Bifunctional Aptamer–Doxorubicin Conjugate Crosses the Blood–Brain Barrier and Selectively Delivers Its Payload to EpCAM-Positive Tumor Cells

Abstract BACKGROUND Glioblastomas are notoriously therapy resistant tumors. As opposed to other tumor entities, no major advances in therapeutic success have been made in the past decades. This has been calling for a deeper biological understanding of the tumor, its growth and resistance patterns. We have been using a xenograft glioma model, where human glioblastoma cells are implanted under chronic cranial windows and studied longitudinally over many weeks and months using multi photon laser scanning microscopy (MPLSM). To test the effect of (new) drugs, a stable and direct delivery system avoiding the blood-brain-barrier has come into our interest. MATERIAL AND METHODS We implanted cranial windows and fluorescently labeled human glioblastoma stem-like cells into NMRI nude mice to follow up on the tumor development in our MPLSM model. After tumor establishment, an Alzet® micropump was implanted to directly deliver agents via a catheter system continuously over 28 days directly under the cranial window onto the brain surface. Using the MPLSM technique, the continuous delivery and infusion of drugs onto the brain and into the tumor was measured over many weeks in detail using MPLSM. RESULTS The establishment of the combined methods allowed reliable concurrent drug delivery over 28 days bypassing the blood-brain-barrier. Individual regions and tumor cells could be measured and followed up before, and after the beginning of the treatment, as well as after the end of the pump activity. Fluorescently labelled drugs were detectable in the MPLSM and its distribution into the brain parenchyma could be quantified. After the end of the micropump activity, further MPLSM measurements offer the possibility to observe long term effects of the applied drug on the tumor. CONCLUSION The combination of tumor observation in the MPSLM and concurrent continuous drug delivery is a feasible and reliable method for the investigation of (novel) anti-tumor agents, especially drugs that are not blood-brain-barrier penetrant. Morphological or even functional changes of individual tumor cells can be measured under and after treatment. These techniques can be used to test new drugs targeting the tumor, its tumor microtubes and tumor cells networks, and measure the effects longitudinally.

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HER-2/neu expression in glioblastoma multiforme (GBM)

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.e13035 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. e13035-e13035

Author(s):

S. Gupta ◽

H. Sheikh ◽

C. Schneider ◽

X. Zhang ◽

A. Padmanabhan ◽

...

Keyword(s):

Glioblastoma Multiforme ◽

Tumor Cells ◽

Blood Brain Barrier ◽

Malignant Gliomas ◽

Human Tumor ◽

Brain Barrier ◽

Breast Cancer Patients ◽

Over Expression ◽

Blood Brain ◽

Her 2

e13035 Background: Glioblastoma multiforme (GBM) is a disease in which very few cytotoxic chemotherapy agents have been shown to have activity. This is partly due to their inability to cross the blood brain barrier. Trials with bevacizumab, a VEGF inhibitor that disrupts tumor angiogenesis, have demonstrated activity against this otherwise chemotherapy resistant disease. This has led to interest in other biologic agents that target angiogenic pathways for the treatment of GBM. Over-expression of HER-2/neu by human tumor cells is closely associated with increased angiogenesis and expression of VEGF. Lapatinib is a recently available low molecular weight immunotherapeutic agent that targets HER-2/neu proteins. In a recent study, breast cancer patients treated with lapatinib and capcitabine had decreased brain metastases indicating that lapatinib may cross the blood brain barrier and thus have potential in the treatment of malignant gliomas. Limited studies have evaluated HER-2/neu gene expression in GBM and the results are inconsistent. We evaluated the expression of Her-2/neu protein in 41 consecutive GBM cases to explore the potential utility of targeting this pathway. Methods: Archival histopathologic sections from 41 patients (age 26–89 years) with a diagnosis of GBM from 2004–2008 were reviewed. The diagnosis was confirmed and optimal sections were selected. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections using the primary antibody against HER-2/neu (clone 4B5, Ventana). The results were evaluated by three independent investigators. Interpretation was performed using the semi-quantitative criteria (Score 0 to 3+) currently used for primary breast carcinomas. Results: 38 out of 41 cases showed no immunohistochemical staining with HER-2/neu antibody (score 0). Three cases demonstrated weak, incomplete membrane staining of rare tumor cells (score 1+) and were interpreted as negative. The positive and negative controls worked properly. Conclusions: Our study indicates that there is no significant immunohistochemical over-expression of HER-2/neu protein in GBM. This suggests that HER-2/neu over-expression is not a significant oncogenic pathway in GBM, and therefore may not be a potential therapeutic target in this disease. No significant financial relationships to disclose.

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Patient derived orthotopic xenograft models of Medulloblastoma lack a functional blood brain barrier

Neuro-Oncology ◽

10.1093/neuonc/noaa266 ◽

2020 ◽

Author(s):

Laura A Genovesi ◽

Simon Puttick ◽

Amanda Millar ◽

Marija Kojic ◽

Pengxiang Ji ◽

...

Keyword(s):

Contrast Enhancement ◽

Tumor Cells ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Preclinical Model ◽

Dynamic Contrast Enhancement ◽

Orthotopic Xenograft ◽

Cellular Analysis ◽

Blood Brain ◽

Genetically Engineered Mouse Model

Abstract Background Novel targeted therapies for children diagnosed with medulloblastoma (MB), the most common malignant pediatric brain tumor, are urgently required. A major hurdle in the development of effective therapies is the impaired delivery of systemic therapies to tumor cells due to a specialized endothelial blood-brain barrier (BBB). Accordingly, the integrity of the BBB is an essential consideration in any preclinical model used for assessing novel therapeutics. This study sought to assess the functional integrity of the BBB in several preclinical mouse models of MB. Methods Dynamic contrast enhancement (DCE) magnetic resonance imaging (MRI) was used to evaluate blood-brain tumour-barrier (BBTB) permeability in a murine genetically engineered mouse model (GEMM) of SHH MB, patient-derived orthotopic xenograft (PDOX) models of MB (SHH and Gp3) and orthotopic transplantation of GEMM tumor cells, enabling a comparison of the direct effects of transplantation on the integrity of the BBTB. Immunofluorescence analysis was performed to compare the structural and sub-cellular features of tumor-associated vasculature in all models. Results Contrast enhancement was observed in all transplantation models of MB. No contrast enhancement was observed in the GEMM despite significant tumor burden. Cellular analysis of BBTB integrity revealed aberrancies in all transplantation models, correlating to the varying levels of BBTB permeability observed by MRI in these models. Conclusions These results highlight functional differences in the integrity of the BBTB and tumor vessel phenotype between commonly utilised preclinical models of MB, with important implications for the preclinical evaluation of novel therapeutic agents for MB.

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Brain Invasion along Perivascular Spaces by Glioma Cells: Relationship with Blood–Brain Barrier

Cancers ◽

10.3390/cancers12010018 ◽

2019 ◽

Vol 12 (1) ◽

pp. 18 ◽

Cited By ~ 2

Author(s):

Simone Pacioni ◽

Quintino Giorgio D’Alessandris ◽

Mariachiara Buccarelli ◽

Alessandra Boe ◽

Maurizio Martini ◽

...

Keyword(s):

Tumor Cells ◽

Blood Brain Barrier ◽

Glioma Cells ◽

Brain Barrier ◽

Perivascular Spaces ◽

Brain Invasion ◽

Blood Brain ◽

Perivascular Invasion ◽

The Brain

The question whether perivascular glioma cells invading the brain far from the tumor bulk may disrupt the blood–brain barrier (BBB) represents a crucial issue because under this condition tumor cells would be no more protected from the reach of chemotherapeutic drugs. A recent in vivo study that used human xenolines, demonstrated that single glioma cells migrating away from the tumor bulk are sufficient to breach the BBB. Here, we used brain xenografts of patient-derived glioma stem-like cells (GSCs) to show by immunostaining that in spite of massive perivascular invasion, BBB integrity was preserved in the majority of vessels located outside the tumor bulk. Interestingly, the tumor cells that invaded the brain for the longest distances traveled along vessels with retained BBB integrity. In surgical specimens of malignant glioma, the area of brain invasion showed several vessels with preserved BBB that were surrounded by tumor cells. On transmission electron microscopy, the cell inter-junctions and basal lamina of the brain endothelium were preserved even in conditions in which the tumor cells lay adjacently to blood vessels. In conclusion, BBB integrity associates with extensive perivascular invasion of glioma cells.

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Protease Responsive Nanogels for Transcytosis across the Blood−Brain Barrier and Intracellular Delivery of Radiopharmaceuticals to Brain Tumor Cells

Advanced Healthcare Materials ◽

10.1002/adhm.202100812 ◽

2021 ◽

pp. 2100812

Author(s):

Smriti Singh ◽

Natascha Drude ◽

Lena Blank ◽

Prachi Bharat Desai ◽

Hiltrud Königs ◽

...

Keyword(s):

Brain Tumor ◽

Tumor Cells ◽

Blood Brain Barrier ◽

Intracellular Delivery ◽

Brain Barrier ◽

Blood Brain ◽

Brain Tumor Cells

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Challenging metastatic breast cancer with the natural defensinPvD1

Nanoscale ◽

10.1039/c7nr05872a ◽

2017 ◽

Vol 9 (43) ◽

pp. 16887-16899 ◽

Cited By ~ 7

Author(s):

Tiago N. Figueira ◽

Filipa D. Oliveira ◽

Inês Almeida ◽

Érica O. Mello ◽

Valdirene M. Gomes ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Brain Metastasis ◽

Tumor Cells ◽

Blood Brain Barrier ◽

Metastatic Breast ◽

Biomechanical Properties ◽

Brain Barrier ◽

Blood Brain ◽

Metastasis Development

PvD1 hampers brain metastasis development by manipulating the biomechanical properties of tumor cells and preventing their adhesion to the blood–brain-barrier.

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SCIDOT-50. A RAT MODEL FOR LASER INTERSTITIAL THERMAL THERAPY FOR GLIOBLASTOMA: EFFECTS ON PERI-ABLATION BLOOD-BRAIN BARRIER AND IMPLICATIONS FOR POST-ABLATION CHEMOTHERAPY

Neuro-Oncology ◽

10.1093/neuonc/noz175.1185 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi282-vi282

Author(s):

Tavarekere Nagaraja ◽

James Ewing ◽

Katelynn Farmer ◽

Grahm Valadie ◽

Stephen Brown ◽

...

Keyword(s):

Tumor Cells ◽

Blood Brain Barrier ◽

Thermal Therapy ◽

Female Rats ◽

Brain Barrier ◽

Laser Interstitial Thermal Therapy ◽

Lethal Temperatures ◽

Blood Brain ◽

U251 Cells ◽

Bbb Opening

Abstract Laser interstitial thermal therapy (LITT) is a minimally invasive tumor cytoreductive treatment for recurrent gliomas, brain tumors in eloquent regions or otherwise inaccessible to traditional open surgery. LITT offers quicker recovery and shorter hospital stays as additional advantages. A laser fiberoptic catheter is positioned in the body of the tumor under magnetic resonance imaging (MRI) guidance via a twist drill hole in the skull. Using MR thermometry, the tumor is ablated by laser heating to lethal temperatures. The peri-ablation zones get heated to sub-lethal temperatures and subsequently recover. It has been reported in humans that opening of the blood-brain barrier (BBB) occurs, lasting several weeks after ablation. We have adapted Visualase®, a clinically available LITT system, for use in a rat glioblastoma model. Athymic female rats (n=7) were implanted with U251 tumor cells in one brain hemisphere. Tumor growth was monitored using MRI. When tumors reached about 4 mm in diameter, they were ablated under supervision of MR diffusion-weighted MRI, sparing the surrounding normal brain tissue. Contrast-enhanced MRI data were obtained before LITT, immediately after and at post-LITT 24 h. The brains were processed for histopathology and stained with hematoxylin and eosin (H&E) for visualizing the extent of the tumor and for major histocompatibility complex (MHC) to identify the human-derived U251 cells. All rats survived the LITT procedure. A ring of contrast enhancement around ablation perimeter was observed immediately after, and also at 24 h. H&E data showed near-complete ablation of the tumors. However, MHC staining showed U251 cells still remaining in the ablation vicinity. These data suggest this model is useful for examining the temporal and spatial development of peri-ablation BBB opening following LITT. Concurrent timing of post-ablation chemotherapy to the development and presence of post-LITT BBB opening may aid in efficient targeting of remaining tumor cells.

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Laser Therapy Enhances Blood-Brain Barrier and Blood-Tumor Barrier Permeability in a Mouse Model of Glioblastoma

Neurosurgery ◽

10.1093/neuros/nyz310_635 ◽

2019 ◽

Vol 66 (Supplement_1) ◽

Author(s):

Afshin Salehi ◽

Mounica Paturu ◽

Matthew Caine ◽

Robyn Klein ◽

Albert H Kim

Keyword(s):

Electron Microscopy ◽

Mouse Model ◽

Tumor Cells ◽

Laser Therapy ◽

Blood Brain Barrier ◽

Antitumor Agents ◽

Fiber Optic ◽

Brain Barrier ◽

Human Igg ◽

Blood Brain

Abstract INTRODUCTION A main challenge in the treatment of glioblastoma is the blood-brain barrier (BBB). In this study, we established a laser interstitial thermotherapy (LITT) platform in mice to test the hypothesis that LITT can increase BBB and blood-tumor barrier (BTB) permeability, which could potentially be exploited to enhance delivery of antitumor agents. METHODS We established a preclinical mouse model for LITT using an Nd-YAG laser coupled to a 600 μm fiber optic and thermocouple probe in C57BL/6 mice. Both the fiber optic and temperature probes were inserted through a small burrhole to target somatosensory cortex. In other experiments, mice were stereotactically implanted with syngeneic GL261 tumor cells prior to LITT. Post-LITT, brain tissue was assessed for BBB and BTB permeability through quantitative measurement of brain fluorescein (FL) after intraperitoneal injection and immunohistochemistry (IHC) after intravenously injected dextran (MWs 10 and 70 kDa) and human IgG (MW 150 kDa) to define spatial characteristics. Electron microscopy and IHC was used to delineate the mechanisms of BBB break down. RESULTS Using BBB permeability tracers, noninjected mice and mice orthotopically implanted with GL261 tumor cells were used to characterize the effect of laser therapy on the BBB and BTB, respectively. We found LITT substantially disrupts the BBB in the first day post-treatment and is sustained for 4 wk. Additionally, tumor-implanted mice demonstrated an increase in Dextran 10 and 70 kDa and human IgG extravasation after IV injection of these tracers. Mechanistically, we provide evidence, by IHC and electron microscopy, that LITT triggers both a decrease in tight junction integrity and an increase in brain endothelial cell transcytosis to increase overall BBB permeability. CONCLUSION Our data suggest that LITT increases BBB and BTB permeability over a defined time window in a mouse model and that large molecular size agents, including human IgG, can infiltrate brain parenchyma and brain tumors after systemic administration. Future experiments will focus on testing delivery of antitumor agents following LITT to enhance survival in brain tumor-bearing mice.

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DDRE-11. SECOND GENERATION IDO INHIBITORS FOR IMPROVING IMMUNOTHERAPEUTIC EFFICACY IN PATIENTS WITH GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.295 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi76-vi76

Author(s):

Lakshmi Bollu ◽

Prashant Bommi ◽

Derek Wainwright ◽

Erik Ladomersky ◽

Lijie Zhai ◽

...

Keyword(s):

Immune Response ◽

Protein Degradation ◽

Tumor Cells ◽

Blood Brain Barrier ◽

Intraperitoneal Administration ◽

Brain Barrier ◽

Patient Treatment ◽

Binary Complex ◽

Blood Brain ◽

Human Gbm

Abstract INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is a rate-limiting enzyme that metabolizes tryptophan and is expressed in >90% of patient-resected glioblastoma (GBM). IDO-mediated tryptophan metabolism has been the proposed mechanism for suppressing the immune response in GBM. Recently, we discovered that IDO also possesses non-enzymic functions that contribute to suppress the anti-GBM immune response. This finding motivated us to develop IDO-Proteolysis Targeting Chimeras (IDO-PROTACs) to degrade IDO protein rather than simply inhibiting IDO enzyme activity. METHODS Western blot analysis was used to determine IDO-PROTAC efficiency of IDO protein degradation among human and mouse GBM cell lines and PDX. Our lead IDO-PROTAC was tested for toxicity, blood brain barrier penetration, and pharmacokinetics (PK) in wild-type C57BL/6 mice. RESULTS IDO-PROTACs degrade IDO protein in both tumor and non-tumor cells with a DC50 value of ~0.5µM in human GBM tumor cells. Biolayer interferometry (BLI) studies show that IDO-PROTAC forms a binary complex with IDO protein with similar affinity comparable to parental compound – BMS986205. IDO-PROTACs induced IDO ubiquitination and the pretreatment with ubiquitin proteasome inhibitors, MG132 or MLN4924, inhibited IDO protein degradation. In vivo toxicity studies showed that treatment with IDO-PROTAC at 25 mg/kg for 3-weeks did not develop any apparent toxicity in C57BL/6 mice. PK analysis revealed that IDO-PROTAC bioavailabilty reached a peak serum and brain concentration within 30 minutes after intraperitoneal administration. CONCLUSIONS This study developed a lead IDO-PROTAC compound that efficiently degrades IDO protein in human GBM cells with a moderate bioavailability and blood-brain barrier (BBB) penetration. Future work will focus on the enhancement of BBB penetration, increased bioavailability, and route of administration to improve IDO-PROTAC potency for combination with other forms of immunotherapy for GBM patient treatment.

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Isolation of Circulating Tumor Cells from Glioblastoma Patients by Direct Immunomagnetic Targeting

Applied Sciences ◽

10.3390/app10093338 ◽

2020 ◽

Vol 10 (9) ◽

pp. 3338 ◽

Cited By ~ 1

Author(s):

David Lynch ◽

Branka Powter ◽

Joseph William Po ◽

Adam Cooper ◽

Celine Garrett ◽

...

Keyword(s):

Cancer Cells ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Blood Brain Barrier ◽

Brain Cancer ◽

Brain Barrier ◽

Blood Samples ◽

Blood Brain

Glioblastoma (GBM) is the most common form of primary brain cancer in adults and tissue biopsies for diagnostic purposes are often inaccessible. The postulated idea that brain cancer cells cannot pass the blood–brain barrier to form circulating tumor cells (CTCs) has recently been overthrown and CTCs have been detected in the blood of GBM patients albeit in low numbers. Given the potential of CTCs to be analyzed for GBM biomarkers that may guide therapy decisions it is important to define methods to better isolate these cells. Here, we determined markers for immunomagnetic targeting and isolation of GBM-CTCs and confirmed their utility for CTC isolation from GBM patient blood samples. Further, we identified a new marker to distinguish isolated GBM-CTCs from residual lymphocytes.

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