitro and in the pancreatic tumors in vivo. Our data show that treatment with 5-FU + IFN-α has significantly decreased tumor volume in comparison with control or 5-FU treatments. This decrease in tumor volume was remarkably abolished by depleting CD8+ T cells or NK cells. The tumors of 5-FU + IFN-α treated mice harbor higher numbers of infiltrating NK cells in comparison with control mice. Furthermore, NK cells isolated from spleens of 5-FU + IFN-α treated mice showed enhanced cytotoxicity towards Panc02 cells. Moreover, 5-FU + IFN-α treatment increased the expression of MHC Class-I and NKG2D-ligands: MULT-1 and Rae-1 in Panc02 cells both in vitro and in vivo that could be the key for the enhanced recognition and per se killing by CD8+ T cells and NK cells respectively. Depletion of CD11c+ DCs did also abrogate the anti-cancer effects mediated by the combination therapy. The ongoing work will give more information about the role of CD11c+ DCs, CD4+ T cells and Tregs in orchestrating the immune response mediated by 5-FU + IFN-α. To conclude, this study gives more insight about the mechanism of action of INF-α in combination with 5-FU and introduces CD8+ T cells and NK cells as main players in the observed anti-cancer immune response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1791. doi:10.1158/1538-7445.AM2011-1791

Abstract B15: Immunochemotherapy with 5-fluorouracil and interferon-enhances innate and adoptive immune responses against pancreatic cancer in vivo

Pancreatic cancer has the poorest prognosis of all cancers urging the necessity of new therapeutic approaches. Recent clinical data show encouraging results for combining Interferon-α (IFN-α) immunotherapy with 5-Fluorouacil (5-FU) chemotherapy. However, the role of IFN-α in this immunochemotherapy scheme is still elusive. Here we investigate in vivo the relevance of different immune subpopulations in the anti-cancer immune response mediated by IFN-α in combination with 5-FU. Luciferase-transfected Panc02 cells were implanted in the pancreas of C57BL/6 mice. Five days later, mice were treated with 5-FU alone, 5-FU + IFN-α or with a vehicle control. In parallel, CD4+ T cells, CD8+ T cells, and NK cells were depleted by neutralizing antibodies. Depletion of CD11c+ dendritic cells (DCs) was performed using transgenic CD11c.DTR mice. On day 21, tumor volume was measured and NK cells were isolated and enriched from spleens of mice then used as effectors against Panc02 cells in a standard chromium release assay. Tumors were frozen for further immunohistochemistry analysis to evaluate the immune cells infiltration. In parallel, we performed flow cytometry analysis to evaluate the effects of different treatments on the expression of MHC-I and NKG2D ligands (MULT-1 and Rae-1) on Panc02 cells. Our data show that treatment with IFN-α + 5-FU has significantly decreased tumor volume in comparison with control or 5-FU treatments. This decrease in tumor volume was remarkably abolished by depleting CD8+ T cells or NK cells. Furthermore, NK cells isolated from spleens of IFN-α + 5-FU treated mice showed enhanced cytotoxicity towards Panc02 cells. Moreover, IFN-α + 5-FU treatment enhances the expression of MHC-I and NKG2D-ligand MULT-1 on Panc02 cells which could be the possible key for the enhanced recognition and per se killing by CD8+ T cells and NK cells respectively. To conclude, this study gives more insight about the mechanism of action of INF-α in combination with 5-FU and introduces CD8+ T cells and NK cells as main players in the observed anti-cancer immune response. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B15

Abstract 5597: NK cells and CD8+ T cells in orthotopic pancreatic cancer model: Mediation of anti-cancer immune response by 5-fluorouracil and interferon-α

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Pancreatic cancer has the poorest prognosis of all cancers urging the necessity of new therapeutic approaches. Recent clinical data show encouraging results for combining Interferon-α (IFN-α) immunotherapy with 5-Fluorouacil (5-FU) chemotherapy. However, the role of IFN-α in this immunochemotherapy scheme is still elusive. Here we investigate in vivo the relevance of different immune subpopulations in the anti-cancer immune response mediated by IFN-α in combination with 5-FU. Luciferase-transfected Panc02 cells were implanted in the pancreas of C57BL/6 mice. Five days later, mice were treated with 5-FU alone, 5-FU + IFN-α or with a vehicle control. In parallel, CD4+ T cells, CD8+ T cells, and NK cells were depleted by neutralizing antibodies. Depletion of CD11c+ dendritic cells (DCs) was performed using transgenic CD11c. DTR mice. On day 21, mice were sacrificed and tumors were measured and frozen for further immunohistochemical analysis to evaluate the immune cells infiltration. NK cells were isolated and enriched from spleens of mice and their lytic activity was tested against Panc02 cells using a standard chromium release assay. Our data show that treatment with IFN-α + 5-FU has significantly decreased tumor volume in comparison with control or 5-FU treatments. On the other hand, this decrease in tumor volume was remarkably abolished by depleting CD8+ T cells or NK cells. Furthermore, NK cells isolated from spleens of IFN-α + 5-FU treated mice showed enhanced cytotoxicity towards pancreatic cancer cells (Panc02). Meanwhile, studying the role of CD4+ T cells and DCs besides immunohistochemical analysis is still ongoing. To conclude, this study gives more insight about the mechanism of action of INF-α in combination with 5-FU and introduces CD8+ T cells and NK cells as main players in the observed anti-cancer immune response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5597.