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Biomedical subjects

R Bjerkvig

Publications and source records attributed to R Bjerkvig.

At least 19 recordsLinked to original sources
PubMed2006

Inhibition of medulloblastoma cell invasion by Slit.

Invasion of brain tumor cells has made primary malignant brain neoplasms among the most recalcitrant to therapeutic strategies. We tested whether the secreted protein Slit2, which guides the projection of axons and developing neurons, could modulate brain tumor cell invasion. Slit2 inhibited the invasion of medulloblastoma cells in a variety of in vitro models. The effect of Slit2 was inhibited by the Robo ectodomain. Time-lapse videomicroscopy indicated that Slit2 reduced medulloblastoma invasion rate without affecting cell direction or proliferation. Both medulloblastoma and glioma tumors express Robo1 and Slit2, but only medulloblastoma invasion is inhibited by recombinant Slit2 protein. Downregulation of activated Cdc42 may contribute to this differential response. Our findings reinforce the concept that neurodevelopmental cues such as Slit2 may provide insights into brain tumor invasion.

Animals↗
PubMed2006

Primary glioma spheroids maintain tumourogenicity and essential phenotypic traits after cryopreservation.

Tumour spheroids initiated from glioma biopsy specimens provide a valuable three-dimensional cell culture system that share several biological features of malignant brain tumours in situ. Upon xenotransplantation in immunodeficient rats, tumours derived from such spheroids exhibit a highly infiltrative growth. Successful cryopreservation of spheroid specimens therefore represents an excellent tool for future comparative studies of tumour growth and progression. Thus, if frozen stocks of human glioma spheroids can be established, similar to those obtained from cancer cell lines, it would ease the planning of biopsy-based experiments. In this context, it is crucial that cryopreservation does not alter the biological behaviour of the tumour spheroids. The biopsy spheroids were frozen to -40 degrees Celsius, stored for 1 week at -196 degrees Celsius, thawed rapidly and cultured for 1 week. The viability of the spheroids was compared against controls using a two-colour fluorescence assay, which demonstrated that cryopreservation was well tolerated. Using an in vitro invasion assay, it is shown that the freezing procedures did not affect the spheroids ability to invade a collagen gel. Cryopreserved and control tumour spheroids were equally tumourogenic, and produced overlapping survival curves when transplanted into the brains of immunocompromised rats. Immunohistochemical analyses showed no significant changes regarding microvessel density or proliferation index. Furthermore, gene expression profiling using a macroarray system detected no significant changes following cryopreservation. The present data show that cryopreservation is well tolerated, and represent a methodologically reliable storage method for biopsy spheroids that can be used in experimental studies at later time points.

Animals↗
PubMed2005

NG2 expression regulates vascular morphology and function in human brain tumours.

Tumour angiogenesis is a tightly regulated process involving cross-talk between tumour cells and the host tissue. The underlying mechanisms that regulate such interactions remain largely unknown. NG2 is a transmembrane proteoglycan whose presence on transformed cells has been demonstrated to increase proliferation in vitro and angiogenesis in vivo. To study the effects of NG2 during tumour growth and progression, we engineered an NG2 positive human glioma cell line (U251-NG2) from parental NG2 negative cells (U251-WT) and implanted both cell types stereotactically into immunodeficient nude rat brains. The tumours were longitudinally monitored in vivo using multispectral MRI employing two differently sized contrast agents (Gd-DTPA-BMA and Gadomer) to assess vascular leakiness, vasogenic oedema, tumour volumes and necrosis. Comparisons of Gd-DTPA-BMA and Gadomer revealed differences in their spatial distribution in the U251-NG2 and U251-WT tumours. The U251-NG2 tumours exhibited a higher leakiness of the larger molecular weight Gadomer and displayed a stronger vasogenic oedema (69.9 +/- 15.2, P = 0.018, compared to the controls (10.7 +/- 7.7). Moreover, immunohistochemistry and electron microscopy revealed that the U251-NG2 tumours had a higher microvascular density (11.81 +/- 0.54; P = 0.0010) compared to controls (5.76 +/- 0.87), with vessels that displayed larger gaps between the endothelial cells. Thus, tumour cells can regulate both the function and structure of the host-derived tumour vasculature through NG2 expression, suggesting a role for NG2 in the cross-talk between tumour-host compartments.

Animals↗
PubMed2004

Cryopreservation of alginate-encapsulated recombinant cells for antiangiogenic therapy.

The potential benefit of continuous local administration of antiangiogenic proteins to CNS tumors in vivo has recently been demonstrated using endostatin-producing recombinant cells encapsulated in alginate beads. Due to the treatment potential of transplanted alginate-encapsulated cells producing therapeutic proteins, we describe a successful method of cryopreservation (CP) of such beads, in which cellular viability, alginate structure, and protein secretion were maintained. Alginate beads containing human embryonic kidney cells (HEK 293 cells) stably transfected with the gene encoding for endostatin were cryopreserved in dimethyl sulfoxide (DMSO) using a slow freezing procedure. Briefly, the DMSO concentration was gradually increased prior to the freezing procedure. The cryotubes were further supercooled to -7.5 degrees C and nucleated. Thereafter, the samples were cooled at a rate of 0.25 degrees C/min and stored in liquid nitrogen. The viability of the encapsulated cells was assessed using confocal microscopy quantification (CLSM) technique and a MTS assay. The cell cycle distribution inside the beads was assessed by DNA flow cytometry and endostatin production was determined by an endostatin-specific ELISA assay, both prior to and after CP. CLSM measurements showed sustained esterase activity in the beads after thawing, with only a slight transient decrease 24 h after CP. The MTS assay verified these findings by displaying similar variations of intracellular dehydrogenase activity. Flow cytometric analyses revealed no cryorelated disturbances in cellular ploidy. Furthermore, ELISA measurements showed a well-preserved endostatin production after CP. In conclusion, this work describes the successful CP of alginate-encapsulated recombinant cells secreting a therapeutic protein. Together with previous published reports, these results further substantiate the feasibility and potential of cell encapsulation therapy in the treatment of malignant tumors.

Alginates↗
PubMed2003

Cost of migration: invasion of malignant gliomas and implications for treatment.

Tumors of glial origin consist of a core mass and a penumbra of invasive, single cells, decreasing in numbers towards the periphery and still detectable several centimeters away from the core lesion. Several decades ago, the diffuse nature of malignant gliomas was recognized by neurosurgeons when super-radical resections using hemispherectomies failed to eradicate these tumors. Local invasiveness eventually leads to regrowth of a recurrent tumor predominantly adjacent to the resection cavity, which is not significantly altered by radiation or chemotherapy. This raises the question of whether invasive glioma cells activate cellular programs that render these cells resistant to conventional treatments. Clinical and experimental data demonstrate that glioma invasion is determined by several independent mechanisms that facilitate the spread of these tumors along different anatomic and molecular structures. A common denominator of this cellular behavior may be cell motility. Gene-expression profiling showed upregulation of genes related to motility, and functional studies demonstrated that cell motility contributes to the invasive phenotype of malignant gliomas. There is accumulating evidence that invasive glioma cells show a decreased proliferation rate and a relative resistance to apoptosis, which may contribute to chemotherapy and radiation resistance. Interestingly, interference with cell motility by different strategies results in increased susceptibility to apoptosis, indicating that this dynamic relationship can potentially be exploited as an anti-invasive treatment paradigm. In this review, we discuss mechanisms of glioma invasion, characteristics of the invasive cell, and consequences of this cellular phenotype for surgical resection, oncologic treatments, and future perspectives for anti-invasive strategies.

Brain Neoplasms↗
PubMed2003

Cell therapy using encapsulated cells producing endostatin.

Despite aggressive surgery and post-operative radiation and chemotherapy, the prognosis is poor for glioblastoma patients. Anti-angiogenic therapy with compounds such as endostatin could delay the onset of relapse. However, the short systemic half-life of this proteins as well as the blood-brain barrier makes the use of this therapy difficult for brain cancer patients. The aim of this project is to develop and implant genetically engineered producer cells secreting endostatin that are encapsulated in calcium cross-linked alginate gel beads. Encapsulation of cells within alginate gels has a potential as a sustained release system in addition to the fact that the encapsulation technology protects the cells from rejection by the immune system. Human embryonal kidney 293 cells have been transfected with the gene for endostatin. These cells have been encapsulated in calcium cross-linked alginate gels and optimized for the secretion of endostatin. Alginate gel beads implanted into rat brain have shown only a moderate loss in cell viability but extended endostatin release for periods of up to 12 months. Visualization of the anti-angiogenic effect on C6 rat glioma growth, tumor vasculature and microhemodynamics has been demonstrated by using intravital video microscopy. The data indicates that endostatin greatly affects tumor-associated microcirculation but does not appear to affect normal microcirculation. The local delivery of endostatin seems to specifically affect tumor-associated microvessels by reduction of the vessel density, diameter and functionality. Tumor cell migration and invasion was greatly reduced in the endostatin treated animals.

Alginates↗
PubMed2002

The glial precursor proteoglycan, NG2, is expressed on tumour neovasculature by vascular pericytes in human malignant brain tumours.

Glial precursor cells express NG2 and GD3 in the developing brain. These antigens are both over-expressed during neoplasia, which suggests they may have specific functions in the malignant progression of human brain tumours. This study describes the expression of NG2 and GD3 in 28 paediatric and adult brain tumours. Glioblastoma biopsy spheroids were also implanted into nude rats to assess the regional distribution of the molecules within the tumour. These xenografts showed extensive infiltration and growth that mimicked the growth patterns of human gliomas in situ. NG2 was identified in 20 out of 28 brain tumours, where the expression was confined to the main mass of the tumour, and was reduced towards the tumour periphery. NG2 was mainly associated with blood vessels on both the pericyte and basement membrane components of the tumour vasculature. Ki67 (MIB-1) labelling indicated that NG2 expression was associated with areas of high cellular proliferation. Conversely, all the tumours expressed GD3, which was present both in the tumour main mass and throughout the periphery. Thus, the expression of NG2 may be indicative of tumour progression and might be an amenable target for future therapeutic interventions.

Adult↗
PubMed2001

Vinblastine and hyperthermia target the neovasculature in BT(4)AN rat gliomas: therapeutic implications of the vascular phenotype.

PURPOSE: The antivascular and antitumor activity of vinblastine and hyperthermia at different tumor volumes were examined in the subcutaneous (s.c.) BT(4)An rat glioma model. METHODS AND MATERIALS: The influence of vinblastine (3 mg/kg) and hyperthermia (44 degrees C/60 min) on tumor growth was assessed in small (100 mm(3)) and large (200 mm(3)) BT(4)An tumors. To disclose how vinblastine and hyperthermia interacted in the neoplasms, tumor blood flow and the extent of vascular damage, hypoxia, cell proliferation, and apoptosis were assessed after treatment. The content of smooth muscle cells/pericytes in the tumor vasculature was examined in small and large tumors to assess how the vascular phenotype changed during tumor growth. RESULTS: In the large tumors, vinblastine reduced the blood flow, but the tumor growth was not affected. The combination of drug and local heating yielded massive vascular damage and a significant tumor response. The small neoplasms had a higher content of smooth muscle cells/pericytes in the vessel walls (host vasculature), and the tumor vasculature displayed a higher resistance to vascular damage than the large neoplasms. Yet, vinblastine alone exhibited a potent antiproliferative activity and induced massive apoptosis in the small tumors, and the drug significantly inhibited tumor growth. The addition of hyperthermia yielded no additional growth delay in the small tumors. CONCLUSION: The antivascular properties of vinblastine and hyperthermia can be exploited to facilitate vascular damage in BT(4)An solid tumors with a low content of host vasculature.

Animals↗
PubMed2001

Intravital microscopy reveals novel antivascular and antitumor effects of endostatin delivered locally by alginate-encapsulated cells.

The current study describes new, antivascular, and antitumor effects of human endostatin. A novel system for continuous, localized delivery of antiangiogenic compounds to brain tumors was used. The delivery system was composed of endostatin-producing 293 cells encapsulated into immuno-isolating sodium alginate. Intravital multifluorescence microscopy was used to assess vascular and antitumor effects of endostatin in C6 glioma spheroids implanted into an ectopic as well as an orthotopic setting. Analysis of total and functional vascular density, microvascular diameters, vessel perfusion, tumor growth, and tumor cell migration were performed repetitively. Tumor growth was reduced by 35% in treated animals. It was of interest that tumor cell invasion into the surrounding tissue was also inhibited. The total vascular density was reduced by 67.6%, perfusion by 67%, and vessel diameters by 37%. This resulted in a significant reduction in tumor perfusion, although the vessel permeability was not influenced. We have demonstrated that human endostatin not only reduces total vascular density, as shown previously, but also greatly reduces the functionality and the diameters of the vessels. Furthermore, we show that this therapeutic approach also inhibits tumor cell invasion, thus supporting the hypothesis that tumor angiogenesis and invasion represent two interrelated processes. Finally, this work further confirms the new therapeutic concept using alginate cell-encapsulation technology for the localized delivery of therapeutic compounds to central nervous system malignancies.

Alginates↗
PubMed2001

Combretastatin A-4 and hyperthermia;a potent combination for the treatment of solid tumors.

BACKGROUND AND PURPOSE: Attacking tumor vasculature is a promising approach for the treatment of solid tumors. The tubulin inhibitor combretastatin A-4 disodium phosphate (CA-4) is a new vascular targeting drug which displays a low toxicity profile. We wanted to investigate how CA-4 influences tumor perfusion in the BT4An rat glioma and how the vascular targeting properties of CA-4 could be exploited to augment hyperthermic damage towards tumor vasculature. MATERIAL AND METHODS: We used the (86)RbCl extraction technique to assess how CA-4 influences tumor perfusion, and the tumor endothelium was examined for morphological changes induced by the drug. We combined CA-4 (50 mg/kg i.p.) with hyperthermia (44 degrees C, 60 min) at different time intervals to evaluate how therapy should be designed to affect tumor growth, and we studied the tumors histologically to assess tissue viability. RESULTS: We found that CA-4 induced a profound, but transient reduction in tumor perfusion 3-6 h postinjection. If hyperthermia was administered 3-6 h after injecting CA-4, massive hemorrhagic necrosis developed, and tumor response was significantly enhanced compared to simultaneous administration of the two treatment modalities (P<0.005). CA-4 alone had no influence on tumor growth and failed to disrupt the vasculature of the BT4An solid tumors. Interestingly though, a mild endothelial edema was observed in some tumor areas 3 h after injecting CA-4. CONCLUSIONS: We conclude that the combination of CA-4 and hyperthermia is a potent therapeutic option for BT4An tumors, but the selection of adequate time intervals between CA-4 and hyperthermia are imperative to obtain tumor response.

Animals↗
PubMed2001

Tumor vasculature is targeted by the combination of combretastatin A-4 and hyperthermia.

BACKGROUND AND PURPOSE: Combretastatin A-4 disodium phosphate (CA-4) enhances thermal damage in s.c. BT(4)An rat gliomas. We currently investigated how CA-4 and hyperthermia affect the tumor microenvironment and neovasculature to disclose how the two treatment modalities interact to produce tumor response. METHODS: By confocal microscopy and immunostaining for von Willebrand factor, we examined the extent of vascular damage subsequent to CA-4 (50 mg/kg) and hyperthermia (waterbath 44 degrees C, 60 min). The influence on tumor oxygenation was assessed using interstitial pO(2)-probes (Licox system) and by immunostaining for pimonidazole. We examined the direct effect of CA-4 on the tumor cell population by flow cytometry (cell cycle distribution) and immunostaining for beta-tubulin (cytoskeletal damage). RESULTS: Whereas slight vascular damage was produced by CA-4 in the BT(4)An tumors, local hyperthermia exhibited moderate anti-vascular activity. In tumors exposed to CA-4 3 h before hyperthermia, massive vascular damage ensued. CA-4 reduced the pO(2) from 36.1 to 17.6 mmHg (P=0.01) in the tumor base, and tumor hypoxia increased slightly in the tumor center (pimonidazole staining). Extensive tumor hypoxia developed subsequent to hyperthermia or combination therapy. Despite a profound influence on beta-tubulin organization in vitro, CA-4 had no significant effect on the cell cycle distribution in vivo. CONCLUSION: Our results indicate that the anti-vascular activity exhibited by local hyperthermia can be augmented by previous exposure to CA-4.

Animals↗
PubMed2001

Local endostatin treatment of gliomas administered by microencapsulated producer cells.

We describe a technique for the treatment of malignant brain tumors based on local delivery of the anti-angiogenic protein endostatin from genetically engineered cells encapsulated in ultrapure sodium alginate. Alginate consists of L-guluronic and D-mannuronic acid, which in the presence of divalent cations forms an extended gel network, in which cells reside and remain immunoisolated, when implanted into the rat brain. Here, we show that endostatin-transfected cells encapsulated in alginate maintain endostatin secretion for at least four months after intracerebral implantation in rats. During the implantation period 70% of the encapsulated cells remained viable, as opposed to 85% in in vitro-cultured capsules. Rats that received transplants of BT4C glioma cells, together with endostatin-producing capsules (0.2 microg/ml per capsule), survived 84% longer than the controls. The endostatin released from the capsules led to an induction of apoptosis, hypoxia, and large necrotic avascular areas within 77% of the treated tumors, whereas all the controls were negative. The encapsulation technique may be used for many different cell lines engineered to potentially interfere with the complex microenvironment in which tumor and normal cells reside. The present work may thus provide the basis for new therapeutic approaches toward brain tumors.

Alginates↗
PubMed2001

The glioma-associated gangliosides 3'-isoLM1, GD3 and GM2 show selective area expression in human glioblastoma xenografts in nude rat brains.

This work describes the in vivo expression and distribution of glioma-associated gangliosides (GD3, GM2, 3'-isoLM1) in a novel human brain tumour nude rat xenograft model. In this model, the tumours, which are established directly from human glioblastoma biopsies, show extensive infiltrative growth within the rat brain. This model therefore provides an opportunity to study ganglioside expression not only within the macroscopic tumour, but also in brain areas with tumour cell infiltration. The ganglioside expression was studied by confocal microscopy of immunostained brain sections using antiganglioside monoclonal antibodies. Xenografts from four human glioblastoma multiformes were established in rats and the brains removed after 3-4 months. Ganglioside GD3 was expressed in the tumour parenchyma while ganglioside 3'-isoLM1 was more abundantly expressed in the periphery of the tumour associated with areas of tumour cell invasion. GM2 expression was only seen in one tumour, where it was located within the main tumour mass. Double staining with a pan antihuman monoclonal antibody (3B4) and the antiganglioside monoclonal antibodies confirmed that the ganglioside expression was associated with tumour cells. This work supports the concept of different biological roles for individual gangliosides and indicates that antibodies or ligands directed against GD3 and 3'-isoLM1 might be complementary when applied in the treatment of human glioblastomas.

Animals↗
PubMed2001

Cell encapsulation technology as a therapeutic strategy for CNS malignancies.

Gene therapy using viral vectors has to date failed to reveal its definitive clinical usefulness. Cell encapsulation technology represents an alternative, nonviral approach for the delivery of biologically active compounds to tumors. This strategy involves the use of genetically engineered producer cells that secrete a protein with therapeutic potential. The cells are encapsulated in an immunoisolating material that makes them suitable for transplantation. The capsules, or bioreactors, permit the release of recombinant proteins that may assert their effects in the tumor microenvironment. During the last decades, there has been significant progress in the development of encapsulation technologies that comprise devices for both macro- and microencapsulation. The polysaccharide alginate is the most commonly used material for cell encapsulation and is well tolerated by various tissues. A wide spectrum of cells and tissues has been encapsulated and implanted, both in animals and humans, indicating the general applicability of this approach for both research and medical purposes, including CNS malignancies. Gliomas most frequently recur at the resection site. To provide local and sustained drug delivery, the bioreactors can be implanted in the brain parenchyma or in the ventricular system. The development of comprehensive analyses of geno- and phenotypic profiles of a tumor (genomics and proteomics) may provide new and important guidelines for choosing the optimal combination of bioreactors and recombinant proteins for therapeutic use.

Alginates↗
PubMed2000

lacZ-neoR transfected glioma cells in syngeneic rats: growth pattern and characterization of the host immune response against cells transplanted inside and outside the CNS.

The rat glioma cell lines BT4C and BT4Cn were stably transfected with the bacterial lacZ-neomycin resistance (neoR) gene construct. Both transfected (BT4ClacZ and BT4CnlacZ) and untransfected cell lines were injected intracerebrally and subcutaneously into rats. Survival time, morphology, growth rate and immunological properties of the tumors were studied. Survival time was significantly prolonged after intracerebral implantation of the transfected cell lines. No similar response was found in nude rats, indicating an immunological response towards the lacZ-neoR-transfected cells in immunocompetent animals. Morphological observations showed that the lacZ-neoR-transfected gliomas were smaller and had a distinct boundary with the normal brain tissue, whereas the parental cell lines revealed a more diffuse growth pattern. Immunostaining showed a higher proportion of immunocompetent cells infiltrating the lacZ-neoR-transfected tumors. After s.c. injection, the lacZ-neoR-transfected BT4C cell line had a prolonged lag phase before assuming a growth rate similar to that of the parental cells. The BT4CnvlacZ tumors initially grew fastest, but then disappeared within 3 weeks. A similar response was observed with mock-transfected tumor cells. A (3)HTdR-incorporation assay on spleen cells from rats transplanted s.c. with BT4CnvlacZ cells showed a 10-fold increase in cell activation as compared with rats with BT4Cn tumors. A humoral response towards the transfected cells was verified by Western-blot analyses.

Animals↗
PubMed2000

Herpes simplex virus thymidine kinase gene therapy in experimental rat BT4C glioma model: effect of the percentage of thymidine kinase-positive glioma cells on treatment effect, survival time, and tissue reactions.

Herpes simplex virus thymidine kinase (HSV-tk) gene transfer and ganciclovir (GCV) administration have been suggested for the treatment of malignant gliomas. To understand tissue responses and possible ways to improve the treatment effect, we studied tumor growth, tissue reactions, and survival time after HSV-tk/GCV treatment in a syngeneic BT4C rat glioma model by mixing various ratios of stably transfected HSV-tk-expressing BT4C-tk glioma cells with wild-type BT4C glioma cells (percentage of BT4C-tk cells: 0%, 1%, 10%, 30%, 50%, and 100%), followed by injection into BDIX rat brains (n = 79). With the exception of some animals with end-stage tumors, very little astroglia or microglia reactivity was detected in the wild-type tumors as analyzed by immunocytochemistry using glial fibrillary acid protein (GFAP)-, vimentin-, human histocompatibility leukocyte antigen-DR-, OX-42-, and CD68-specific monoclonal antibodies. After 14 days of GCV treatment, tumors induced with > or = 10% BT4C-tk cells showed a significant reduction in tumor size (P < .05) and prolonged survival time (P < .01). Astrogliosis, as indicated by a strong GFAP and vimentin immunoreactivity, was seen in the tumor scar area. GFAP and vimentin reactivity was already present after the GCV treatment in tumors induced with 1% BT4C-tk cells. Much less human histocompatibility leukocyte antigen-DR-positive microglia was seen in the treated animals, indicating low microglia reactivity and immunoactivation against the tumor. However, GCV-treated tumors were positive for apoptosis, indicating that apoptosis is an important mechanism for cell death in the BT4C-tk glioma model. Our results suggest that > or = 10% transfection efficiency is required for a successful reduction in BT4C glioma tumor size with HSV-tk/GCV treatment in vivo. Tissue reactions after 14 days of GCV treatment are characterized by astrogliosis and apoptosis, whereas microglia response and immunoactivation of the brain cells appear to play a minor role. Stimulation of the microglia response by gene transfer or other means might improve the efficacy of the HSV-tk/GCV treatment in vivo.

Animals↗
PubMed1999

Infection of rat brain cell aggregates with neurovirulent and nonneurovirulent strains of herpes simplex virus type 1.

Rat brain cell aggregates represent a three-dimensional tissue culture system of brain tissue in the form of small, multicellular spheroids. In the present work, we have infected these "minibrains" with neurovirulent, nonneurovirulent, and nonreplicating strains of HSV-1. The neurovirulent strains 17(+) and KOS(M) spread rapidly through the aggregates, while the nonreplicating ICP4 deletion mutant KD6 infected cells only at the periphery of the aggregates. Spread and replication of the nonneurovirulent strains RE6 and tk-7, and to some extent also of R13/1, were restricted. The interaction between different strains of HSV-1 and the rat brain cell aggregates is thus comparable to that seen in the brain, suggesting that the aggregates represent a useful tool for studying HSV-1 infection of brain tissue in vitro.

Animals↗