P11.57 A 3D brain organoid coculture system delineates the invasive cell components in glioblastoma

Abstract BACKGROUND Glioblastoma (GBM) cell infiltration into the surrounding normal brain tissue where the blood brain barrier is intact, represents a major problem for clinical management and therapy. There is a vital need to understand the molecular mechanism that drives tumor cell invasion into the surrounding brain. We have previously developed a 3D coculture model where mature brain organoids are confronted with patient-derived glioblastoma stem-like cells (GSCs). In such a coculture system, single cell invasion into the normal brain tissue can be studied in detail. Here, we first describe in detail, by RNA-seq and proteomics, the differentiation of various neural cell lineages into mature brain organoids as well as their cellular organization. By real-time confocal microscopy and imaging analyses we also determine the speed of tumor cell invasion into the brain. Finally, we used this coculture system to delineate in detail the cellular heterogeneity within the invasive compartment and their gene expression. MATERIAL AND METHODS Immunohistochemistry and immunofluorescence were used to determine the expression and distribution of mature neurons, astrocytes, oligodendrocytes, and microglia within the brain organoids. Proteomics and RNA-seq were used to determine brain development ex-vivo. To assess the clonal composition of the GBM-invasive compartment, we used cellular (RGB) barcoding technology. By advanced imaging, we tracked in real time the invasion of barcoded cells into the brain organoids. Finally, we isolated invasive cells and non-invasive cells from our coculture system and used single cell sequencing to analyze their gene expression profiles and molecular phenotypes. RESULTS Immunohistochemistry and immunofluorescence showed that brain organoids, after 21 days of differentiation, display a highly cellular and structural organization. RNA-seq and proteomics, performed at different time points of organoid differentiation, revealed that the brain organoids develop into mature brain structures after 21 days as verified by a comparative analysis to normal rat brain development in vivo. Imaging analyses showed that multiple clones within the GBMs have the capacity to invade into the brain tissue with an average speed of ~ 20 μm/h. RNA-sec analysis of the invasive compartment revealed a strong up-regulation of genes and pathways associated with anaerobic respiration (glycolysis). CONCLUSION We describe a highly standardized brain organoid coculture system that can be used to delineate GBM invasion ex-vivo. We demonstrate that this platform can be used to unravel the mechanisms that drive GBM invasion into the normal brain.

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Multiple Irradiation Affects Cellular and Extracellular Components of the Mouse Brain Tissue and Adhesion and Proliferation of Glioblastoma Cells in Experimental System In Vivo

International Journal of Molecular Sciences ◽

10.3390/ijms222413350 ◽

2021 ◽

Vol 22 (24) ◽

pp. 13350

Author(s):

Maxim O. Politko ◽

Alexandra Y. Tsidulko ◽

Oxana A. Pashkovskaya ◽

Konstantin E. Kuper ◽

Anastasia V. Suhovskih ◽

...

Keyword(s):

Brain Tissue ◽

Mouse Brain ◽

Glial Cells ◽

Ex Vivo ◽

Experimental System ◽

Normal Brain ◽

Biosynthetic Activity ◽

Normal Brain Tissue

Intensive adjuvant radiotherapy (RT) is a standard treatment for glioblastoma multiforme (GBM) patients; however, its effect on the normal brain tissue remains unclear. Here, we investigated the short-term effects of multiple irradiation on the cellular and extracellular glycosylated components of normal brain tissue and their functional significance. Triple irradiation (7 Gy*3 days) of C57Bl/6 mouse brain inhibited the viability, proliferation and biosynthetic activity of normal glial cells, resulting in a fast brain-zone-dependent deregulation of the expression of proteoglycans (PGs) (decorin, biglycan, versican, brevican and CD44). Complex time-point-specific (24–72 h) changes in decorin and brevican protein and chondroitin sulfate (CS) and heparan sulfate (HS) content suggested deterioration of the PGs glycosylation in irradiated brain tissue, while the transcriptional activity of HS-biosynthetic system remained unchanged. The primary glial cultures and organotypic slices from triple-irradiated brain tissue were more susceptible to GBM U87 cells’ adhesion and proliferation in co-culture systems in vitro and ex vivo. In summary, multiple irradiation affects glycosylated components of normal brain extracellular matrix (ECM) through inhibition of the functional activity of normal glial cells. The changed content and pattern of PGs and GAGs in irradiated brain tissues are accompanied by the increased adhesion and proliferation of GBM cells, suggesting a novel molecular mechanism of negative side-effects of anti-GBM radiotherapy.

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Isavuconazole Diffusion in Infected Human Brain

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02474-18 ◽

2019 ◽

Vol 63 (10) ◽

Cited By ~ 8

Author(s):

Claire Rouzaud ◽

Vincent Jullien ◽

Anne Herbrecht ◽

Bruno Palmier ◽

Simona Lapusan ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Plasma Concentration ◽

Brain Tissue ◽

Radical Surgery ◽

Neurological Complication ◽

Lymphocytic Leukemia ◽

Normal Brain ◽

Granulomatous Disease ◽

Normal Brain Tissue ◽

The Brain

ABSTRACT We report the cases of a 39-year-old woman with chronic lymphocytic leukemia and a 21-year-old man with chronic granulomatous disease treated for cerebral aspergillosis. The patients required radical surgery for infection progression despite adequate isavuconazole plasma concentration or neurological complication. We thus decided to measure the brain isavuconazole concentration. These results suggest that the concentrations of isavuconazole obtained in the infected brain tissue clearly differ from those obtained in the normal brain tissue and the cerebrospinal fluid.

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Elucidating the kinetics of sodium fluorescein for fluorescence-guided surgery of glioma

Journal of Neurosurgery ◽

10.3171/2018.4.jns172644 ◽

2019 ◽

Vol 131 (3) ◽

pp. 724-734 ◽

Cited By ~ 7

Author(s):

Margaret Folaron ◽

Rendall Strawbridge ◽

Kimberley S. Samkoe ◽

Caroline Filan ◽

David W. Roberts ◽

...

Keyword(s):

Brain Tissue ◽

Sodium Fluorescein ◽

Normal Brain ◽

Normal Brain Tissue ◽

Guided Surgery ◽

Fluorescence Guided Surgery ◽

Kinetics Of ◽

The Brain ◽

Pegylated Form ◽

Tissue Kinetics

OBJECTIVEThe use of the optical contrast agent sodium fluorescein (NaFl) to guide resection of gliomas has been under investigation for decades. Although this imaging strategy assumes the agent remains confined to the vasculature except in regions of blood-brain barrier (BBB) disruption, clinical studies have reported significant NaFl signal in normal brain tissue, limiting tumor-to-normal contrast. A possible explanation arises from earlier studies, which reported that NaFl exists in both pure and protein-bound forms in the blood, the former being small enough to cross the BBB. This study aims to elucidate the kinetic binding behavior of NaFl in circulating blood and its effect on NaFl accumulation in brain tissue and tumor contrast. Additionally, the authors examined the blood and tissue kinetics, as well as tumor uptake, of a pegylated form of fluorescein selected as a potential optical analog of gadolinium-based MRI contrast agents.METHODSCohorts of mice were administered one of the following doses/forms of NaFl: 1) high human equivalent dose (HED) of NaFl, 2) low HED of NaFl, or 3) pegylated form of fluorescein. In each cohort, groups of animals were euthanized 15, 30, 60, and 120 minutes after administration for ex vivo analysis of fluorescein fluorescence. Using gel electrophoresis and fluorescence imaging of blood and brain specimens, the authors quantified the temporal kinetics of bound NaFl, unbound NaFl, and pegylated fluorescein in the blood and normal brain tissue. Finally, they compared tumor-to-normal contrast for NaFl and pegylated-fluorescein in U251 glioma xenografts.RESULTSAdministration of NaFl resulted in the presence of unbound and protein-bound NaFl in the circulation, with unbound NaFl constituting up to 70% of the signal. While protein-bound NaFl was undetectable in brain tissue, unbound NaFl was observed throughout the brain. The observed behavior was time and dose dependent. The pegylated form of fluorescein showed minimal uptake in brain tissue and improved tumor-to-normal contrast by 38%.CONCLUSIONSUnbound NaFl in the blood crosses the BBB, limiting the achievable tumor-to-normal contrast and undermining the inherent advantage of tumor imaging in the brain. Dosing and incubation time should be considered carefully for NaFl-based fluorescence-guided surgery (FGS) of glioma. A pegylated form of fluorescein showed more favorable normal tissue kinetics that translated to higher tumor-to-normal contrast. These results warrant further development of pegylated-fluorescein for FGS of glioma.

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Fibrin As a Target for Glioblastoma Detection and Treatment

Blood ◽

10.1182/blood-2019-124089 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3630-3630 ◽

Cited By ~ 1

Author(s):

Lynn M Knowles ◽

Carolin Wolter ◽

Ralf Ketter ◽

Steffi Urbschat ◽

Stefan Linsler ◽

...

Keyword(s):

Extracellular Matrix ◽

Brain Tissue ◽

Ex Vivo ◽

Normal Brain ◽

High Grade ◽

Green Fluorescence ◽

Normal Brain Tissue ◽

Integrin Β3 ◽

3D Matrix

Glioblastoma is a highly aggressive brain tumor characterized by diffuse growth and resistance to therapy. Angiogenesis in glioblastoma is poorly organized and therefore, tends to be associated with tumor cell necrosis, hemorrhage and thrombosis. This leads to the formation of a fibrin-rich extracellular matrix, which could provide important adhesive cues for glioblastoma growth and proliferation. To establish that blood clotting takes place in the extracellular matrix of malignant brain tumors, we assessed fibrin formation in tumor samples from patients with astrocytoma and glioblastoma using immunohistochemistry. Compared to normal brain tissue, which is essentially fibrin-free, this analysis revealed a marked upregulation of clot formation in the interstitial spaces of patients with high-grade tumors (i.e. astrocytoma 3 and GBM). The low-grade astrocytoma 2, however, expressed 3-3.5 fold less fibrin than was found in tissues from patients with astrocytoma 3 and GBM indicating that the degree of clot formation positively correlates with tumor grade. Paralleling these data, we found that primary GBM cells, that were freshly isolated from patients after tumor surgery, infiltrated and proliferated strongly after embedding in a three dimensional (3D) matrix of clotted plasma ex vivo. Primary tumor cells from patients with astrocytoma 2 and 3, on the other hand, infiltrated clot but were unable to proliferate in 3D. GBM proliferation in 3D depended on fibrin, which mediated upregulation of the stem cell marker nestin, whereas culturing glioblastoma cells in a 3D matrix of matrigel™ failed to promote nestin expression as well as glioblastoma proliferation. Therefore, these data suggest that the presence of clotted plasma in the tumor extracellular matrix represents a niche for glioblastoma stem cells and, as such, contributes to GBM progression. To determine the interaction of GBM cells with fibrin on a molecular basis, we transfected GBM cells with siRNA against integrin β3, which completely abolished invadopodia formation and, at the same time, caused a sustained growth inhibition. GBM cell proliferation in 3D fibrin also depended on the formation of a fibronectin matrix as knockdown of fibronectin led to complete growth arrest. These findings appear to be clinically relevant since freshly isolated tumor cells from patients with glioblastoma colonized 3D fibrin most efficiently when they express fibronectin in combination with integrin β3. This suggests that fibrin stimulates adhesive interactions between integrin β3 and fibronectin and that these interactions in turn support glioblastoma stemness. To assess fibrin formation in glioblastoma in vivo, mice with orthotopic U87MG xenografts were injected intravenously with the fluorescein-coupled dekapeptide CGLKIQKNEC, which is a derivative of the clot-binding peptide CLT1. Using a fluorescence endoscope in situ, we detected strong green fluorescence over the parietal lobe of the right cerebral hemisphere, where tumor growth had been established by MRI beforehand. Subsequently, we confirmed tumor binding of the peptide in isolated brain tissue by fluorescence microscopy ex vivo, which demonstrated specific green fluorescence in the tumor xenograft while adjacent normal brain tissue as well as tissues from distant organs only exhibited background fluorescence. Together, our data demonstrate a specific upregulation of fibrin in high-grade astrocytoma, which promotes infiltration and proliferation of glioblastoma stem cells via integrin β3 and fibronectin. Moreover, we present a strategy to identify fibrin in the tumor extracellular matrix as a possible means to identify astrocytoma progression in vivo. Disclosures Eichler: Novo Nordisk: Membership on an entity's Board of Directors or advisory committees. Pilch:CSL Behring: Other: Grants (investigator initiated), Speakers Bureau; ASPIRE Award/Pfizer: Other: Grants (investigator initiated); Bayer: Consultancy, Speakers Bureau; Roche: Consultancy.

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Neuroinflammatory changes of the normal brain tissue in cured mice following combined radiation and anti-PD-1 blockade therapy for glioma

Scientific Reports ◽

10.1038/s41598-021-84600-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mariano Guardia Clausi ◽

Alexander M. Stessin ◽

Zirun Zhao ◽

Stella E. Tsirka ◽

Samuel Ryu

Keyword(s):

Brain Tissue ◽

Cranial Irradiation ◽

Subcortical White Matter ◽

Glioma Cell Line ◽

Hippocampal Neurogenesis ◽

Normal Brain ◽

Long Term Effects ◽

Normal Brain Tissue ◽

Molecule Inhibitor

AbstractThe efficacy of combining radiation therapy with immune checkpoint inhibitor blockade to treat brain tumors is currently the subject of multiple investigations and holds significant therapeutic promise. However, the long-term effects of this combination therapy on the normal brain tissue are unknown. Here, we examined mice that were intracranially implanted with murine glioma cell line and became long-term survivors after treatment with a combination of 10 Gy cranial irradiation (RT) and anti-PD-1 checkpoint blockade (aPD-1). Post-mortem analysis of the cerebral hemisphere contralateral to tumor implantation showed complete abolishment of hippocampal neurogenesis, but neural stem cells were well preserved in subventricular zone. In addition, we observed a drastic reduction in the number of mature oligodendrocytes in the subcortical white matter. Importantly, this observation was evident specifically in the combined (RT + aPD-1) treatment group but not in the single treatment arm of either RT alone or aPD-1 alone. Elimination of microglia with a small molecule inhibitor of colony stimulated factor-1 receptor (PLX5622) prevented the loss of mature oligodendrocytes. These results identify for the first time a unique pattern of normal tissue changes in the brain secondary to combination treatment with radiotherapy and immunotherapy. The results also suggest a role for microglia as key mediators of the adverse treatment effect.

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Quantitative Measurements of Regional Glucose Utilization and Rate of Valine Incorporation into Proteins by Double-Tracer Autoradiography in the Rat Brain Tumor Model

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.1989.12 ◽

1989 ◽

Vol 9 (1) ◽

pp. 87-95 ◽

Cited By ~ 22

Author(s):

Michihiro Kirikae ◽

Mirko Diksic ◽

Y. Lucas Yamamoto

Keyword(s):

Protein Synthesis ◽

Brain Tumor ◽

Rat Brain ◽

Brain Tissue ◽

Glucose Utilization ◽

Tumor Model ◽

Normal Brain ◽

Normal Brain Tissue ◽

Rat Brain Tumor ◽

Brain Tumor Model

We examined the rate of glucose utilization and the rate of valine incorporation into proteins using 2-[18F]fluoro-2-deoxyglucose and L-[1-14C]-valine in a rat brain tumor model by quantitative double-tracer autoradiography. We found that in the implanted tumor the rate of valine incorporation into proteins was about 22 times and the rate of glucose utilization was about 1.5 times that in the contralateral cortex. (In the ipsilateral cortex, the tumor had a profound effect on glucose utilization but no effect on the rate of valine incorporation into proteins.) Our findings suggest that it is more useful to measure protein synthesis than glucose utilization to assess the effectiveness of antitumor agents and their toxicity to normal brain tissue. We compared two methods to estimate the rate of valine incorporation: “kinetic” (quantitation done using an operational equation and the average brain rate coefficients) and “washed slices” (unbound labeled valine removed by washing brain slices in 10% thrichloroacetic acid). The results were the same using either method. It would seem that the kinetic method can thus be used for quantitative measurement of protein synthesis in brain tumors and normal brain tissue using [11C]-valine with positron emission tomography.

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PET Study of Methionine Accumulation in Glioma and Normal Brain Tissue

Journal of Computer Assisted Tomography ◽

10.1097/00004728-198703000-00002 ◽

1987 ◽

Vol 11 (2) ◽

pp. 208-213 ◽

Cited By ~ 71

Author(s):

Mats Bergström ◽

Kaj Ericson ◽

Lars Hagenfeldt ◽

Mikael Mosskin ◽

Hans von Holst ◽

...

Keyword(s):

Brain Tissue ◽

Normal Brain ◽

Normal Brain Tissue

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Quantitative Analysis of Mobile Proteins in Normal Brain Tissue by Amide Proton Transfer Imaging

Journal of Computer Assisted Tomography ◽

10.1097/rct.0000000000001141 ◽

2021 ◽

Vol Publish Ahead of Print ◽

Author(s):

Kazuaki Sugawara ◽

Tosiaki Miyati ◽

Ryo Ueda ◽

Daisuke Yoshimaru ◽

Masanobu Nakamura ◽

...

Keyword(s):

Quantitative Analysis ◽

Proton Transfer ◽

Brain Tissue ◽

Amide Proton ◽

Normal Brain ◽

Normal Brain Tissue ◽

Amide Proton Transfer ◽

Amide Proton Transfer Imaging

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Glutathione S-Transferases and Cytochrome P450 Enzyme Expression in Patients with Intracranial Tumors: Preliminary Report of 55 Patients

Medical Principles and Practice ◽

10.1159/000494496 ◽

2018 ◽

Vol 28 (1) ◽

pp. 56-62

Author(s):

Cahit Kural ◽

Arzu Kaya Kocdogan ◽

Gulcin Güler Şimşek ◽

Serpil Oğuztüzün ◽

Pınar Kaygın ◽

...

Keyword(s):

Cytochrome P450 ◽

Brain Tissue ◽

Pituitary Adenomas ◽

Normal Brain ◽

Cytochrome P450 Enzyme ◽

Intracranial Tumors ◽

Glutathione S Transferase ◽

Normal Brain Tissue ◽

Tissue Samples ◽

P450 Enzyme

Objective: Intracranial tumors are one of the most frightening and difficult-to-treat tumor types. In addition to surgery, protocols such as chemotherapy and radiotherapy also take place in the treatment. Glutathione S-transferase (GST) and cytochrome P450 (CYP) enzymes are prominent drug-metabolizing enzymes in the human body. The aim of this study is to show the expression of GSTP1, GSTM1, CYP1A1, and CYP1B1 in different types of brain tumors and compare our results with those in the literature. Subjects and Methods: The expression of GSTP1, GSTM1, CYP1A1, and CYP1B1 was analyzed using immunostaining in 55 patients with intracranial tumors in 2016–2017. For GST and CYP expression in normal brain tissue, samples of a portion of surrounding normal brain tissue as well as a matched far neighbor of tumor tissue were used. The demographic features of the patients were documented and the expression results compared. Results: The mean age of the patients was 46.72 years; 29 patients were female and 26 were male. Fifty-seven specimens were obtained from 55 patients. Among them, meningioma was diagnosed in 12, metastases in 12, glioblastoma in 9, and pituitary adenoma in 5. The highest GSTP1, GSTM1, and CYP1A1 expressions were observed in pituitary adenomas. The lowest GSTP1 expression was detected in glioblastomas and the lowest CYP1B1 expression in pituitary adenomas. Conclusion: GSTP1 and CYP expression is increased in intracranial tumors. These results should be confirmed with a larger series and different enzyme subtypes.

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