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ABSTRACT: TRAIL selectively kills cancer cells while bispecific antibody EpCAMxCD3 guides effector lymphocytes to cancer cells. Arming of ex vivo constructed TRAIL-lymphocytes with EpCAMxCD3 enhances contact time and affinity between lymphocytes and tumor cells and enforces tumor elimination. This boosts endogenous immune responses and augments the effect of cytotoxic tumor therapy.
Oncoimmunology. 08/2012; 1(5):741-743.
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Ariane Groth,
Alexei V Salnikov,
Sabine Ottinger,
Jury Gladkich,
Li Liu,
Georgios Kallifatidis,
Olga Salnikova,
Eduard Ryschich,
Nathalia Giese,
Thomas Giese,
Frank Momburg,
Markus W Büchler,
Gerhard Moldenhauer,
Ingrid Herr
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ABSTRACT: To enhance T-cell responsiveness toward cancer cells, we overexpressed TRAIL in lymphocytes, as this death ligand induces tumor-specific apoptosis. To increase contact time of lymphocytes with tumor cells and thereby of TRAIL with its death receptors, lymphocytes were linked to the CD3 arm of bispecific antibody EpCAMxCD3, to guide the lymphocytes to tumor cells positive for the cancer stem cell marker EpCAM/ESA.
Lymphocytes were transduced with TRAIL lentivirus and the antitumor effect in presence and absence of EpCAMxCD3 was evaluated in vitro and in xenograft studies using epithelial cell adhesion molecule (EpCAM)-positive pancreatic and prostate cancer cells.
Compared with control lymphocytes, TRAIL-lymphocytes increased cytotoxicity and further induced expression of several apoptosis-related molecules. Cotransplantation of TRAIL-lymphocytes and tumor cells in mice or peritumoral injection of TRAIL-lymphocytes in larger xenografts retarded growth and induced apoptosis. Combination of TRAIL-lymphocytes with EpCAMxCD3 potentiated tumor eradication by enhancing antiapoptotic and antiproliferative signaling and by decreasing tumor vasculature. Intratumoral cyst formation was involved and associated with enhanced chemokine secretion and infiltration of mouse macrophages, suggesting contribution of an inflammatory host response. Most importantly, tumorigenicity of pancreatic cancer cells with cancer stem cell features resistant to conventional chemotherapy was strongly reduced.
This gene-immunotherapeutic approach may be a new tool to support endogenous immune responses toward cancer even in its advanced stages.
Clinical Cancer Research 02/2012; 18(4):1028-38. · 7.74 Impact Factor
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Vanessa Rausch,
Li Liu,
Anja Apel,
Theresa Rettig,
Jury Gladkich,
Sabrina Labsch,
Georgios Kallifatidis,
Adam Kaczorowski, Ariane Groth,
Wolfgang Gross,
Martha M Gebhard,
Peter Schemmer,
Jens Werner,
Alexei V Salnikov,
Hanswalter Zentgraf,
Markus W Büchler,
Ingrid Herr
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ABSTRACT: Involvement of dysregulated autophagy in cancer growth and progression has been shown in different tumour entities, including pancreatic ductal adenocarcinoma (PDA). PDA is an extremely aggressive tumour characterized by a small population of highly therapy-resistant cancer stem cells (CSCs) capable of self-renewal and migration. We examined whether autophagy might be involved in the survival of CSCs despite nutrition and oxygen deprivation typical for the hypoxic tumour microenvironment of PDA. Immunohistochemistry revealed that markers for hypoxia, CSCs and autophagy are co-expressed in patient-derived tissue of PDA. Hypoxia starvation (H/S) enhanced clonogenic survival and migration of established pancreatic cancer cells with stem-like properties (CSC(high)), while pancreatic tumour cells with fewer stem cell markers (CSC(low)) did not survive these conditions. Electron microscopy revealed more advanced autophagic vesicles in CSC(high) cells, which exhibited higher expression of autophagy-related genes under normoxic conditions and relative to CSC(low) cells, as found by RT-PCR and western blot analysis. LC3 was already fully converted to the active LC3-II form in both cell lines, as evaluated by western blot and detection of accumulated GFP-LC3 protein by fluorescence microscopy. H/S increased formation of autophagic and acid vesicles, as well as expression of autophagy-related genes, to a higher extent in CSC(high) cells. Modulation of autophagy by inhibitors and activators resensitized CSC(high) to apoptosis and diminished clonogenicity, spheroid formation, expression of CSC-related genes, migratory activity and tumourigenicity in mice. Our data suggest that enhanced autophagy levels may enable survival of CSC(high) cells under H/S. Interference with autophagy-activating or -inhibiting drugs disturbs the fine-tuned physiological balance of enhanced autophagy in CSC and switches survival signalling to suicide.
The Journal of Pathology 01/2012; 227(3):325-35. · 6.32 Impact Factor
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Ariane Groth,
Sabine Ottinger,
Christian Kleist,
Elisabeth Mohr,
Mohammad Golriz,
Daniel Schultze,
Helge Bruns,
Arianeb Mehrabi,
Peter Schemmer,
Markus W Büchler,
Ingrid Herr
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ABSTRACT: Mesenchymal stem cell (MSC) transplantation is suggested for therapy of end-stage liver disease, due to e.g. liver cancer and metastasis. Liver transplantation is the only therapeutic option so far but donor organs are short. Also, the availability of allogeneic human MSCs for liver regeneration is limited. Therefore, we evaluated the suitability of porcine bone marrow MSCs from semi-adult pigs and found that morphology, surface expression pattern and multilineage differentiation are similar to those of human MSCs. Porcine MSCs differentiated to a hepatocyte-like phenotype and expressed porcine mRNA of typical liver proteins. However, hepatocyte-like MSCs failed to express the corresponding proteins and did not produce glycogen and urea as primary porcine hepatocytes do. Porcine MSCs were immunotolerated, since they did not activate resting human PBMCs, and were not attacked by human activated PBMCs. However, porcine MSCs led to enhanced proliferation of human pre-activated PBMCs suggesting that immunotoleration of porcine MSCs in the human system has limitations. Together, the potential of porcine MSCs for xenogenous use in human liver therapy is promising but needs further evaluation prior to clinical use.
International Journal of Oncology 09/2011; 40(2):391-401. · 2.40 Impact Factor
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ABSTRACT: Human natural killer (NK) cells recognize and efficiently eliminate MHC class I low or negative malignant targets and virally infected host cells, without requirement for prior sensitization. However, viruses and various tumor cells display elaborate adaptations to evade and overcome immunosurveillance. The current review focuses on escape mechanisms of viruses and malignantly transformed 'stressed' cells to evade from NK cell cytotoxicity. A general overview of recent clinical studies using allogeneic donor NK cells is given, summarizing first data about a possible benefit for patients suffering from high-risk leukemia and solid tumors. Finally, the review discusses the future perspectives and hypotheses aiming to improve therapeutic NK cell strategies against tumor immune escape mechanisms.
Journal of Innate Immunity 01/2011; 3(4):344-54. · 4.21 Impact Factor
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ABSTRACT: Patients with pancreatic cancer have a poor survival rate, and new therapeutic strategies are needed. Epithelial cell adhesion molecule (EpCAM), suggested as a marker for cancer stem cells, is over-expressed on most pancreatic tumour cells but not on normal cells and may be an ideal therapeutic target. We evaluated the anti-tumour efficiency of bispecific EpCAMxCD3 antibody linking tumour cells and T lymphocytes. In NOD SCID mice, EpCAMxCD3 had a long serum half-life (t(1/2) approximately 7 days). EpCAMxCD3 significantly retarded growth of BxPC-3 pancreatic carcinoma xenografts. For mimicking a pancreatic cancer microenvironment in vitro, we used a three-dimensional tumour reconstruct system, in which lymphocytes were co-cultured with tumour cells and fibroblasts in a collagen matrix. In this in vivo-like system, EpCAMxCD3 potently stimulated production of the effector cytokines IFN-gamma and TNF-alpha by extracorporally pre-activated lymphocytes. Moreover, compared with a bivalent anti-CD3 antibody, EpCAMxCD3 more efficiently activated the production of TNF-alpha and IFN-gamma by non-stimulated peripheral blood mononuclear cells. Most excitingly, we demonstrate for the first time that EpCAMxCD3 induces prolonged contacts between lymphocytes and tumour cells, which may be the main reason for the observed anti-tumour effects. As an important prerequisite for future use in patients, EpCAMxCD3 did not alter lymphocyte migration as measured by time-lapse video microscopy. Our data may open a way to improve the immune response and treatment outcome in patients with pancreatic cancer.
Journal of Cellular and Molecular Medicine 09/2009; 13(9B):4023-33. · 4.13 Impact Factor
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ABSTRACT: Great hope is set in the use of mesenchymal stem cells for gene therapy and regenerative medicine. Since the frequency of this subpopulation of stem cells in bone marrow is low, mesenchymal stem cells are expanded ex vivo and manipulated prior to experimental or clinical use. Different methods for isolation and expansion are available, but the particular effect on the stem cell character is unclear. While the isolation of mesenchymal stem cells by density centrifugation followed by selection of the plastic adherent fraction is frequently used, the composition of expansion media differs. Thus, in the present study we cultured mesenchymal stem cells isolated from five healthy young volunteers in three widely used expansion media and performed a detailed analysis of the effect on morphology, proliferation, clonogenicity, passaging, differentiation and senescence. By this way we clearly show that the type of expansion medium used determines the stem cell character and time of senescence which is critical for future gene therapeutic and regenerative approaches using mesenchymal stem cells.
Experimental Cell Research 01/2009; 315(3):498-507. · 3.58 Impact Factor
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G Kallifatidis,
V Rausch,
B Baumann,
A Apel,
B M Beckermann, A Groth,
J Mattern,
Z Li,
A Kolb,
G Moldenhauer,
P Altevogt,
T Wirth,
J Werner,
P Schemmer,
M W Büchler,
A V Salnikov,
I Herr
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ABSTRACT: Emerging evidence suggests that highly treatment-resistant tumour-initiating cells (TICs) play a central role in the pathogenesis of pancreatic cancer. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a novel anticancer agent; however, recent studies have shown that many pancreatic cancer cells are resistant to apoptosis induction by TRAIL due to TRAIL-activated nuclear factor-kappaB (NF-kappaB) signalling. Several chemopreventive agents are able to inhibit NF-kappaB, and favourable results have been obtained--for example, for the broccoli compound sulforaphane-in preventing metastasis in clinical studies. The aim of the study was to identify TICs in pancreatic carcinoma for analysis of resistance mechanisms and for definition of sensitising agents.
TICs were defined by expression patterns of a CD44(+)/CD24(-), CD44(+)/CD24(+) or CD44(+)/CD133(+) phenotype and correlation to growth in immunodeficient mice, differentiation grade, clonogenic growth, sphere formation, aldehyde dehydrogenase (ALDH) activity and therapy resistance.
Mechanistically, specific binding of transcriptionally active cRel-containing NF-kappaB complexes in TICs was observed. Sulforaphane prevented NF-kappaB binding, downregulated apoptosis inhibitors and induced apoptosis, together with prevention of clonogenicity. Gemcitabine, the chemopreventive agents resveratrol and wogonin, and the death ligand TRAIL were less effective. In a xenograft model, sulforaphane strongly blocked tumour growth and angiogenesis, while combination with TRAIL had an additive effect without obvious cytotoxicity in normal cells. Freshly isolated patient tumour cells expressing markers for TICs could be sensitised by sulforaphane for TRAIL-induced cytotoxicity.
The data provide new insights into resistance mechanisms of TICs and suggest the combination of sulforaphane with TRAIL as a promising strategy for targeting of pancreatic TICs.
Gut 11/2008; 58(7):949-63. · 10.11 Impact Factor
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ABSTRACT: Hepatocellular carcinoma is one of the most aggressive solid tumours associated with poor prognosis. Despite its significance, there is only an elemental understanding of the mechanisms that drive disease pathogenesis, and there are just limited therapy options. The medical community is currently experiencing a wave of enthusiasm for clinical trials, in which adult stem/progenitor cells are used for liver regeneration. This is based on promising results in animal models and encouraging reports from some initial clinical studies. On the other hand, several essential precautions are not being fully addressed. Stem cells may contribute to fibrosis or give rise to hepatic cancer stem cells as a source of hepatocellular carcinoma. This review outlines the current state of knowledge in progression of liver disease and highlights the function of adult stem cells in disease and therapy.
International Journal of Cancer 12/2007; 121(9):1875-82. · 5.44 Impact Factor
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Ariane Groth
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ABSTRACT: The aim of this project was to test the efficacy of a novel therapeutic approach for cancer therapy. This approach combines immunotherapy with bispecific antibody (bsAb)EpCAMxCD3 and gene therapy with TRAIL. The potential efficacy of this approach was tested in experimental models of pancreatic and prostate carcinoma. Specific aims were i) to construct a lentiviral vector suitable for the transduction of human lymphocytes and the over-expression of TRAIL. The next aim was ii) to establish and to evaluate suitable xenograft models for the study of the chosen approach, iii) to test the efficacy and the anti-tumor potential of TRAIL-over-expressing lymphocytes and bsAb EpCAMxCD3 in vivo and iv) to elucidate the functional mechanisms underlying observed anti-tumor effects of TRAIL-over-expressing lymphocytes and bsAb EpCAMxCD3 in vivo and in vitro. In conclusion, this is the first study, which shows the effective lentiviral transduction of human lymphocytes with the death ligand TRAIL. The vector pV3TP2A effectively transduced human lymphocytes to over-express TRAIL. The majority of these TRAIL-over-expressing lymphocytes were CD8+ T cells and retained their cytotoxic functions, their migratory and proliferative properties after the transduction. Furthermore, this study showed that EpCAMxCD3 possesses a potent anti-tumor activity in vivo. In NOD/SCID mice, EpCAMxCD3 had a long serum half-life (t1/2 ∼ 7 days). In two mouse models the combination of EpCAMxCD3 with lymphocytes significantly retarded the growth of BxPc-3 pancreatic and PC-3 prostate cancer xenografts. For mimicking a pancreatic cancer microenvironment in vitro a 3D tumor reconstruct system was used, in which lymphocytes were co-cultured with EpCAM-expressing tumor cells and fibroblasts in a collagen matrix. In this in vivo-like system EpCAMxCD3 potently stimulated the production of the effector cytokines IFN-γ and TNF-α by extracorporally pre-activated lymphocytes. Moreover, compared with a bivalent anti-CD3 antibody, EpCAMxCD3 more efficiently activated production of TNF-α and IFN-γ by non-stimulated PBMCs. We demonstrate for the first time that EpCAMxCD3 induces prolonged contacts between lymphocytes and tumor cells, which may be one of the main reason for the observed anti-tumor effects. The combination with lymphocytes over-expressing the death ligand TRAIL could effectively enhance the anti-tumor effects of bsAb EpCAMxCD3. The combination with TRAIL-over-expressing lymphocytes enabled us to strongly reduce the dose of EpCAMxCD3 necessary for an anti-tumor effect. This is a great advantage, which could minimize potential side effects of bsAb treatment. EpCAMxCD3 did not alter lymphocyte migration as measured by time-lapse video microscopy, which is an important prerequisite for future use in patients. Apoptosis induction in tumor cells by TRAIL-over-expressing lymphocytes was confirmed in vitro in 3D tumor reconstructs. In cytotoxic killing assays, the anti-tumor effect of TRAIL-over-expressing lymphocytes was significantly enhanced by EpCAMxCD3. In 3D gels, lymphocytes were effective producers of IFN-γ, TNF-α and chemokines, which could also be detected in vivo from tumor lysates. An increased infiltration of the tumor islets with macrophages and granulocytes was observed upon treatment with TRAIL-over-expressing lymphocytes and EpCAMxCD3. A potential explanation for the demonstrated anti-tumor effect of EpCAMxCD3 and TRAIL-over-expressing lymphocytes combination therapy, is an enhancement of the observed effects of single treatment, namely the reduced proliferation in tumor cells, decreased blood vessels formation, local production of cytokines and chemokines, apoptosis and an increased infiltration of tumor tissue with macrophages. Conclusively, the combination of TRAIL-over-expressing lymphocytes and the bispecific antibody EpCAMxCD3 was very efficient in pancreatic and prostate xenograft models in vivo. This combination of gene therapy with immunotherapy could potentially be a possible therapeutic option for the clinical application in the future. Ziel dieser Studie war einen neuartigen Therapieansatz für die Krebstherapie zu testen. Dieser Ansatz kombiniert die Immuntherapie mit dem bispezifischem Antikörper (bsAk) EpCAMxCD3 mit einer Gentherapie mit TRAIL. Die potentielle Wirksamkeit dieser Therapie wurde in experimentellen Modellen des Prostata und des Pankreaskarzinoms getestet. Konkrete Ziele dabei waren i) die Generierung eines lentiviralen Vektors, der geeignet für die Transduktion von humanen Lymphozyten ist und die Überexpression des Todesliganden TRAIL ermöglicht, ii) die Etablierung und die Evaluierung eines geeigneten Xenograft Models, iii) die Untersuchung der Effektivität und der Anti-Tumor Wirkung TRAIL- überexprimierender Lymphozyten mit bsAk EpCAMxCD3 in vivo und in vitro und iv) die Analyse des Funktionsmechanismus der beobachteten Anti-Tumor Wirkung von TRAIL-überexprimierender Lymphozyten und EpCAMxCD3 in vivo und in vitro. Zusammenfassend zeigte diese Studie dass EpCAMxCD3 eine potente Anti-Tumor-Wirkung in vivo besitzt. In NOD/SCID Mäusen besaß EpCAMxCD3 eine lange Serum Halbwertszeit von ungefähr 7 Tagen. In zwei unabhängigen Mausmodellen reduzierte EpCAMxCD3 zusammen mit Lymphozyten effizient das Tumorwachstum von pankreatischen BxPc-3 und Prostata PC-3 Xenografts. Um die Mikroumgebung eines Karzinoms in vitro nachzuahmen, wurde ein 3D Tumor Rekonstrukt benutzt in dem Lymphozyten zusammen mit EpCAM- exprimierenden Tumorzellen in Fibroblasten in einer Kollagenmatrix co-kultiviert wurden. In diesem in vivo artigem System stimulierte EpCAMxCD3 die Produktion der Effektorzytokine IFN-γ und TNF-α in voraktivierten Lymphozyten. Verglichen mit bivalenten anti-CD3 Antikörpern, stimulierte EpCAMxCD3 sogar die Produktion von IFN-γ und TNF-α von unstimulierten Lymphozyten. Wir demonstrieren hier erstmalig dass EpCAMxCD3 die Kontaktzeit zwischen Lymphozyten und Tumorzellen verlängert, was die Grundlage für die Anti-Tumor Wirkung sein könnte. Als eine wichtige Vorraussetzung für eine mögliche klinische Anwendung hatte EpCAMxCD3 keinen Einfluß auf die Migration von Lymphozyten, was mit Time-lapse Video Mikroskopie demonstriert wurde. Die Kombination mit Lymphozyten die den Todesliganden TRAIL überexprimierten verstärkte die Anti- Tumor-Wirkung von EpCAMxCD3 sehr effizient. Schon geringe Dosen von EpCAMxCD3 genügten um eine Anti-Tumor Wirkung zu erzielen in dieser Kombination mit TRAIL- überexprimierenden Lymphozyten. Diese Tatsache könnte vorteilhaft sein um mögliche Nebeneffekte der Behandlung zu minimieren. Die Induktion von Apoptose in Tumorzellen konnte in 3D Tumor Rekonstrukten in vitro gezeigt werden. In Versuchen zur Zytotoxizität von Lymphozyten auf Tumorzellen wurde die Wirkung von TRAIL-überexprimieren Lymphozyten noch durch EpCAMxCD3 signifikant gesteigert. In 3D Tumor Rekonstrukten wurden neben den Zytokinen IFN-γ und TNF-α auch Chemokine produziert, die sowohl in vitro als auch in Tumor Lysaten detektiert werden konnten. Diese Chemokine stehen höchstwahrscheinlich im Zusammenhang mit der vermehrten Infiltrierung des Tumorgewebes mit Makrophagen und Granulozyten nach der Behandlung mit TRAIL-überexprimierenden Lymphozyten und mit EpCAMxCD3. Eine mögliche Erklärung für die gezeigte Anti-Tumor Wirkung von TRAIL-überexprimierenden Lymphozyten und EpCAMxCD3 ist eine Reduktion der Blutgefäße, die lokale Produktion von Zytokinen und Chemokinen mit einer vermehrten Einwanderung von Makrophagen in das Tumor Gewebe. Zusammenfassend konnte die Kombination aus TRAIL-überexprimierenden Lymphozyten und dem bispezifischen Antikörper EpCAMxCD3 erfolgreich für die Therapie experimenteller Pankreas und Prostata Tumor Xenografts in vivo getestet werden.
