Human Metastatic Brain Research Articles

Neuropathological Applications of Microscopy with Ultraviolet Surface Excitation (MUSE): A Concordance Study of Human Primary and Metastatic Brain Tumors.

Whereas traditional histology and light microscopy require multiple steps of formalin fixation, paraffin embedding, and sectioning to generate images for pathologic diagnosis, Microscopy using Ultraviolet Surface Excitation (MUSE) operates through UV excitation on the cut surface of tissue, generating images of high resolution without the need to fix or section tissue and allowing for potential use for downstream molecular tests. Here, we present the first study of the use and suitability of MUSE microscopy for neuropathological samples. MUSE images were generated from surgical biopsy samples of primary and metastatic brain tumor biopsy samples (n = 27), and blinded assessments of diagnoses, tumor grades, and cellular features were compared to corresponding hematoxylin and eosin (H&E) images. A set of MUSE-treated samples subsequently underwent exome and targeted sequencing, and quality metrics were compared to those from fresh frozen specimens. Diagnostic accuracy was relatively high, and DNA and RNA integrity appeared to be preserved for this cohort. This suggests that MUSE may be a reliable method of generating high-quality diagnostic-grade histologic images for neuropathology on a rapid and sample-sparing basis and for subsequent molecular analysis of DNA and RNA.

Phenotypic diversity of T cells in human primary and metastatic brain tumors revealed by multiomic interrogation

The immune-specialized environment of the healthy brain is tightly regulated to prevent excessive neuroinflammation. However, after cancer development, a tissue-specific conflict between brain-preserving immune suppression and tumor-directed immune activation may ensue. To interrogate potential roles of T cells in this process, we profiled these cells from individuals with primary or metastatic brain cancers via integrated analyses on the single-cell and bulk population levels. Our analysis revealed similarities and differences in T cell biology between individuals, with the most pronounced differences observed in a subgroup of individuals with brain metastasis, characterized by accumulation of CXCL13-expressing CD39+ potentially tumor-reactive T (pTRT) cells. In this subgroup, high pTRT cell abundance was comparable to that in primary lung cancer, whereas all other brain tumors had low levels, similar to primary breast cancer. These findings indicate that T cell-mediated tumor reactivity can occur in certain brain metastases and may inform stratification for treatment with immunotherapy.

A simple metastatic brain cancer model using human embryonic stem cell-derived cerebral organoids.

Every year, hundreds of thousands of people die because of metastatic brain cancer. Most metastatic cancer research uses 2D cell culture or animal models, but they have a few limitations, such as difficulty reproducing human tissue structures. This study developed a simple 3D in vitro model to better replicate brain metastasis using human cancer cells and human embryonic stem cell-derived cerebral organoids (metastatic brain cancer cerebral organoid [MBCCO]). The MBCCO model successfully reproduced metastatic cancer processes, including cell adhesion, proliferation, and migration, in addition to cell-cell interactions. Using the MBCCO model, we demonstrated that lung-specific X protein (LUNX) plays an important role in cell proliferation and migration or invasion. We also observed astrocyte accumulation around and their interaction with cancer cells through connexin 43 in the MBCCO model. We analyzed whether the MBCCO model can be used to screen drugs by measuring the effects of gefitinib, a well-known anticancer agent. We also examined the toxicity of gefitinib using normal cerebral organoids (COs). Therefore, the MBCCO model is a powerful tool for modeling human metastatic brain cancer in vitro and can also be used to screen drugs.

P03.14 Validation of microscopy with ultraviolet surface excitation - MUSE - imaging of human primary and metastatic brain tumors

P03.14 Validation of microscopy with ultraviolet surface excitation - MUSE - imaging of human primary and metastatic brain tumors

Homology analysis detects topological changes of Iba1 localization accompanied by microglial activation

The state of microglial activation provides important information about the central nervous system. However, a reliable index of microglial activation in histological samples has yet to be established. Here, we show that microglial activation induces topological changes of Iba1 localization that can be detected by analysis based on homology theory. Analysis of homology was applied to images of Iba1-stained tissue sections, and the 0-dimentional Betti number (b0: the number of solid components) and the 1-dimentional Betti number (b1: the number of windows surrounded by solid components) were obtained. We defined b1/b0 as the Homology Value (HV), and investigated its validity as an index of microglial activation using cerebral ischemia model mice. Microglial activation was accompanied by changes to Iba1 localization and morphology of microglial processes. In single microglial cells, the change of Iba1 localization increased b1. Conversely, thickening or retraction of microglial processes decreased b0. Consequently, microglial activation increased the HV. The HV of a tissue area increased with proximity to the ischemic core and showed a high degree of concordance with the number of microglia expressing activation makers. Furthermore, the HV of human metastatic brain tumor tissue also increased with proximity to the tumor. These results suggest that our index, based on homology theory, can be used to correctly evaluate microglial activation in various tissue images.

Anti-cancer Antibody Trastuzumab-Melanotransferrin Conjugate (BT2111) for the Treatment of Metastatic HER2+ Breast Cancer Tumors in the Brain: an In-Vivo Study.

The ability of human melanotransferrin (hMTf) to carry a therapeutic concentration of trastuzumab (BTA) in the brain after conjugation (in the form of trastuzumab-melanotransferrin conjugate, BT2111 conjugate) was investigated by measuring the reduction of the number and size of metastatic human HER2+ breast cancer tumors in a preclinical model of brain metastases of breast cancer. Human metastatic brain seeking breast cancer cells were injected in NuNu mice (n = 6-12 per group) which then developed experimental brain metastases. Drug uptake was analyzed in relation to metastasis size and blood-tumor barrier permeability. To investigate in-vivo activity against brain metastases, equimolar doses of the conjugate, and relevant controls (hMTf and BTA) in separate groups were administered biweekly after intracardiac injection of the metastatic cancer cells. The trastuzumab-melanotransferrin conjugate (BT2111) reduced the number of preclinical human HER2+ breast cancer metastases in the brain by 68% compared to control groups. Tumors which remained after treatment were 46% smaller than the control groups. In contrast, BTA alone had no effect on reducing number of metastases, and was associated with only a minimal reduction in metastasis size. The results suggest the novel trastuzumab-melanotransferrin conjugate (BT2111) may have utility in treating brain metastasis and validate hMTf as a potential vector for antibody transport across the Blood Brain Barrier (BBB).

Multiparametric MR imaging of tumor response to intraarterial chemotherapy in orthotopic xenograft models of human metastatic brain tumor.

The purpose of our study was to investigate the therapeutic efficacy of intraarterial (IA) chemotherapy via multiparametric magnetic resonance imaging (MRI) analysis in orthotopic mouse brain tumor models. Stereotactic-guided intracranial inoculation of MDA-MB-231 cells was performed in nude mice. Thirty tumor bearing mice were randomized into three groups, and each group received either IA docetaxel administration (n = 10), intravenous (IV) docetaxel administration (n = 10), or IA solvent injection (n = 10) as control. Treatment response was monitored by diffusion-weighted imaging and dynamic contrast enhanced-MRI obtained 1 day before and 8 days after therapy initiation. Imaging results were correlated with histopathology. In the results, IA chemotherapy showed a significant decrease in tumor volume (86.5 ± 15.6 %) compared to the IV chemotherapy (121.1 ± 39.6%) and control (126.2 ± 22.0%) 8 days after therapy (p < 0.05). Furthermore, IA chemotherapy resulted in a significant increase in mean tumor apparent diffusion coefficient (ADC) values (116.8 ± 44.9%); in contrary IV chemotherapy (66.6 ± 26.9%) and control (69.1 ± 29.5%) showed a significant decrease in ADC values corresponding to further tumor growth (p < 0.05). However, there was no significant difference in perfusion parameters including initial area under the curve, K(trans), K(ep), and V(e) between the groups (p > 0.05). Histopathology confirmed necrosis and necroptosis in the tumors after IA chemotherapy. In conclusion, IA chemotherapy may lead to effective inhibition of tumor cell proliferation and offer potential benefit of inducing higher degree of treatment response than IV chemotherapy.

Abstract 5208: Neovascularization in brain metastasis is through both angiogenesis and vasculogenesis

Abstract Metastatic brain tumors are the most common brain tumor in adults with an incidence 10 times greater than primary brain tumors. A variety of malignancies eventually spread to the brain, with a majority arising from cancers of the lung, breast, kidney, and skin. Experimental studies using breast and melanoma cell lines have recently shown that growth of metastatic brain tumors may occur by utilizing pre-existing blood vessel, or by co-opting rather than inducing new vessel formation. Whether the same mechanisms of vascular utilization and expansion play a role in human metastatic brain tumors is unknown and is the focus of the present study. We examined the immuno-phenotype of blood vessels in primary and metastatic human brain tumors to determine the presence of new blood vessels formation. Glioblastoma multiforme (n = 12) and secondary tumors from melanoma (n = 10), breast (n = 10), kidney (n = 5), and lung (n = 13) all showed extensive immunoreactivity for endothelial marker CD31. We also found a significant number of endothelial cells expressing Ki-67, most notably among breast carcinoma, glioblastoma multiforme and melanoma cases, indicating microvessel proliferation and angiogenesis. Interestingly, both primary and secondary metastatic tumors also showed vascular endothelial cells concomitantly expressing stem cell markers, such as Oct4, suggesting that some of these microvessels are derived through vasculogenesis. These findings show that both proliferation of endothelial cells and de novo endothelial differentiation may underlie metastatic tumor growth to different degrees depending on their site of primary origin. Currently, our laboratory is determining the exact contribution of angiogenesis and vasculogenesis in brain metastasis. Our findings show that neo-vessels are evident in brain metastasis. Elucidating the source of these vessels may uncover new treatment targets for patients with brain metastasis. Citation Format: Stephanie Mok, Lee-Cyn Ang, Christopher J. Howlett, Zia A. Khan. Neovascularization in brain metastasis is through both angiogenesis and vasculogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5208. doi:10.1158/1538-7445.AM2015-5208

Abstract 2653: Anti-cancer antibody trastuzumab-melanotransferrin conjugate (BT2111) for the treatment of metastatic HER2+ breast cancer tumors in the brain: An in vivo study

Abstract Background: Approximately 20% of patients with Her2+ metastatic breast cancer may be diagnosed with brain metastases. Trastuzumab, has been used successfully to treat peripheral Her2+ breast cancers, but has shown limited efficacy in brain metastases. Human melanotransferrin analog (MTf) is a vector to transport drugs across the blood brain barrier. In the current work, we investigated brain and brain metastases uptake of a MTf-trastuzumab conjugate (BT2111), and determined if the conjugate had efficacy in reducing the number and size of metastatic lesions in a preclinical model of brain metastases of breast cancer. Methods: Human metastatic brain seeking breast cancer cells (MDA-MB-231-Her2) were injected intracardially in 8 week old female NuNu mice (n=6-12 per group), allowed to circulate, and eventually develop experimental brain metastases. To examine drug uptake, radiolabeled conjugate (BT2111), trastuzumab and MTf was injected intravenously, and allowed to circulate for 30 min, 2 and 8 hours. Serial blood samples were drawn during circulation and terminal brain and blood samples were obtained. Drug uptake (Kin) was analyzed in relation to metastasis size and blood-tumor barrier permeability. To investigate in-vivo activity against brain metastases, we administered equimolar doses of the conjugate, and relevant controls in separate groups, biweekly starting 21 days after intracardiac injection of the metastatic cancer cells. Efficacy was assessed by determining both the number and size of metastases in brain at defined terminal endpoints. Results: Uptake of trastuzumab in “normal” brain (brain distant to tumor; BDT) was 0.17 + 0.004 x 10-6 mL/s/g and nearly 10 fold higher on average in metastatic lesions (1.1 + 0.04 x 10-6 mL/s/g). MTf uptake into BDT (18.4 + 0.5 x 10-6 mL/s/g) and brain metastases (53.0 + 3.1 x 10-6 mL/s/g) was significantly higher (p&amp;lt;0.01) than that of the antibody alone. BT2111 was nearly 30 fold higher (p&amp;lt;0.01) than that of the antibody alone in BDT (3.91 + 0.3 x 10-6 mL/s/g) and 10 fold higher (p&amp;lt;0.01) in metastases (10.7 + 0.43 x 10-6 mL/s/g). BT2111 reduced the number of preclinical human HER2+ breast cancer metastases in the brain of control animals (85 ± 6.3) by 68% (27.6 ± 3.9; P&amp;lt;0.001). Further, lesions in control animals were 54% larger (1.7 ± 0.7 µm2) than those in the BT2111 treated animals (0.78 ± 0.6 µm2; P&amp;lt;0.001). In contrast, trastuzumab alone had no effect on reducing the number of metastases (74 ± 5.0, P&amp;gt;0.05) and was associated with only a minimal (21%) reduction in metastasis size (1.34 ± 0.04 µm2). Conclusions: These results provide evidence that the MTf-trastuzumab conjugate, BT2111, has efficacy in treating brain metastasis in preclinical models and validates the role MTf has as a vector for the transport of antibodies across the BBB. Citation Format: Mohamed Nounou, Chris Adkins, Tori R. Terrell, Afroz Mohamed, Tim Vitalis, Reinhard Gabathuler, Paul R. Lockman. Anti-cancer antibody trastuzumab-melanotransferrin conjugate (BT2111) for the treatment of metastatic HER2+ breast cancer tumors in the brain: An in vivo study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2653. doi:10.1158/1538-7445.AM2014-2653

Efficacy of bispecific targeted immunotoxin DTATEGF against NSCLC brain metastatic tumor PC9-BrM3 cells

To investigate the in vitro and in vivo anticancer efficacy of the immunotoxin DTATEGF against human NSCLC brain metastatic tumor PC9-BrM3 cell line. The effect of the immunotoxin DTATEGF was tested for its ability to inhibit the proliferation of PC9-BrM3 cells in vitro by MTT assay. The cell cycle and the apoptosis of cells with 1 pmol/L DTATEGF were examined by flow cytometry. In vivo, 2 μg of DTATEGF or control Bickel3 was given intratumor to nude mice with established PC9-BrM3 xenografts on their hips, and tumor volumes were measured and tumor samples were investigated by immunchistochemistry SABC method. The microvessel density (MVD) was measured in each group. In vitro, DTATEGF killed PC9-BrM3 cells and showed an IC50 of 1 pmol/L. The apoptotic rate in the 1 pmol/L DTATEGF group was (64.0±0.5)% , significantly higher than that in the control group (1.5±0.4)% (P<0.01). The cell cycle was obviously inhibited by DTATEGF in a dose-dependent manner. The percentage of cells treated with 1 pmol/L DTATEGF in SubG0/G1 phase was (32.0±1.5)%, significantly higher than that in the control group (2.0±0.4)% (P<0.01). In vivo, DTATEGF significantly inhibited the growth of PC9-BrM3 hip tumors (P<0.05). The MVD of the DTATEGF group was (15.6±4.6)/mm2, significantly lower than that of the control group (31.2±5.4)/mm2 (P<0.01). DTATEGF inhibits the growth of the PC9-BrM3 cell line and induces its apoptosis. It is highly efficacious against human metastatic NSCLC brain tumor and against neovascularization.

Evaluation of a novel bispecific targeted toxin in a mouse model of a human metastatic breast cancer

Objective To investigate the anticancer effect of the immunotoxin DTATEGF in vitro and in vivo when delivered in mouse model of a human metastatic breast cancer brain tumors.Methods The effects of the immunotoxin were tested for their ability to inhibit the proliferation of 231Br cell line in vitro by methyl thiazol tetrazolium (MTT) assay.In vivo,2 μg of DTATEGF or control Bickel3 was givenby intraperitoneal injection to nude mice with established 231 Br xenografts on their flanks,tumor volumes were measured.On a xenograft intracranial model,1 μg of DTATEGF or control Bickel3 was delivered intracranially,the bioluminescent imaging was performed and Kaplan-Meier survival curves were generated.Results In vitro,DTATEGF could kill 231Br-luc cells and showed an 50％ inhibitory dose (IC50) less than 0.01 nmol/L.In vivo,DTATEGF significantly inhibited the growth of 231Br flank tumors (P ＜0.05),mice with intracranial tumors were treated was successful in providing a survival benefit significantly prolonged relative to controls (87 vs.58 days,P ＜0.01).Conclusion DTATEGF kills the 231Br-luc cell line in vitro,and it is highly efficacious against human metastatic breast cancer brain tumors. Key words: Immunotherapy; Breast carcinoma; Metastatic brain tumors; Model,animal

A novel brain metastases model developed in immunodeficient rats closely mimics the growth of metastatic brain tumours in patients

brain metastasis is a common cause of mortality in cancer patients, and associated with poor prognosis. Our objective was to develop a clinically relevant animal model by transplanting human biopsy spheroids derived from metastatic lesions into brains of immunodeficient rats. nine different patient brain metastases from four different primary cancers were implanted into brains of immunodeficient rats. The xenografts were compared with patient tumours by magnetic resonance imaging, histochemistry, immunohistochemistry and DNA copy number analysis. after transplantation, tumour growth was achieved in seven out of nine human brain metastases. Spheroids derived from four of the metastases initiated in the rat brains were further serially transplanted into new animals and a 100% tumour take was observed during second passage. Three of the biopsies were implanted subcutaneously, where no tumour take was observed. The animal brain metastases exhibited similar radiological features as observed clinically. Histological comparisons between the primary tumours from the patients, the patient brain metastases and the derived xenografts showed striking similarities in histology and growth patterns. Also, immunohistochemistry showed a strong marker expression similarity between the patient tumours and the corresponding xenografts. DNA copy number analysis between the brain metastases, and the corresponding xenografts revealed strong similarities in gains and losses of chromosomal content. we have developed a representative in vivo model for studying the growth of human metastatic brain cancers. The model described represents an important tool to assess responses to new treatment modalities and for studying mechanisms behind metastatic growth in the central nervous system.

The sodium pump α1 sub‐unit: a disease progression–related target for metastatic melanoma treatment

Melanomas remain associated with dismal prognosis because they are naturally resistant to apoptosis and they markedly metastasize. Up-regulated expression of sodium pump α sub-units has previously been demonstrated when comparing metastatic to non-metastatic melanomas. Our previous data revealed that impairing sodium pump α1 activity by means of selective ligands, that are cardiotonic steroids, markedly impairs cell migration and kills apoptosis-resistant cancer cells. The objective of this study was to determine the expression levels of sodium pump α sub-units in melanoma clinical samples and cell lines and also to characterize the role of α1 sub-units in melanoma cell biology. Quantitative RT-PCR, Western blotting and immunohistochemistry were used to determine the expression levels of sodium pump α sub-units. In vitro cytotoxicity of various cardenolides and of an anti-α1 siRNA was evaluated by means of MTT assay, quantitative videomicroscopy and through apoptosis assays. The in vivo activity of a novel cardenolide UNBS1450 was evaluated in a melanoma brain metastasis model. Our data show that all investigated human melanoma cell lines expressed high levels of the α1 sub-unit, and 33% of human melanomas displayed significant α1 sub-unit expression in correlation with the Breslow index. Furthermore, cardenolides (notably UNBS1450; currently in Phase I clinical trials) displayed marked anti-tumour effects against melanomas in vitro. This activity was closely paralleled by decreases in cMyc expression and by increases in apoptotic features. UNBS1450 also displayed marked anti-tumour activity in the aggressive human metastatic brain melanoma model in vivo. The α1 sodium pump sub-unit could represent a potential novel target for combating melanoma.

CTNNB1 (β-catenin) mutation is rare in brain tumours but involved as a sporadic event in a brain metastasis

The Wnt signaling pathway has been implicated in colon and other cancers. Nevertheless, few or no mutations of CTNNB1 (beta-catenin) have so far been described in brain cancer. We therefore examined the prevalence of constitutive activation of the Wnt signaling pathway in brain cancer specimens as well as cancer cell lines. We used polymerase chain reaction PCR and direct sequencing methods to investigate whether mutations in the CTNNB1 phosphorylation sites S33, S37, S41 and T45 were present in 68 brain tumours, including meningioma, astrocytoma, pituitary adenoma, neuroblastoma, metastasis to the brain, and cell lines. CTNNB1 gene mutations were not found in either the original brain tumour specimens or the cell lines. However, a missense mutation of CTNNB1 was identified at residue 33, TCT (Ser) --> TGT (Cys) in a patient with lung metastasis to brain. In addition, in vitro functional assay showed that the S33C mutant of beta-catenin did affect transcriptional activity in a TCF-4-luciferase reporter construct. These results indicate that the mutation of exon 3 of the CTNNB1 gene in brain tumours may be a rare event and yet may be required for a small subset of human metastatic brain tumours.

Fluorescence-guided resection of metastatic brain tumors using a 5-aminolevulinic acid-induced protoporphyrin IX: pathological study

We performed a pathological study to identify the locus of production of protoporphyrin IX (PPIX) in human metastatic brain tumors. Patients with metastatic brain tumors (n = 11) received 1 g of 5-aminolevulinic acid (5-ALA) perorally 2 h before undergoing surgery. The target region was exposed to laser light with a peak wavelength of 405 +/- 1 nm and an output of 40 mW. Tissue samples from the tumor bulk and surrounding areas were examined by histological and fluorescence methods. Of the 11 tumors, 9 manifested PPIX fluorescence in the tumor bulk and peritumoral brain tissue. Our findings indicate that PPIX fluorescence can be observed in peritumoral edematous areas that are free of neoplastic cells, because PPIX produced by neoplastic cells leaks into the surrounding edematous area.

Calcium-activated potassium channels mediated blood-brain tumor barrier opening in a rat metastatic brain tumor model

BackgroundThe blood-brain tumor barrier (BTB) impedes the delivery of therapeutic agents to brain tumors. While adequate delivery of drugs occurs in systemic tumors, the BTB limits delivery of anti-tumor agents into brain metastases.ResultsIn this study, we examined the function and regulation of calcium-activated potassium (KCa) channels in a rat metastatic brain tumor model. We showed that intravenous infusion of NS1619, a KCa channel agonist, and bradykinin selectively enhanced BTB permeability in brain tumors, but not in normal brain. Iberiotoxin, a KCa channel antagonist, significantly attenuated NS1619-induced BTB permeability increase. We found KCa channels and bradykinin type 2 receptors (B2R) expressed in cultured human metastatic brain tumor cells (CRL-5904, non-small cell lung cancer, metastasized to brain), human brain microvessel endothelial cells (HBMEC) and human lung cancer brain metastasis tissues. Potentiometric assays demonstrated the activity of KCa channels in metastatic brain tumor cells and HBMEC. Furthermore, we detected higher expression of KCa channels in the metastatic brain tumor tissue and tumor capillary endothelia as compared to normal brain tissue. Co-culture of metastatic brain tumor cells and brain microvessel endothelial cells showed an upregulation of KCa channels, which may contribute to the overexpression of KCa channels in tumor microvessels and selectivity of BTB opening.ConclusionThese findings suggest that KCa channels in metastatic brain tumors may serve as an effective target for biochemical modulation of BTB permeability to enhance selective delivery of chemotherapeutic drugs to metastatic brain tumors.

Decreased expression of heparanase in glioblastoma multiforme

The authors investigated the presence of endoglycosidase heparanase in human glioblastoma multiforme (GBM) and metastatic brain tumors as well as in healthy brain tissue to explore the relationship between the biological characteristics of GBM and the role of heparanase. Heparanase messenger (m)RNA was almost undetectable in GBMs in vivo, whereas it was frequently seen in metastatic brain tumors according to results of reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical analysis of paraffin-embedded tissue sections showed that neoplastic cells in metastatic brain tumors, especially in cells that invaded blood vessels, exhibit intense heparanase immunoreactivity. Heparanase was present in two highly invasive glioma cell lines, U87MG and U251MG, in vitro. These cell lines did not have metastatic capability, which was tested in an experimental pulmonary metastases model in mice. The activity of heparanase in these cell lines was almost the same as that in the highly metastatic melanoma cell line B16-F1. After nude mice were inoculated with U87MG cells, however, heparanase was no longer detected in subcutaneous or intracerebral experimental glioma in vivo based on results of immunohistochemical analysis. According to results of real-time quantitative PCR, there was a 10-fold increase in heparanase mRNA in U87MG glioma cells in vitro compared with that in experimental U87MG glioma tissue in vivo in nude mice. These results indicate that the expression of heparanase was downregulated in GBM in vivo, which rarely metastasizes to distant organs outside the central nervous system. Heparanase is not implicated in the invasiveness of GBM to surrounding healthy brain tissue in vivo.

Proliferative activity and in vitro replication of HSV1716 in human metastatic brain tumours

The neurotropic herpes simplex virus mutant HSV1716 lacks the gene encoding the virulence factor ICP34.5 and cannot replicate in non-dividing cells where proliferating cell nuclear antigen (PCNA) is not actively engaged in cellular DNA synthesis. In the brain, tumoral expression of PCNA therefore confers on it oncolytic specificity and may determine its efficacy. Three phase I trials in glioma patients and one in metastatic melanoma patients have established that HSV1716 is safe and replicates selectively in tumour tissue. Here we examine the in situ PCNA profiles of common human metastatic brain tumours and determine their in vitro permissivity for HSV1716 replication to ascertain their suitability for HSV1716 therapy. Histological sections of tumour biopsies obtained from patients undergoing craniotomies were stained for PCNA expression. The replicative ability of HSV wild-type (17(+)) and mutant (1716) viruses was assessed in tissue cultures of the same tumour biopsies and in cancer cell lines by plaque assay. Biopsies of all 10 metastatic tumours (3 melanoma, 4 carcinoma and 3 adenocarcinoma) as well as 4 glioblastoma multiforme were positive for PCNA immunoexpression and supported the replication of HSV1716. The PCNA-positive cells in the metastatic tumours were distributed comparatively in larger and more contiguous areas than in glioblastoma (1.69 +/- 1.61 mm(2) vs. 0.73 +/- 0.77 mm(2)) and numbered 29.0 +/- 12.4 and 12.6 +/- 4.7%, respectively. The results show that human cerebral metastatic tumours have generally larger and more contiguous proliferative areas, support efficient HSV1716 replication, and are thus potential candidates for such oncolytic viral therapy.

Consistent and Selective Expression of the Discoidin Domain Receptor-1 Tyrosine Kinase in Human Brain Tumors

Few molecular targets are both consistently and selectively expressed in a majority of central nervous system (CNS) neoplasms. Receptor tyrosine kinases have been implicated in brain tumor oncogenesis. We previously isolated one such receptor, discoidin domain receptor-1 (DDR1), from high-grade pediatric brain tumors. Here, we analyze the cellular origin and distribution of DDR1 expression in human brain tumors and its expression in tumor cells relative to surrounding brain.By use of a digoxigenin-labeled DDR1 riboprobe, we investigated the expression of DDR1 messenger ribonucleic acid in a prospective series of 30 resected human primary and metastatic brain neoplasms, nonneoplastic human brain, and mouse embryonic brain, as well as a mouse glioblastoma model, by in situ hybridization.All the high-grade primary brain and metastatic brain tumors showed unequivocal, intense DDR1 expression within the majority of tumor cells, whereas expression was not observed in hyperplastic tumor blood vessels, normal brain blood vessels, inflammatory cells, or in the normal brain tissue that surrounded the tumor. Receptor expression was limited to tumor cells located within solid tumor tissue. Overall, 27 of 29 resected CNS tumors exhibited tumor cell-specific DDR1 expression, whereas one specimen composed of isolated glioblastoma cells within invaded brain parenchyma showed no detectable staining for this receptor. DDR1 was also expressed preferentially in the ventricular zone (a region of highly proliferating precursor cells) of mice at embryonic Day 15.5.We found that DDR1 is consistently expressed in all high-grade brain neoplasms studied and is selective for tumor cells in the specimens analyzed. The expression of DDR1 by tumor cells of CNS neoplasms and by primitive cells of the embryonic ventricular zone suggests that DDR1 is a potentially useful marker of tumor cells within the CNS.

Consistent and Selective Expression of the Discoidin Domain Receptor-1 Tyrosine Kinase in Human Brain Tumors

Few molecular targets are both consistently and selectively expressed in a majority of central nervous system (CNS) neoplasms. Receptor tyrosine kinases have been implicated in brain tumor oncogenesis. We previously isolated one such receptor, discoidin domain receptor-1 (DDR1), from high-grade pediatric brain tumors. Here, we analyze the cellular origin and distribution of DDR1 expression in human brain tumors and its expression in tumor cells relative to surrounding brain. By use of a digoxigenin-labeled DDR1 riboprobe, we investigated the expression of DDR1 messenger ribonucleic acid in a prospective series of 30 resected human primary and metastatic brain neoplasms, nonneoplastic human brain, and mouse embryonic brain, as well as a mouse glioblastoma model, by in situ hybridization. All the high-grade primary brain and metastatic brain tumors showed unequivocal, intense DDR1 expression within the majority of tumor cells, whereas expression was not observed in hyperplastic tumor blood vessels, normal brain blood vessels, inflammatory cells, or in the normal brain tissue that surrounded the tumor. Receptor expression was limited to tumor cells located within solid tumor tissue. Overall, 27 of 29 resected CNS tumors exhibited tumor cell-specific DDR1 expression, whereas one specimen composed of isolated glioblastoma cells within invaded brain parenchyma showed no detectable staining for this receptor. DDR1 was also expressed preferentially in the ventricular zone (a region of highly proliferating precursor cells) of mice at embryonic Day 15.5. We found that DDR1 is consistently expressed in all high-grade brain neoplasms studied and is selective for tumor cells in the specimens analyzed. The expression of DDR1 by tumor cells of CNS neoplasms and by primitive cells of the embryonic ventricular zone suggests that DDR1 is a potentially useful marker of tumor cells within the CNS.