The growth of glioblastoma orthotopic xenografts in nude mice is directly correlated with impaired object recognition memory

Cognitive dysfunction is found in patients with brain tumors and there is a need to determine whether it can be replicated in an experimental model. In the present study, the object recognition (OR) paradigm was used to investigate cognitive performance in nude mice, which represent one of the most important animal models available to study human tumors in vivo. Mice with orthotopic xenografts of the human U87MG glioblastoma cell line were trained at 9, 14, and 18days (D9, D14, and D18, respectively) after implantation of 5×10(5) cells. At D9, the mice showed normal behavior when tested 90min or 24h after training and compared to control nude mice. Animals at D14 were still able to discriminate between familiar and novel objects, but exhibited a lower performance than animals at D9. Total impairment in the OR memory was observed when animals were evaluated on D18. These alterations were detected earlier than any other clinical symptoms, which were observed only 22-24days after tumor implantation. There was a significant correlation between the discrimination index (d2) and time after tumor implantation as well as between d2 and tumor volume. These data indicate that the OR task is a robust test to identify early behavior alterations caused by glioblastoma in nude mice. In addition, these results suggest that OR task can be a reliable tool to test the efficacy of new therapies against these tumors.

Rag2-/-;gammac-/- immunodeficient mice, a new preclinical model to study antitumor approaches

Animal models have been relevant to study the molecular mechanisms of cancer and to develop new antitumor agents. Anyway, the huge divergence in mouse and human evolution made difficult the translation of the gained achievements in preclinical mouse based studies. The generation of clinically relevant murine models requires their humanization both concerning the creation of transgenic models and the generation of humanized mice in which to engraft a functional human immune system, and reproduce the physiological effects and molecular mechanisms of growth and metastasization of human tumors. In particular, the availability of genotypically stable immunodepressed mice able to accept tumor injection and allow human tumor growth and metastasization would be important to develop anti-tumor and anti-metastatic strategies. Recently, Rag2-/-;gammac-/- mice, double knockout for genes involved in lymphocyte differentiation, had been developed (CIEA, Central Institute for Experimental Animals, Kawasaki, Japan). Studies of human sarcoma metastasization in Rag2-/-; gammac-/- mice (lacking B, T and NK functionality) revealed their high metastatic efficiency and allowed the expression of human metastatic phenotypes not detectable in the conventionally used nude murine model. In vitro analysis to investigate the molecular mechanisms involved in the specific pattern of human sarcomas metastasization revealed the importance of liver-produced growth and motility factors, in particular the insulin-like growth factors (IGFs). The involvement of this growth factor was then demonstrated in vivo through inhibition of IGF signalling pathway. Due to the high growth and metastatic propensity of tumor cells, Rag2-/-;gammac-/- mice were used as model to investigate the metastatic behavior of rhabdomyosarcoma cells engineered to improve the differentiation. It has been recently shown that this immunodeficient model can be reconstituted with a human immune system through the injection of human cord blood progenitor cells. The work illustrated in this thesis revealed that the injection of different human progenitor cells (CD34+ or CD133+) showed peculiar engraftment and differentiation abilities. Experiments of cell vaccination were performed to investigate the functionality of the engrafted human immune system and the induction of specific human immune responses. Results from such experiments will allow to collect informations about human immune responses activated during cell vaccination and to define the best reconstitution and experimental conditions to create a humanized model in which to study, in a preclinical setting, immunological antitumor strategies.

Changes in tumor stiffness for early prediction of tumor response to sorafenib: a proof-of-concept study with elastosonography in an animal model of Hepatocellular Carcinoma (HCC)

Background and aims: Sorafenib is the reference therapy for advanced Hepatocellular Carcinoma (HCC). No method exists to predict in the very early period subsequent individual response. Starting from the clinical experience in humans that subcutaneous metastases may rapidly change consistency under sorafenib and that elastosonography a new ultrasound based technique allows assessment of tissue stiffness, we investigated the role of elastonography in the very early prediction of tumor response to sorafenib in a HCC animal model. Methods: HCC (Huh7 cells) subcutaneous xenografting in mice was utilized. Mice were randomized to vehicle or treatment with sorafenib when tumor size was 5-10 mm. Elastosonography (Mylab 70XVG, Esaote, Genova, Italy) of the whole tumor mass on a sagittal plane with a 10 MHz linear transducer was performed at different time points from treatment start (day 0, +2, +4, +7 and +14) until mice were sacrified (day +14), with the operator blind to treatment. In order to overcome variability in absolute elasticity measurement when assessing changes over time, values were expressed in arbitrary units as relative stiffness of the tumor tissue in comparison to the stiffness of a standard reference stand-off pad lying on the skin over the tumor. Results: Sor-treated mice showed a smaller tumor size increase at day +14 in comparison to vehicle-treated (tumor volume increase +192.76% vs +747.56%, p=0.06). Among Sor-treated tumors, 6 mice showed a better response to treatment than the other 4 (increase in volume +177% vs +553%, p=0.011). At day +2, median tumor elasticity increased in Sor-treated group (+6.69%, range –30.17-+58.51%), while decreased in the vehicle group (-3.19%, range –53.32-+37.94%) leading to a significant difference in absolute values (p=0.034). From this time point onward, elasticity decreased in both groups, with similar speed over time, not being statistically different anymore. In Sor-treated mice all 6 best responders at day 14 showed an increase in elasticity at day +2 (ranging from +3.30% to +58.51%) in comparison to baseline, whereas 3 of the 4 poorer responders showed a decrease. Interestingly, these 3 tumours showed elasticity values higher than responder tumours at day 0. Conclusions: Elastosonography appears a promising non-invasive new technique for the early prediction of HCC tumor response to sorafenib. Indeed, we proved that responder tumours are characterized by an early increase in elasticity. The possibility to distinguish a priori between responders and non responders based on the higher elasticity of the latter needs to be validated in ad-hoc experiments as well as a confirmation of our results in humans is warranted.

Respiratory chain complex I dysfunction in tumorigenesis

Diseases due to mutations in mitochondrial DNA probably represent the most common form of metabolic disorders, including cancer, as highlighted in the last years. Approximately 300 mtDNA alterations have been identified as the genetic cause of mitochondrial diseases and one-third of these alterations are located in the coding genes for OXPHOS proteins. Despite progress in identification of their molecular mechanisms, little has been done with regard to the therapy. Recently, a particular gene therapy approach, namely allotopic expression, has been proposed and optimized, although the results obtained are rather controversial. In fact, this approach consists in synthesis of a wild-type version of mutated OXPHOS protein in the cytosolic compartment and in its import into mitochondria, but the available evidence is based only on the partial phenotype rescue and not on the demonstration of effective incorporation of the functional protein into respiratory complexes. In the present study, we took advantage of a previously analyzed cell model bearing the m.3571insC mutation in MTND1 gene for the ND1 subunit of respiratory chain complex I. This frame-shift mutation induces in fact translation of a truncated ND1 protein then degraded, causing complex I disassembly, and for this reason not in competition with that allotopically expressed. We show here that allotopic ND1 protein is correctly imported into mitochondria and incorporated in complex I, promoting its proper assembly and rescue of its function. This result allowed us to further confirm what we have previously demonstrated about the role of complex I in tumorigenesis process. Injection of the allotopic clone in nude mice showed indeed that the rescue of complex I assembly and function increases tumor growth, inducing stabilization of HIF1α, the master regulator of tumoral progression, and consequently its downstream gene expression activation.

Transcriptional regulation and functional characterization of the tumor suppressor genes Hugl-1 and Hugl-2

Cancer is a multi-step process in which both the activation of oncogenes and the inactivation of tumor suppressor genes alter the normal cellular programs to a state of proliferation and growth. The regulation of a number of tumor suppressor genes and the mechanism underlying the tumor suppression have been intensively studied. Hugl-1 and Hugl-2, the human homologues of Drosophila lgl are shown to be down-regulated in a variety of cancers including breast, colon, lung and melanoma, but the mechanism responsible for loss of expression is not yet known. The regulation of gene expression is influenced by factors inducing or repressing transcription. The present study was focused on the identification and characterization of the active promoters of Hugl-1 and Hugl-2. Further, the regulation of the promoter and functional consequences of this regulation by specific transcription factors was analyzed. Experiments to delineate the function of the mouse homologue of Hugl-2, mgl2 using transgenic mice model were performed. This study shows that the active promoter for both Hugl-1 and Hugl-2 is located 1000bp upstream of transcription start sites. The study also provides first insight into the regulation of Hugl-2 by an important EMT transcriptional regulator, Snail. Direct binding of Snail to four E-boxes present in Hugl-2 promoter region results in repression of Hugl-2 expression. Hugl-1 and Hugl-2 plays pivotal role in establishment and maintenance of cell polarity in a diversity of cell types and organisms. Loss of epithelial cell polarity is a prerequisite for cancer progression and metastasis and is an important step in inducing EMT in cells. Regulation of Hugl-2 by Snail suggests one of the initial events towards loss of epithelial cell polarity during Snail-mediated EMT. Another important finding of this study is the induction of Hugl-2 expression can reverse the Snail-driven EMT. Inducing Hugl-2 in Snail expressing cells results in the re-expression of epithelial markers E-cadherin and Cytokeratin-18. Further, Hugl-2 also reduces the rate of tumor growth, cell migration and induces the epithelial phenotype in 3D culture model in cells expressing Snail. Studies to gain insight into the signaling pathways involved in reversing Snail-mediated EMT revealed that induction of Hugl-2 expression interferes with the activation of extracellular receptor kinase, Erk. Functional aspects of mammalian lgl in vivo was investigated by establishing mgl2 conditional knockout mice. Though disruption of mgl2 gene in hepatic tissues did not alter the growth and development, ubiquitous disruption of mgl2 gene causes embryonic lethality which is evident by the fact that no mgl2-/- mice were born.

Host immune response in immunodeficient mice against infection with Cryptosporidium parvum

Immunantwort von immundefizienten Mäusen gegenüber Infektionen mit Cryptosporidium parvum. Cryptosporidium parvum ist ein intrazellulärer, protozoischer Krankheitserreger, der im immunkompromittierten Wirt zu lebensbedrohender Enteritis führen kann. CD4+ T-Zellen und Interferon (IFN)-γ spielen wesentliche Rollen bei der Wirtsimmunantwort gegen die Infektion. Dennoch sind die Effektormechanismen, die zur Resistenz führen nur wenig verstanden. In dieser Studie wurde die Immunantwort von IFN-γ- und Interleukin (IL)-12-Defektmäusen parallel zu Wildtypmäusen analysiert. Die Ergebnisse identifizierten IFN-γ als Schlüsselzytokin bei der natürlichen und erworbenen Immunität während der Erst- und Folgeinfektion mit C. parvum. Tumornekrosefaktor (TNF)-α ist möglicherweise ein Induktor der frühen IFN-γ-Antwort in IL-12 Knockout-Mäusen. Weiterhin tragen offenbar sowohl Th1- als auch Th2-Zytokine zur Überwindung der Primärinfektion bei, die ersten mehr als die letztgenannten. Zytokingene waren am Ort der Infektion (Ileum) dramatisch verändert, nicht aber in den lokalen Lymphknoten und der Milz. Nach Folgeinfektion ergab sich in Abwesenheit von IFN-γ eine signifikante Erhöhung der Th2-Zytokine IL-5 and IL-13. Die Ergebnisse zeigten weiterhin, dass das Th1-Zytokin IL-18 zur Resistenz gegenüber C. parvum beiträgt, möglicherweise durch verschiedene Immunfunktionen, wie der Regulation von Serum-IFN-γ während der Infektion und/oder der Erhaltung der Homeostase der Th1/Th2-Zytokine durch Regulation der Th2-Zytokine. Weiterhin zeigten diese Untersuchungen den Transfer von Resistenz gegenüber C. parvum von infizierten auf naïve Mäuse mittels stimulierter intraepithelialer Lymphozyten und CD4+ T-Zellen. Diese Ergebnisse weisen auf die Gegenwart von C. parvum-spezifischen CD4+ T-Zellen in anderen lymphatischen Geweben neben der Darmmukosa hin. Eine Stimulation der Spendertiere durch Infektion war notwendig für eine übertragbare schützende Immunität. Dennoch konnte die übertragene Immunität nicht die Infektion der Empfängertiere vollständig verhindern; eine Verdopplung der Spenderzellen führte zu keinem besseren Ergebnis. Weiterhin ergab der Transfer von CD4+ und CD8+ T-Zellen (Pan-T-Zellen) keinen erhöhten Schutz der naiven Empfängertiere als der alleinige Transfer von CD4+ T-Zellen. Dies weist auf die fehlende Bedeutung der CD8+ T-Zellen beim Schutz vor C. parvum-Infektion hin.

Synthese von tumor-assoziierten MUC1-Mucin-Glycopeptid-Vakzinen und deren immunologische Evaluierung

Eine alternative Methode zur Therapie von Tumorerkrankungen bestünde in einer Immuntherapie ausgelöst durch synthetische Antitumor-Vakzine. Ein vielversprechendes Zielmolekül für eine solche Aktivimmunisierung ist das Glycoprotein MUC1, das auf nahezu allen Epithelgeweben exprimiert und auf Tumorgeweben stark überexprimiert wird. Seine extrazelluläre Domäne enthält eine Vielzahl von Tandem-Repeat-Sequenzen der Art: HGVTSAPDTRPAPGSTAPPA mit fünf potentiellen O-Glycosylierungs-Positionen. Da die Form der Glycosylierung des MUC1 in Tumorzellen stark von der auf normalen Zellen abweicht, liegen auf Tumorzellen eine Reihe tumor-assoziierter Saccharidantigene und Peptidepitope vor.rnIn dieser Arbeit wurden tumor-assoziierte Glycopeptidantigene aus der MUC1-Tandem-Repeat-Region hergestellt. Die synthetisierten MUC1-Glycopeptide tragen in verschiedenen Positionen eine Glycosylierung mit den tumor-assoziierten Tn- und STn-Saccharid-Antigenen. Zur Gewinnung von Vakzinen wurden diese Glycopeptid-Antigene über einen Spacer mit immunstimulierenden Komponenten verknüpft. Als Immunstimulanzien wurden ein T-Zell-Epitop aus dem Ovalbumin (OVA323-339) sowie die Carrier-Proteine Rinderserumalbumin (BSA) und Tetanus-Toxoid (TTox) verwendet. rnDie synthetischen MUC1-Glycopeptide wurden durch Immunisierung von Mäusen einer immunologischen Evaluierung unterzogen. Insbesondere die synthetischen MUC1-Glycopeptid-TTox-Vakzine lösen sehr starke Immunantworten aus. Es konnte gezeigt werden, dass die induzierten Antikörper stark an Tumorzellen und auch an Mammakarzinom-Gewebe binden, was für die Entwicklung von Antitumor-Vakzinen als vielversprechend einzustufen ist.

Development of a highly potent bispecific antibody format targeting the novel tumor-specific antigen CLDN18.2

Due to multiple immune evasion mechanisms of cancer cells, novel therapy approaches are required to overcome the limitations of existing immunotherapies. Bispecific antibodies are potent anti-cancer drugs, which redirect effector T cells for specific tumor cell lysis, thus enabling the patient’s immune system to fight cancer cells. The antibody format used in this proof of concept study–bispecific ideal monoclonal antibodies termed BiMAB–is a tailor-made recombinant protein, which consists of two fused scFv antibodies recognizing different antigens. Both are arranged in tandem on a single peptide chain and the individual variable binding domains are separated by special non-immunogenic linkers. The format is comprised of a scFv targeting CLDN18.2–a gastric cancer tumor associated antigen (TAA) –while the second specificity binds the CD3 epsilon (CD3ε) subunit of the T cell receptor (TCR) on T cells. For the first time, we compared in our IMAB362-based BiMAB setting, four different anti-CD3-scFvs, respectively derived from the mAbs TR66, CLB-T3, as well as the humanized and the murine variant of UCHT1. In addition, we investigated the impact of an N- versus a C-terminal location of the IMAB362-derived scFv and the anti-CD3-scFvs. Thus, nine CLDN18.2 specific BiMAB proteins were generated, of which all showed a remarkably high cytotoxicity towards CLDN18.2-positive tumor cells. Because of its promising effectiveness, 1BiMAB emerged as the BiMAB prototype. The selectivity of 1BiMAB for its TAA and CD3ε, with affinities in the nanomolar range, has been confirmed by in vitro assays. Its dual binding depends on the design of an N-terminally positioned IMAB362 scFv and the consecutive C-terminally positioned TR66 scFv. 1BiMAB provoked a concentration and target cell dependent T cell activation, proliferation, and upregulation of the cytolytic protein Granzyme B, as well as the consequent elimination of target cells. Our results demonstrate that 1BiMAB is able to activate T cells independent of elements that are usually involved in the T cell recognition program, like antigen presentation, MHC restriction, and co-stimulatory effector molecules. In the first in vivo studies using a subcutaneous xenogeneic tumor mouse model in immune incompetent NSG mice, we could prove a significant therapeutic effect of 1BiMAB with partial or complete tumor elimination. The initial in vitro RIBOMAB experiments correspondingly showed encouraging results. The electroporation of 1BiMAB IVT-RNA into target or effector cells was feasible, while the functionality of translated 1BiMAB was proven by induced T cell activation and target cell lysis. Accordingly, we could show that the in vitro RIBOMAB approach was applicable for all nine BiMABs, which proves the RIBOMAB concept. Thus, the CLDN18.2-BiMAB strategy offers great potential for the treatment of cancer. In the future, administered either as protein or as IVT-RNA, the BiMAB format will contribute towards finding solutions to raise and sustain tumor-specific cellular responses elicited by engaged and activated endogenous T cells. This will potentially enable us to overcome immune evasion mechanisms of tumor cells, consequently supporting current solid gastric cancer therapies.

Hepatic gene expression profile in mice perorally infected with Echinococcus multilocularis eggs

Alveolar echinococcosis (AE) is a severe chronic hepatic parasitic disease currently emerging in central and eastern Europe. Untreated AE presents a high mortality (>90%) due to a severe hepatic destruction as a result of parasitic metacestode proliferation which behaves like a malignant tumor. Despite this severe course and outcome of disease, the genetic program that regulates the host response leading to organ damage as a consequence of hepatic alveolar echinococcosis is largely unknown.

Absence of somatostatin SST(2) receptor internalization in vivo after intravenous SOM230 application in the AR42J animal tumor model

Among clinically relevant somatostatin functions, agonist-induced somatostatin receptor subtype 2 (sst(2)) internalization is a potent mechanism for tumor targeting with sst(2) affine radioligands such as octreotide. Since, as opposed to octreotide, the second generation multi-somatostatin analog SOM230 (pasireotide) exhibits strong functional selectivity, it appeared of interest to evaluate its ability to affect sst(2) internalization in vivo. Rats bearing AR42J tumors endogenously expressing somatostatin sst(2) receptors were injected intravenously with SOM230 or with the [Tyr(3), Thr(8)]-octreotide (TATE) analog; they were euthanized at various time points; tumors and pancreas were analyzed by immunohistochemistry for the cellular localization of somatostatin sst(2) receptors. SOM230-induced sst(2) internalization was also evaluated in vitro by immunofluorescence microscopy in AR42J cells. At difference to the efficient in vivo sst(2) internalization triggered by intravenous [Tyr(3), Thr(8)]-octreotide, intravenous SOM230 did not elicit sst(2) internalization: immunohistochemically stained sst(2) in AR42J tumor cells and pancreatic cells were detectable at the cell surface at 2.5min, 10min, 1h, 6h, or 24h after SOM230 injection while sst(2) were found intracellularly after [Tyr(3), Thr(8)]-octreotide injection. The inability of stimulating sst(2) internalization by SOM230 was confirmed in vitro in AR42J cells by immunofluorescence microscopy. Furthermore, SOM230 was unable to antagonize agonist-induced sst(2) internalization, neither in vivo, nor in vitro. Therefore, SOM230 does not induce sst(2) internalization in vivo or in vitro in AR42J cells and pancreas, at difference to octreotide derivatives with comparable sst(2) binding affinities. These characteristics may point towards different tumor targeting but also to different desensitization properties of clinically applied SOM230.

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Small-animal PET/CT for monitoring the development and response to chemotherapy of thymic lymphoma in Trp53-/- mice

Transgenic mouse models of human cancers represent one of the most promising approaches to elucidate clinically relevant mechanisms of action and provide insights into the treatment efficacy of new antitumor drugs. The use of Trp53 transgenic mice (Trp53 knockout [Trp53(-/-)] mice) for these kinds of studies is, so far, restricted by limitations in detecting developing tumors and the lack of noninvasive tools for monitoring tumor growth, progression, and treatment response.

Effect of electroporation-mediated diphtheria toxin A expression on PSA positive human prostate xenograft tumors in SCID mice

Current therapies to treat prostate cancer are often limited. Since it has been shown that very low concentrations of diphtheria toxin A (DT-A) result in abrogation of protein synthesis and apoptosis of cells, DT-A might serve as an efficient killer in cancer gene therapy. For this purpose we investigated in a quantitative manner using a stereological approach the apoptotic effect of DT-A in androgen receptor (AR) and prostate specific antigen (PSA) expressing cells after tumor formation in both flanks of SCID mice.

Self-sufficient control of urate homeostasis in mice by a synthetic circuit

Synthetic biology has shown that the metabolic behavior of mammalian cells can be altered by genetic devices such as epigenetic and hysteretic switches, timers and oscillators, biocomputers, hormone systems and heterologous metabolic shunts. To explore the potential of such devices for therapeutic strategies, we designed a synthetic mammalian circuit to maintain uric acid homeostasis in the bloodstream, disturbance of which is associated with tumor lysis syndrome and gout. This synthetic device consists of a modified Deinococcus radiodurans-derived protein that senses uric acids levels and triggers dose-dependent derepression of a secretion-engineered Aspergillus flavus urate oxidase that eliminates uric acid. In urate oxidase-deficient mice, which develop acute hyperuricemia, the synthetic circuit decreased blood urate concentration to stable sub-pathologic levels in a dose-dependent manner and reduced uric acid crystal deposits in the kidney. Synthetic gene-network devices providing self-sufficient control of pathologic metabolites represent molecular prostheses, which may foster advances in future gene- and cell-based therapies.

Synthesis and biological activity of new functionalized epothilones for prodrug design and tumor targeting

Epothilones are potent antiproliferative agents, which have served as successful lead structures for anticancer drug discovery. However, their therapeutic efficacy would benefit greatly from an increase in their selectivity for tumor cells, which may be achieved through conjugation with a tumor-targeting moiety. Three novel epothilone analogs bearing variously functionalized benzimidazole side chains were synthesized using a strategy based on palladium-mediated coupling and macrolactonization. The synthesis of these compounds is described and their in vitro biological activity is discussed with respect to their interactions with the tubulin/microtubule system and the inhibition of human cancer cell proliferation. The additional functional groups may be used to synthesize conjugates of epothilone derivatives with a variety of tumor-targeting moieties.