Evaluation of brain tumor therapy by multi-modal small animal imaging

Multi-modal small animal imaging for brain tumor therapy assessment Malignant gliomas are the most common tumors arising in the central nervous system. Despite advances in neurosurgery and adjuvant radio - and chemotherapy, these patients however still face a dismal prognosis. A major reason for their bad prognosis is the infiltrative growth pattern of these tumors, with single tumor cells migrating distantly fromthe solid tumor mass and infiltrating normal brain tissue, where they can cause recurrent tumors. One promising therapeutic approach for gliomais the delivery of therapeutic agents by engrafted cells that have the capacity to specifically migrate to pathogenic foci and in particular totrack infiltrating tumor cells. In so called bystander mediated killingapproaches, therapeutic cells produce enzymes that are responsible for the cell death of tumor cells (for example thymidine kinase (TK) expressing cells). The approach of using stem cells as suitable cellular vehicles for glioma therapy relies however on assumptions of the migration potential and biodistribution of these cells. These therapies often lack knowledge based administration of required pro drugs and therefore knowledge based initiation of therapy. This promising method can be optimizedby labelling therapeutic cells in vitro with an MRI contrast agent, followed by non - invasive monitoring of their tumor infiltration using MR imaging after implantation and administration of the pro - drug at the appropriate time point. In this study, stem cells willbe labelled using non - toxic, superparamagnetic nano -particles as MRIcontrast agent. Once the cells are labelled with nanoparticles and transduced with lentiviral vectors containing the HSV-TK and a fluorescent protein, the cells will be implanted in the rat and / or mouse brain. Thetherapeutic effect will be achieved by expression of suicide genes and bystander - mediated glioma cell killing. The thymidine kinase of human cells, unlike that of the herpes simplex virus, is unable to transform the pro drug gancyclovir into its toxic derivative. This transformationthus results in a product, which is only toxic for the implanted stem cells. A process called bystander mediated cell killing, which involves gap junction formation, ensures that also tumor cells adjacent to these stem cells are destroyed. To achieve a maximal therapeutic effect though,the pro drug has to be delivered at the time of completed migration of the stem cells to the pathogenic focus and in particular to infiltrating tumor cells to avoid tumor reoccurrence. MRI will be used to determine the optimal time for pro drug administration, while the effect of therapy will be followed up by optical and PET imaging. This project will thus aim to overcome remaining shortcomings of apromising therapy approach by combining the highly successful bystanderkilling effect of stem cells with the non invasive guidanceof the pro drug administration.status: publishe

Monitoring the Bystander Killing Effect of Human Multipotent Stem Cells for Treatment of Malignant Brain Tumors

Tumor infiltrating stem cells have been suggested as a vehicle for the delivery of a suicide gene towards otherwise difficult to treat tumors like glioma. We have used herpes simplex virus thymidine kinase expressing human multipotent adult progenitor cells in two brain tumor models (hU87 and Hs683) in immune-compromised mice. In order to determine the best time point for the administration of the codrug ganciclovir, the stem cell distribution and viability were monitored in vivo using bioluminescence (BLI) and magnetic resonance imaging (MRI). Treatment was assessed by in vivo BLI and MRI of the tumors. We were able to show that suicide gene therapy using HSV-tk expressing stem cells can be followed in vivo by MRI and BLI. This has the advantage that (1) outliers can be detected earlier, (2) GCV treatment can be initiated based on stem cell distribution rather than on empirical time points, and (3) a more thorough follow-up can be provided prior to and after treatment of these animals. In contrast to rodent stem cell and tumor models, treatment success was limited in our model using human cell lines. This was most likely due to the lack of immune components in the immune-compromised rodents

Monitoring the bystander killing effect of human multipotent stem cells for treatment of malignant brain tumors

Tumor infiltrating stem cells have been suggested as a vehicle for the delivery of a suicide gene towards otherwise difficult to treat tumors like glioma. We have used herpes simplex virus thymidine kinase expressing human multipotent adult progenitor cells in two brain tumor models (hU87 and Hs683) in immune-compromised mice. In order to determine the best time point for the administration of the codrug ganciclovir, the stem cell distribution and viability were monitored in vivo using bioluminescence (BLI) and magnetic resonance imaging (MRI). Treatment was assessed by in vivo BLI and MRI of the tumors. We were able to show that suicide gene therapy using HSV-tk expressing stem cells can be followed in vivo by MRI and BLI. This has the advantage that (1) outliers can be detected earlier, (2) GCV treatment can be initiated based on stem cell distribution rather than on empirical time points, and (3) a more thorough follow-up can be provided prior to and after treatment of these animals. In contrast to rodent stem cell and tumor models, treatment success was limited in our model using human cell lines. This was most likely due to the lack of immune components in the immune-compromised rodents.status: publishe

Assessment of bystander killing-mediated therapy of malignant brain tumors using a multimodal imaging approach

INTRODUCTION: In this study, we planned to assess if adult stem cell-based suicide gene therapy can efficiently eliminate glioblastoma cells in vivo. We investigated the therapeutic potential of mouse Oct4(-) bone marrow multipotent adult progenitor cells (mOct4(-) BM-MAPCs) in a mouse glioblastoma model, guided by multimodal in vivo imaging methods to identify therapeutic windows. METHODS: Magnetic resonance imaging (MRI) of animals, wherein 5 × 10(5) syngeneic enhanced green fluorescent protein-firefly luciferase-herpes simplex virus thymidine kinase (eGFP-fLuc-HSV-TK) expressing and superparamagnetic iron oxide nanoparticle labeled (1 % or 10 %) mOct4(-) BM-MAPCs were grafted in glioblastoma (GL261)-bearing animals, showed that labeled mOct4(-) BM-MAPCs were located in and in close proximity to the tumor. Subsequently, ganciclovir (GCV) treatment was commenced and the fate of both the MAPCs and the tumor were followed by multimodal imaging (MRI and bioluminescence imaging). RESULTS: In the majority of GCV-treated, but not phosphate-buffered saline-treated animals, a significant difference was found in mOct4(-) BM-MAPC viability and tumor size at the end of treatment. Noteworthy, in some phosphate-buffered saline-treated animals (33 %), a significant decrease in tumor size was seen compared to sham-operated animals, which could potentially also be caused by a synergistic effect of the immune-modulatory stem cells. CONCLUSIONS: Suicide gene therapy using mOct4(-) BM-MAPCs as cellular carriers was effective in reducing the tumor size in the majority of the GCV-treated animals leading to a longer progression-free survival compared to sham-operated animals. This treatment could be followed and guided noninvasively in vivo by MRI and bioluminescence imaging. Noninvasive imaging is of particular interest for a rapid and efficient validation of stem cell-based therapeutic approaches for glioblastoma and hereby contributes to a better understanding and optimization of a promising therapeutic approach for glioblastoma patients.status: publishe

The capsaicin receptor TRPV1 is a crucial mediator of the noxious effects of mustard oil.

Mustard oil (MO) is a plant-derived irritant that has been extensively used in experimental models to induce pain and inflammation. The noxious effects of MO are currently ascribed to specific activation of the cation channel TRPA1 in nociceptive neurons. In contrast to this view, we show here that the capsaicin receptor TRPV1 has a surprisingly large contribution to aversive and pain responses and visceral irritation induced by MO. Furthermore, we found that this can be explained by previously unknown properties of this compound. First, MO has a bimodal effect on TRPA1, producing current inhibition at millimolar concentrations. Second, it directly and stably activates mouse and human recombinant TRPV1, as well as TRPV1 channels in mouse sensory neurons. Finally, physiological temperatures enhance MO-induced TRPV1 stimulation. Our results refute the dogma that TRPA1 is the sole nocisensor for MO and motivate a revision of the putative roles of these channels in models of MO-induced pain and inflammation. We propose that TRPV1 has a generalized role in the detection of irritant botanical defensive traits and in the coevolution of multiple mammalian and plant species

Controlling and monitoring stem cell safety in vivo in an experimental rodent model

Adult stem cells have been investigated increasingly over the past years for multiple applications. Although they have a more favorable safety profile compared to pluripotent stem cells, they are still capable of self-renewal and differentiate into several cell types. We investigated the behavior of Oct4-positive (Oct4(+) ) and Oct4-negative (Oct4(-) ) murine or rat bone marrow (BM)-derived stem cells in the healthy brain of syngeneic mice and rats. Engraftment of mouse and rat Oct4-positive BM-derived hypoblast-like stem cells (m/rOct4(+) BM-HypoSCs) resulted in yolk-sac tumor formation in the healthy brain which was monitored longitudinally using magnetic resonance imaging (MRI) and bioluminescence imaging (BLI). Contrast enhanced MRI confirmed the disruption of the blood brain barrier. In contrast, m/r Oct4-negative BM-derived multipotent adult progenitor cells (m/rOct4(-) BM-MAPCs) did not result in mass formation after engraftment into the brain. mOct4(+) BM-HypoSCs and mOct4(-) BM-MAPCs were transduced to express enhanced green fluorescent protein, firefly luciferase (fLuc), and herpes simplex virus-thymidine kinase to follow up suicide gene expression as a potential "safety switch" for tumor-forming stem cells by multimodal imaging. Both cell lines were eradicated efficiently in vivo by ganciclovir administration indicating successful suicide gene expression in vivo, as assessed by MRI, BLI, and histology. The use of suicide genes to prevent tumor formation is in particular of interest for therapeutic approaches where stem cells are used as vehicles to deliver therapeutic genes. Stem Cells 2014;32:2833-2844.status: publishe

Additional file 1: Figure S1. of Assessment of bystander killing-mediated therapy of malignant brain tumors using a multimodal imaging approach

High magnification images from brain sections of representative animals for all groups and subgroups. Massson’s Trichrome staining (top) of sham-operated and PBS-injected, control animals showed very large tumors with some bleeding. Prussian blue (bottom) staining was also performed, which indicated presence of iron (*) in PBS-injected, control animals that did not respond to treatment. For these animals, positive iron staining was seen both at the tumor border (left Prussian blue staining) and inside the tumor mass (right Prussian blue staining). GCV-treated responders showed high iron content as the cells remained more localized around the much smaller tumor lesions. The presence of high amounts of iron is an indication for the presence of SPIO from engrafted, labeled stem cells. Finally, Iba1 staining (middle) was performed which showed microglial activation in the GCV-treated responding group. Microglial activation was also pronounced around the tumor in sham-operated and PBS-injected, control animals. In general, Prussian blue and Iba1 staining appears more intense in animals that responded to treatment due to the small (former) lesion site. (TIFF 6465 kb