Tuulia Huhtala

Human umbilical cord blood cells do not improve sensorimotor or cognitive outcome following transient middle cerebral artery occlusion in rats

The present study investigated effects of human umbilical cord blood (HUCB) cells on sensorimotor, cognitive, and histological outcome in rats subjected to transient middle cerebral artery occlusion (MCAO). Halothane anesthetized adult male Wistar rats were subjected to transient MCAO for 2 h. HUCB cells (mononuclear 1-5x10(7) or Lin(-) cells 1-5x10(5)) were administered intravenously after 24 h recovery. The limb-placing test was performed on postoperative days 2, 4, 6, 9, 12, 16, and 20. In addition, beam-walking and cylinder tests were used to assess sensorimotor function at baseline, and on postoperative days 4, 12, and 20. Morris water-maze was used to assess cognitive performance on postoperative days 22-24. Subsequently, rats were perfused for measurement of infarct volumes and detection of HUCB cells by immunohistochemistry (MAB1281). MCAO rats showed a partial spontaneous recovery in sensorimotor function during the follow-up. However, the recovery profile was similar in MCAO controls and in MCAO rats that received HUCB cells. HUCB did not affect impaired water-maze performance of MCAO rats. Only few human nuclei-specific MAB1281-positive cells were detected in the ipsilateral hemisphere in MCAO rats that received HUCB cells. Infarct volumes did not differ between the experimental groups. A group of additional rats were used to further study biodistribution of intravenously given (111)In-oxine-labelled mononuclear HUCB cells in MCAO and sham-operated rats. SPECT imaging data indicated a high tracer uptake in the lung, liver, spleen, and kidney, but not in the brain immediately after administration or 24 h post-administration. The present study suggests that HUCB cells do not improve functional recovery or histological outcome in MCAO rats after systemic administration because of limited migration of cells in the ischemic brain.

The SPECT imaging shows the accumulation of neural progenitor cells into internal organs after systemic administration in middle cerebral artery occlusion rats

The regenerative potential of stem cells from various sources has been under intense investigation in the experimental models of cerebral ischemia. To end up with a restorative therapeutic treatment, it is crucial to get the cell transplants to the site of injury. Here, we evaluated the feasibility of small animal SPECT/CT in assessing the definite accumulation of (111)In-oxine-labeled human embryonic stem (ES) cell-derived neural progenitors and rat hippocampal progenitors after intravenous or intra-arterial administration (femoral vein vs. common carotid artery) in middle cerebral artery occlusion (MCAO) and sham-operated rats. Cell detection was carried out immediately and 24h after the infusion using a SPECT/CT device. The results showed that after intravenous injections both cell types accumulated primarily into internal organs, instead of brain. In contrast, after intra-arterial injection, a weak signal was detected in the ischemic hemisphere. Additional studies showed that the detection sensitivity of SPECT/CT device was approximately 1000 (111)In-oxine-labeled cells and labeling did not affect the cell viability. In conclusion, a small animal SPECT is powerful technique to study the whole body biodistribution of cell-based therapies. Our data showed that intravenous administration is not an optimal route to deliver neural progenitor cell-containing transplants into the brain after MCAO in rats.

Intra-arterial infusion of human bone marrow-derived mesenchymal stem cells results in transient localization in the brain after cerebral ischemia in rats

Cell therapies from various sources have been under intense research in stroke. Efficient homing of the cells to the injured brain without complications is necessary to realize the therapeutic potential of cell therapy. Intra-arterial (IA) infusion of cells bypasses the filtering organs and directs the cells to the target area more efficiently. Here we studied the biodistribution of human bone marrow-derived mesenchymal stromal/stem cells (BMMSCs) after a direct infusion into the external carotid artery (ECA) in rats. Cells, which were cultured without animal-derived agents and also treated with a proteolytic enzyme to transiently modify cell surface adhesion proteins, were infused 24 h after transient middle cerebral artery occlusion (MCAO). SPECT imaging was used immediately after cell infusion and 24 h thereafter to track (111)In-oxine-labeled BMMSC in sham-operated and MCAO rats. IA infusion of BMMSCs in rats resulted in immediate cell entrapment in the brain, but the majority of the signal disappeared during the next 24 h and relocated to the internal organs. In MCAO rats, radioactivity counts 24 h after infusion were higher in the ischemic hemisphere compared to the contralateral hemisphere. Our results showed that IA infusion through ECA is a safe and efficient administration route for BMMSCs resulting in a transient localization of cells in the rat brain.

Interferon-β Sensitivity of Tumor Cells Correlates With Poor Response to VA7 Virotherapy in Mouse Glioma Models

In our recent study, replicative alphaviral vector VA7 was found to be effective against orthotopic human U87-glioma xenografts in an athymic mouse model eradicating the tumors with single intravenous (i.v.) injection. Here, we tested the efficacy of VA7 in immunocompetent orthotopic GL261 and CT-2A glioma models of C57BL/6 mouse in vivo. The cell lines were susceptible to VA7 infection in vitro, but GL261 infection was highly restricted in confluent cell cultures, and mouse interferon-β (IFNβ) pretreatment prevented the replication of VA7 in both cell lines. When mice bearing orthotopic GL261 or CT-2A tumors were administered neurotropic VA7, either i.v. or intracranially (i.c.), the vector was unable to infect the tumor and no survival benefit was achieved. Pretreatments with immunosuppressive cyclophosphamide (CPA) and rapamycin markedly lowered serum-neutralizing antibodies (NAbs) but had no effect on tumor infection or survival. Intracranial GL261 tumors were refractory also in athymic C57BL/6 mice, which have serious defects in their adaptive immunity. Implanted VA7-infected GL261 cells formed tumors with only slightly delayed kinetics and without improving survival thus excluding the participation of physical barriers and indicating robust host IFN action. Mouse and human IFNβ do not seem be species cross-reactive, which might limit the translational relevance of xenograft models in oncolytic virotherapy.

In vivo SPECT/CT imaging of human orthotopic ovarian carcinoma xenografts with 111In-labeled monoclonal antibodies☆

UNLABELLED Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 3 (VEGFR-3) are expressed in the tumor area during the progression of ovarian carcinoma. Monoclonal antibodies developed against these receptors are potential diagnostic molecules for in vivo imaging of ovarian carcinoma. METHODS Biodistribution of the monoclonal antibodies cetuximab against EGFR and mF4-31C1 against VEGFR-3 was studied in nude mice with orthotopic SKOV-3m human ovarian carcinoma xenografts. The biodistribution of (111)Indium-labeled antibodies was followed up to 48 h postinjection using combined SPECT and CT imaging modality. Organ samples were collected postmortem and specific organ activity was measured. Accumulation of the intravenously injected antibodies in the tumor tissue and lymph nodes was verified using immunohistology. RESULTS Imaging studies with SPECT/CT showed clear accumulation of both antibodies into tumor area. The tumor uptake was 8.78 ± 0.74 %ID/g for cetuximab and 5.77 ± 0.62 %ID/g for mF4-31C1 after 48 h postinjection. Cetuximab had lower liver tropism and faster tumor homing rate. In addition, after 48 h two of five tumor-bearing mice showed a clear accumulation of the In-labeled mF4-31C1 at the left axillary area. Both intravenously administered antibodies could also be detected from the tumor sections by immunohistological staining but only mF4-31C1 forms in the lymph nodes. CONCLUSION These results demonstrate the accumulation of EGFR- and VEGFR-3-specific antibodies in orthotopic ovarian carcinoma tumors. Systemically administered they had slow pharmacokinetics which is typical for antibodies. Accumulation of mF4-31C1 antibody in the lymph nodes suggests the remote activation of VEGFR-3 by the primary tumor.

Native and Complexed IGF-1: Biodistribution and Pharmacokinetics in Infantile Neuronal Ceroid Lipofuscinosis

Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disorder of childhood characterized by selective death of cortical neurons. Insulin-like growth factor 1 (IGF-1) is important in embryonic development and is considered as a potential therapeutic agent for several disorders of peripheral and central nervous systems. In circulation IGF-1 is mainly bound to its carrier protein IGFBP-3. As a therapeutic agent IGF-1 has shown to be more active as free than complexed form. However, this may cause side effects during the prolonged treatment. In addition to IGFBP-3 the bioavailability of IGF-1 can be modulated by using mesoporous silicon nanoparticles (NPs) which are optimal carriers for sustained release of unstable peptide hormones like IGF-1. In this study we compared biodistribution, pharmacokinetics, and bioavailability of radiolabeled free IGF-1, IGF-1/IGFBP-3, and IGF-1/NP complexes in a Cln1-/- knockout mouse model. IGF-1/NP was mainly accumulated in liver and spleen in all studied time points, whereas minor and more constant amounts were measured in other organs compared to free IGF-1 or IGF-1/IGFBP-3. Also concentration of IGF-1/NP in blood was relatively high and stable during studied time points suggesting continuous release of IGF-1 from the particles.

Targeted magnetic resonance imaging of Scavidin‐receptor in human umbilical vein endothelial cells in vitro

Current therapeutic approaches to treat cancer are often hampered by the lack of specificity of the drugs used for therapy. Scavidin, a novel fusion protein expressed on cell membranes, could be utilized for targeting of therapeutic molecules. Scavidin exploits the high binding affinity between avidin and biotin and is capable of mediating endocytosis of a bound ligand. In the current study we evaluated the efficiency of biotinylated ultrasmall superparamagnetic iron oxide (USPIO) particles in Scavidin‐expressing human umbilical vein endothelial cell (HUVEC) cultures in vitro as a novel receptor‐targeted magnetic resonance imaging contrast agent. Biotinylated USPIO (bUSPIO) were targeted to Scavidin adenovirus‐transduced HUVECs in vitro. Scavidin expressing cells were capable of binding and mediating endocytosis of the bUSPIO in vitro, which led to a significant decrease in T2 relaxation times, and a loss of signal intensity in comparison to controls. The findings were confirmed with Prussian blue staining for iron and detection of Scavidin by bound biotinylated horseradish peroxidase. Our data shows that biotinylated ligands target specifically to Scavidin‐expressing HUVEC in vitro. The utilization of Scavidin gene transfer ex vivo thus constitutes a platform for potential ligand delivery via cell therapy and time‐independent imaging of biologic processes. Magn Reson Med, 2006.

Novel Target for Peptide-Based Imaging and Treatment of Brain Tumors

Malignant gliomas are associated with high mortality due to infiltrative growth, recurrence, and malignant progression. Even with the most efficient therapy combinations, median survival of the glioblastoma multiforme (grade 4) patients is less than 15 months. Therefore, new treatment approaches are urgently needed. We describe here identification of a novel homing peptide that recognizes tumor vessels and invasive tumor satellites in glioblastomas. We demonstrate successful brain tumor imaging using radiolabeled peptide in whole-body SPECT/CT imaging. Peptide-targeted delivery of chemotherapeutics prolonged the lifespan of mice bearing invasive brain tumors and significantly reduced the number of tumor satellites compared with the free drug. Moreover, we identified mammary-derived growth inhibitor (MDGI/H-FABP/FABP3) as the interacting partner for our peptide on brain tumor tissue. MDGI was expressed in human brain tumor specimens in a grade-dependent manner and its expression positively correlated with the histologic grade of the tumor, suggesting MDGI as a novel marker for malignant gliomas. Mol Cancer Ther; 13(4); 996–1007. ©2014 AACR.

Biodistribution and antitumor effect of Cetuximab-targeted lentivirus

Viral vectors are central tools for gene therapy. Targeting of the vector to desired tissues followed by expression of the therapeutic gene forms one of the most critical points in effective therapy. In this study we used streptavidin-displaying lentivirus conjugated to biotinylated anti-epidermal growth factor receptor (EGFR) antibody (Cetuximab) to target vector specifically to ovarian tumors. Biodistribution of the targeted virus was studied in nude mice with orthotropic SKOV-3m human ovarian carcinoma xenografts. Radiolabeled antibodies were conjugated to streptavidin-displaying lentiviruses and biodistribution of the virus after the intravenous delivery to tumor-bearing mice was monitored up to 6 days using combined SPECT/CT imaging modality. Organ samples were collected post mortem and specific organ activities were measured. The integration of lentivirus vectors in collected tissue samples was analyzed using qPCR and the expression of green fluorescent protein (GFP)-transgene was tested by enzyme-linked immunosorbent assay. Our results showed that lentiviruses conjugated to Cetuximab (Cet-LV) or control human IgG (IgG-LV) accumulated mainly to the liver and spleen of the mice and to lower extent to lung, kidneys and tumors. Strikingly, in 50% of the mice injected with cetuximab-targeted lentivirus no tumor tissue was found, whereas the remaining half showed a significant decrease in tumor size. We hypothesize/present data that lentivirus-mediated INF-αβ production together with tumor targeting could function as an effective antitumor treatment.

Toward a More Effective Intravascular Cell Therapy in Stroke

Cerebral ischemia remains the main cause of adult disability in Western countries. More than 50% of stroke survivors are left with a motor disability, causing a huge burden for patients, relatives and healthcare systems (Bonita et al., 1997). Cell-based therapies have emerged as some of the most promising experimental approaches to restore brain function after stroke (Bliss et al., 2010; Banerjee et al., 2011; Lindvall & Kokaia, 2011). A wide variety of cell types have been studied, such as neural progenitors from different sources, including bone marrowand blood-derived stem cells. Preclinical data with cell therapies are promising (Bliss et al., 2007; Hicks & Jolkkonen, 2009; Hicks et al., 2009a; Janowski et al., 2010). The understanding of how transplanted cells exert their therapeutic effect is, however, not clear, but it is believed that the positive outcome is due to paracrine effects with an improved protective cellular environment (e.g., reduced inflammation, neuroprotection, reduced apoptosis, activation of endogenous repair) rather than as a consequence of neuronal differentiation and cell replacement (Zhang & Chopp, 2009).