Human Brain Metastases Research Articles

Effects of Roundabout 5 on adhesion, invasion and potential motility of human tongue carcinoma Tb cells.

Roundabout 5 (R5) is a monoclonal antibody which can neutralize the binding of Roundabout 1 (Robo1) to Slit2. Oral squamous cell carcinoma angiogenesis was significantly inhibited when R5 blocked slit-robo signaling pathway. However, the effect of R5 on the invasion of tongue cancer cells has not been investigated clearly. In this study, we treated human brain metastasis of tongue cancer cell lines (Tb cells) with R5 at different concentrations, and the control Tb cells were treated with 10 mg/ml immunoglobin G 2b (IgG2b). The effect of R5 on the proliferation, adhension, invasion and motility of Tb cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, cell attachment assay on fibronectin (FN), wound assay and chemotaxis assay, respectively. And gelatin-incorporated sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to investigate the activity of matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9). R5 had no effect on the proliferation of Tb cells. However, R5 could significantly inhibit the motility, attachment and chemotaxis of Tb cells to FN, and it could also significantly inhibit the activity of MMP2 and MMP9 in Tb cells. R5 can inhibit the adhesion, invasion and motility of human tongue carcinoma Tb cells.

Abstract 1477: Tumor cell-leukocyte fusion in human cancer: A melanoma brain metastasis with a donor-patient hybrid genome following allogeneic BMT

Abstract Tumor cell fusion with motile bone marrow-derived cells (BMDCs) has long been proposed as a mechanism for metastasis, but while such fusion has been demonstrated in animal models little is known of this in human cancer. Here we report the first genetic evidence for BMDC-tumor fusion and genomic hybridization in a human melanoma brain metastasis from a patient with a prior allogeneic bone marrow transplant (male-male, sibling). STR analyses of DNA from melanoma cells throughout the tumor revealed the presence of both donor and patient alleles, with common aberrant allelic patterns at multiple chromosomal loci indicating a clonal origin of the tumor. Methods. All procedures were in accordance with HIPAA regulations and approved by the Yale HIC. Donor and patient DNAs were obtained from frozen lymphocytes and from a patient buccal swab. Formalin-fixed, paraffin-embedded tumor specimens were stained by immuniohistochemistry with common leucocyte antigen (CLA) in order to avoid leucocytes during microdissection of tumor cells. LCA-negative tumor cells were isolated with an Arturus XT laser dissection microscope system. DNA was extracted and analyzed for donor and patient alleles using forensic STR analyses at 13 chromosomal loci and amelogenin for the X and Y. Tissue sections were analyzed from 14 areas throughout the tumor. Results. DNA from donor and patient lymphocytes showed balanced diploidy at all loci and the allelic patterns were consistent with the sibling relationship. On the other hand, DNA from tumor cells contained a mixture of donor and host alleles at some loci, only donor or patient alleles at others, and numerous allelic imbalances throughout–particularly for the X and Y (X:Y = 2.8:1) that pointed to a probable tumor genotype of XXXY. With few exceptions, aberrant genetic profiles seen in one tumor section were seen in all, demonstrating a clonal origin of the tumor. Conclusions. The results provide the first genetic proof of leucocyte-tumor fusion and genomic hybridization in human cancer and indicate that the tumor was generated from a single fusion event. While this report concerns only one case, we demonstrate that forensic genetic analyses of tumors arising as secondary malignancies represents a fruitful area for research in tumor cell fusion in human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1477. doi:10.1158/1538-7445.AM2011-1477

Abstract 2667: L1CAM neural adhesion molecule is overexpressed in human brain metastases of breast cancer and contributes to colony formation efficiency

Abstract To investigate the molecular mechanisms responsible for breast cancer metastasis to the brain, a gene expression analysis was performed using formalin-fixed, paraffin embedded sections of matched primary breast tumors and resected brain metastases. The neuronal cell adhesion molecule L1CAM was upregulated by approximately 1.57 fold in the brain metastases relative to the primary tumors (p=0.037). This trend was most apparent in Her-2+ tumors (p=0.044) and did not extend to ER+ patients (p=0.42). L1CAM has been correlated to decreased patient survival in breast cancer. MDA-MB-231 breast cancer cells selected to grow in the brain (231-BR) expressed 6.4 fold greater L1CAM on western blots than the parental cell line (231-P). Using three shRNA constructs, L1CAM expression was decreased by 1.76, 3.82, and 63.3 fold in the 231-BR cell line. Decreased L1CAM expression inhibited the clonogenic formation of 231-BR cells by 40-50% (p<0.002); however, increased proliferation by MTT assay by 30-60% (p<0.002). The most efficient L1CAM knockdown also showed a 30% decrease in migration via scratch assay relative to the non-targeting control, suggesting that L1CAM may be involved in 231-BR cell migration (p<0.03). Studies are underway to determine the effect of L1CAM down-regulation on the development of breast cancer brain metastases. These results suggest L1CAM may be a potential novel therapeutic target for metastatic breast cancer in the brain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2667. doi:10.1158/1538-7445.AM2011-2667

Abstract 3561: Pazopanib inhibits a subpopulation of PDGFR+ astrocytes in the neuro-inflammatory microenvironment of breast cancer brain metastases

Abstract Brain metastases occur in approximately 35% of metastatic breast cancer patients whose tumors overexpress HER2. Using a quantitative model system to study breast cancer brain metastasis, HER2 overexpression increased the brain colonization of a brain seeking subline of MDA-MB-231 cells (231-BR-HER2). Since HER2 has been shown to induce overexpression of VEGF, we hypothesized that the anti-angiogenic drug pazopanib would reduce the metastatic outgrowth of breast cancer cells in the brain. Pazopanib is a multispecific tyrosine kinase inhibitor targeting VEGFR-1, -2, and -3, PDGFR alpha and beta, and c-kit. We previously showed that pazopanib directly targets the tumor cell through the disruption of B-Raf pathway. Pazopanib prevented the outgrowth of 231-BR-HER2 large brain metastasis by 73% (p<0.0001). Metastasis has been described as an interaction of tumor cells and their environment. Here we evaluated the effect of pazopanib on the brain neuro-inflammatory microenvironment. Previously, activated microglia and activated GFAP-expression astrocytes were observed around and within experimental brain metastases as well as from resected human brain metastases of breast cancer. In our experiments, the 231-BR-HER2 cell line was injected into the left cardiac ventricle of immunocompromised mice; mice were treated with vehicle, 30mg/kg or 100mg/kg pazopanib twice a day beginning on day 3 post-injection. Brain metastases were counted using H&E staining and brain slides were immunostained with markers for brain micro-environment components. In the brain metastases, using an antibody to the phosphorylation site tyrosine 751 of PDGFR beta, a subpopulation of GFAP+ astrocytes was observed, previously undescribed. P-PDGFR beta + astrocytes were only detected around the brain metastases, suggesting a specific activation of astrocytes by tumor cells. Pazopanib treatment resulted in a trend, 76% (p=0.13) and 80% (p=0.070) decrease of the pY751 PDGFR beta + astrocyte population, at 30 and 100mg/kg respectively. In contrast, Pazopanib did not have any effect on NG2+pericyte, collagen IV or microglia expression. The data suggest a specific effect of pazopanib on the brain microenvironment. Collectively, the data identify an effect of pazopanib on the brain microenvironment, as well as tumor cells, which makes this drug an interesting candidate for brain metastasis prevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3561. doi:10.1158/1538-7445.AM2011-3561

Abstract 2846: Pigment epithelium-derived factor (PEDF) functions as a brain metastasis suppressor of breast cancer

Abstract Brain metastases occur in 10-20% of metastatic breast cancer patients. The one year survival rate is approximately 20%. Brain metastasis is typically diagnosed late in breast cancer progression; however, studies suggest that the incidence of brain metastasis may be increasing under current chemotherapeutic regimens. We have investigated changes in gene expression associated with the acquisition of brain metastatic potential by breast cancer. A DNA microarray analysis comparing human brain metastases and a cohort of unlinked primary breast carcinomas identified Pigment Epithelium-Derived Factor (PEDF) as down-regulated in the brain metastases relative to the primary tumors (mean ∼14X lower by QPCR validation). The data suggest the hypothesis that PEDF may have a negative impact on brain metastatic progression. PEDF, a secreted factor, has been shown to act as a tumor suppressor, has strong anti-angiogenic activity, but supports the proliferation/viability/ differentiation of neural tissue. We hypothesize that the multiple roles of PEDF make it a potent agent which targets not only tumor cells, but also the microenvironment of the brain. In vitro experiments demonstrated that PEDF can promote death of breast cancer cells. Conversely, PEDF promoted neuronal survival, in vitro. In a xenograft model system (231BR, a brain-tropic derivative of human MDA-MB-231 breast cancer cells) PEDF overexpression inhibited formation of large brain metastases (∼2-fold, p=0.001) and reduced proliferation of PEDF-expressing breast cancer cells in mouse brain metastases (11% Ki67-positive, PEDF-positive vs. >56% Ki67-positive, PEDF-negative). In an intracranial implantation model, expression of PEDF by breast cancer cells lead to a rapid reduction in brain metastatic tumor volume. Histological examination suggests that PEDF reduces gliosis and protects neurons concomitant to suppression of metastatic growth. The data suggest the intriguing possibility that PEDF overexpression may concurrently inhibit brain metastasis of breast cancer cells but preserve neuronal function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2846. doi:10.1158/1538-7445.AM2011-2846

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.

Neural cell adhesion molecule isoform 140 declines with rise of WHO grade in human gliomas and serves as indicator for the invasion zone of multiform glioblastomas and brain metastases

Gliomas are highly invasive neuroepithelial tumors with a propensity of malignant transformation and very restricted treatment options. The neural cell adhesion molecule (NCAM) modulates cellular migration, proliferation, and synaptic plasticity by homophilic and heterophilic interactions. Hereby, we investigated its relevance as a glioma tissue marker for the biological aggressiveness of these tumors and compared these features with the carcinoma brain metastasis invasion zone. We analyzed 194 human brain samples. Human tumor-free brain specimens served as control for the white and gray matter. In addition to that, we used human glioblastomas from nude rats. All tissues were investigated immunohistochemically for the expression of the NCAM isoform 140. Additionally, the multiplanar MRI-CT fusion neuronavigation-guided serial stereotactic biopsy was performed and completed by histopathological workup. Human gliomas loose NCAM-140 with the rise of their WHO grade. Meningiomas are NCAM-140 negative. As the most striking feature, human brain metastases and the majority of human glioblastomas of our patients and of nude rats were totally NCAM-140 negative. This NCAM negativity led us to the conclusion of three different main glioblastoma invasion patterns. Surprisingly, the majority of brain metastasis samples that contained surrounding brain parenchyma demonstrated invasive tumor cell nests beyond the sharply demarcated metastasis border. We also found invasive metastatic cell nests outside the contrast enhancing tumor zone by means of the MRI-CT fusion neuronavigation-guided serial stereotactic biopsy. The expression of NCAM-140 inversely correlates with the WHO grade of human gliomas. The lost expression of NCAM-140 in human glioblastomas and in brain metastases enables the investigation of the brain-tumor interface and the definition of glioblastoma invasion patterns and shows that brain metastases are more invasive than ever thought.

Abstract 2373: Heterogeneity of permeability in a preclinical model of brain metastases of breast cancer does not correlate with vascular density, HIF-1α expression, or tumor survival

Abstract Introduction: High vascular density of brain metastases of breast cancer has been correlated with both poor patient prognosis and with potential chemotherapeutic success. Inversely a low vascular density implies there would be decreased distribution of anti-cancer chemotherapeutics, and over time reduced efficacy of tumor kill. Given that there is heterogeneity in response to chemotherapeutics in human brain metastases and metastases are different than primary tumors; we used a novel brain metastases of breast cancer model to characterize vascular density, and corresponding changes in tumor permeability, hypoxia and survival. Methods: Brain seeking metastatic tumor lines MDA-MB-231-Br and 4T1-Br5 were injected via the left cardiac ventricle in separate experiments and brain metastases developed for ∼28 days and ∼15 days respectively. After development, both 14C-AIB and indocyanine green (ICG) were injected and allowed to circulate for 10 and 1 min respectively. Near infrared imaging was used to identify ICG filled vessels to quantify vascular density of lesions compared to normal brain. To determine permeability, concentrations of 14C-AIB in blood and brain tissue were calculated using autoradiography. Immunofluorescent analysis was performed using anti-HIF-1α and anti Ki67 antibodies and co-stained with DAPI. Results: A significant amount of metastatic lesions were observed to have developed in each model. Lesions varied in size from 0.028 to 2.3mm2. Vascular density was significantly decreased (MDA 176 ± 96 and 4T1 201 ± 76) compared to control brain (429 ± 25) and to an RG2 implanted glioma used as a positive control (736 ± 168). Based on accumulation of 14C-AIB, metastases (range: 22 nCi/g to 1682 nCi/g) were considered to be either permeable (3 fold or greater uptake compared to adjacent control tissue) or non-permeable. There was no strong correlation between lesion size and permeability (r2=0.23) or vascular density (r2 = 0.06). HIF-1α was expressed in both permeable and non-permeable lesions. Lastly, no significant difference was seen in the percentage of Ki67 positive cells in the MDA (permeable 41 ± 2.9, n=7; and non-permeable 40.8 ± 2.7, n=14) and 4T1 model (permeable 52.0 ± 4.6, n=6; and non-permeable 45.0 ± 2.8, n=6). Discussion: This data provides a correlation of permeability to vascular density, HIF-1α and Ki67 in a brain metastases model. While, both brain model systems exhibited a high degree of heterogeneous permeability, in neither model did permeability strongly correlate with vascular density, lesion size, HIF-1α expression and positivity for Ki67. Based upon the data, this experimental model may reflect the heterogeneity seen in human brain metastases of breast cancer and suggests there are other biological factors besides vascular density which correlate with drug distribution and or tumor/survival growth. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2373.

Patupilone for the treatment of recurrent/progressive brain metastases in patients (pts) with non-small cell lung cancer (NSCLC): An open-label phase II study

2033 Background: Preclinical data have shown that patupilone, a novel microtubule stabilizer, crosses the intact blood brain barrier in mice, rats and dogs and has antitumor activity in brain tumor models. This study evaluates patupilone's efficacy in NSCLC pts with brain metastases and progression after chemotherapy, surgery and/or radiation to the brain. Methods: This open-label, single-arm, multicenter, phase II, Simon 2-stage (St) study (25 pts per St) uses a multinomial design (response (R), early progression (EP)). Primary efficacy endpoints were a combination of EP (PD or death before cycle 1 Day 21) and R (progression free at 9 wks). Criteria to proceed to St 2 were >3 R and <11 EP in 25 pts. Eligible pts had confirmed NSCLC and ≥1 recurrent, measurable intracranial lesion ≥2 cm. Patupilone was given as 10 mg/m2 in a single 20-min IV infusion every 3 wks. Results: So far, 25 pts in St I and 2 pts in St II (median age 62 yrs, range 40–74, 59% male) have received 78 cycles (median 2 cycles). All pts received prior radiotherapy and 10/27 (37%) had prior chemotherapy for the brain. Karnofsky status in 25/27 ≥70% and in 2/27 <70%. Common G1/2 adverse events (AEs) were as follows: diarrhea 59%, nausea 19%, fatigue and peripheral neuropathy, each 11%. There were 21 G3 AEs (44%); 10 were related to study drug (diarrhea in 6). Eight G4 AEs (19%) were not related and 1 G4 AE (colitis) was suspected. Investigator reported efficacy data from St 1 (n=25) showed 8 responders (median duration of response 192 days, range 191–273); 7 had stable disease, 9 EP and assessment was unknown in 1 pt. Investigator reported median progression free survival is 1.2 mos. Three of the responding patients continued ≥8 cycles. Conclusions: The study met the statistical criteria to proceed to St 2. Proof of concept of patupilone activity in human brain metastases has been reached. These results warrant further investigation of patupilone as a treatment for CNS metastases from NSCLC. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis Novartis Novartis AstraZeneca, Aventis, Eli Lilly, Genentech™ BioOncology, Pfizer Merck, Novartis

Biological Pathways Contributing to Organ-Specific Phenotype of Brain Metastatic Cells

Secondary to the increased survival following chemotherapy, brain metastases have recently become a significant clinical problem for breast cancer patients. The aim of this study was to characterize those functional phenotypes that might enhance brain metastasis in breast cancer cells. We first analyzed by two-dimensional electrophoresis (2DE-DIGE) differences in protein expression between parental MDA-MB 435 cells and the brain metastatic variant 435-Br1, obtaining 19 identified proteins by peptide mass fingerprinting, 11 under-expressed (<2-fold) and 8 overexpressed (>2-fold) in 435-Br1. We created and analyzed protein interaction networks with a bioinformatic program (PIANA) from protein data, and it allowed us to associate 34/67-laminin receptor functionally with HSP 27, through a chaperone glucose-regulated protein GRP 94. Moreover, HSP 27 had the largest amount of direct and indirect protein interactions, forming a cluster of chaperones and cochaperones, associated through kinases to a set of intermediated filament proteins. In addition, functional groups of proteins identified were peptidase, DNA binding transcription factors, ATP synthase complex, anion transporters, and carbohydrate metabolism. Further functional analyses in cells, expression analyses in experimental tissues, and in human brain metastasis were addressed to validate the biological pathways contributing to organ-specific phenotype of brain metastasis.

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.

Activation of Stat3 in Human Melanoma Promotes Brain Metastasis

Brain metastasis is a major cause of morbidity and mortality in patients with melanoma. The molecular changes that lead to brain metastasis remain poorly understood. In this study, we developed a model to study human melanoma brain metastasis and found that Stat3 activity was increased in human brain metastatic melanoma cells when compared with that in cutaneous melanoma cells. The expression of activated Stat3 is also increased in human brain metastasis specimens when compared with that in the primary melanoma specimens. Increased Stat3 activation by transfection with a constitutively activated Stat3 enhanced brain metastasis, whereas blockade of Stat3 activation by transfection with a dominant-negative Stat3 suppressed brain metastasis of human melanoma cells in animal models. Furthermore, altered Stat3 activity profoundly affected melanoma angiogenesis in vivo and melanoma cell invasion in vitro and significantly affected the expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-2 (MMP-2) in vivo and in vitro. Finally, Stat3 activity transcriptionally regulated the promoter activity of bFGF in addition to VEGF and MMP-2 in human melanoma cells. These results indicated that Stat3 activation plays an important role in dysregulated expression of bFGF, VEGF, and MMP-2 as well as angiogenesis and invasion of melanoma cells and contributes to brain metastasis of melanoma. Therefore, Stat3 activation might be a new potential target for therapy of human melanoma brain metastases.

Proteolytic Cleavage of the CD44 Adhesion Molecule in Multiple Human Tumors

Cell surface adhesion molecules are crucial for the development and/or pathogenesis of various diseases including cancer. CD44 has received much interest as a major adhesion molecule that is involved in tumor progression. We have previously demonstrated that the ectodomain of CD44 undergoes proteolytic cleavage by membrane-associated metalloproteases in various tumor cell lines. The remaining membrane-bound CD44 cleavage product can be detected using antibodies against the cytoplasmic domain of CD44 (anti-CD44cyto antibody). However, the cleavage of CD44 in primary human tumors has not been investigated. Using Western blots with anti-CD44cyto antibody to assay human tumor tissues, we show that the CD44 cleavage product can be detected in 58% (42 of 72) of gliomas but not in normal brain. Enhanced CD44 cleavage was also found in 67% (28 of 42) of breast carcinomas, 45% (5 of 11) of non-small cell lung carcinomas, 90% (9 of 10) of colon carcinomas, and 25% (3 of 12) of ovarian carcinomas. Tumors expressing a CD44 splice variant showed a significantly higher incidence of enhanced CD44 cleavage. The wide prevalence of CD44 cleavage suggests that it plays an important role in the pathogenesis of human tumors.

Paucity of retinoic acid receptor alpha (RAR alpha) nuclear immunostaining in gliomas and inability of retinoic acid to influence neural cell adhesion molecule (NCAM) expression.

Neural cell adhesion molecule (NCAM) is down-regulated during periods of embryological cell migration and may be important in local tumor migration or metastases. Conflicting information exists in the literature about NCAM expression in human glial tumors and little is known about its expression in human brain metastases. We immunohistochemically stained a panel of 43 primary human brain tumors and their cultured counterparts for NCAM including glioblastoma multiformes, anaplastic astrocytomas, oligodendrogliomas, and contrasted their staining with a panel of 3 meningiomas, 11 brain metastases, and 5 normal brain samples utilizing the monoclonal antibody NKH-1. Most gliomas and metastatic melanomas and lung carcinomas showed a high percentage of cells positive for NCAM expression while NCAM staining was negative for other carcinomas. No difference was seen between intensity or percentage of cells that were NCAM positive, based on tumor grade or type. In glioma cell lines, NCAM expression was lost upon passage. In 15 glioma cell lines we also determined NCAM isoform expression by reverse transcription/polymerase chain reaction (RT/PCR) and found that 6 of 15 had message for NCAM 180, 8 of 15 for NCAM 140, and only 3 of 15 had message for NCAM 120. Normal brains always contained message for the 180 isoform and usually had mRNA for all 3 isoforms. Using monoclonal antibodies for retinoic acid receptor alpha (RAR alpha), we found nuclear staining in melanomas and lung carcinomas metastatic to brain and only rarely in gliomas. Neither the relative antigen density of NCAM nor the percent of NCAM-positive cells appreciably changed upon incubation with retinoic acid (RA), as measured by flow cytometry. RAR alpha was not found at a level measurable by immunohistochemistry in nuclei of most glial tumors, providing an explanation for why RA might not induce NCAM expression. Whether paucity of RAR alpha on primary gliomas might also correlate with results from clinical trials showing limited efficacy of RA in treatment of human gliomas awaits further study.

Immunocytochemical characterization of Cd44 molecules expressed in human brain metastases

CD44v adhesion molecules have been known to be related with carcinogenesis and metastasis of some human cancers including brain metastases of various origin. In this study, we demonstrated further that CD44v expression in brain metastases is extremely heterogeneous. Generally, v5/v6 is homogeneously expressed in the cell populations, while up-regulated v7-v10 could be usually found in a proportion of tumor cells, especially those close to necrosis, at the tumor border and with strong tendency of invasiveness. Our data thus suggest that heterogeneous CD44v expression may be determined not only by intrinsic nature of the cells but also by the surrounding micro-environment. Expression of multiple isoforms of CD44v may confer metastatic cells certain unique abilities, allowing them to localize, then to form metastatic colony in the brain.

Expression of the epidermal growth factor receptor gene in human brain metastases.

Biopsy specimens of human brain metastases were examined for amplification and expression of the proto-oncogene c-erbB1 (located on chromosome 7) encoding the epidermal growth factor receptor (EGFR). Moreover, the tumour DNA was also examined for amplification of other cancer-related genes on this chromosome: the proto-oncogene c-met, the gene for platelet-derived growth factor A-chain, and the gene for plasminogen activator inhibitory type 1. All 18 brain metastases demonstrated positive binding of biotinylated EGF on cryosections. Three out of 18 metastases had amplification of the EGFR gene; the other chromosome-7 genes tested were not amplified. Thus, an increased EGFR gene expression seems to be a general finding in a wide range of carcinomas metastatic to the brain, whereas we found only occasional selective EGFR gene amplifications in single cases.

Absence of Laminin Deposition in Breast Cancer and Metastases Except to the Brain

Laminin, a major glycoprotein of basement membrane has been found to play significant roles during invasion and metastases. In this study, we have examined the distribution of laminin in several human brain carcinoma metastases, human breast cancers, skin and lymph node metastases of breast cancer as well as in an in vitro and an in vivo model of invasion. A laminin accumulation was demonstrated a) at the border between human metastatic carcinoma cells and surrounding neural tissue; b) at the invasive edge between MO4 cells (a highly malignant cell line which synthesizes large amounts of laminin) and host tissues of syngenic mice; c) at the front of invasion between MO4 cells and precultured heart fragments in an in vitro model of invasion. Laminin, but not type IV collagen, promoted attachment of MO4 cells. This attachment was inhibited by preincubation of laminin matrix support with (+)-catechin, a flavonoid which also prevented invasion of the precultured heart fragment in vitro. Our data demonstrate that laminin accumulates between malignant cells and host tissue in human brain metastases and in an in vitro and an in vivo model of invasion. In these later models, accumulation of laminin is the consequence, at least in part, of its biosynthesis by MO4 cells. Since laminin promotes attachment of malignant cells in vitro, increases invasiveness and metastatic activities of murine malignant cells, it is tempting to speculate that laminin synthesized by invasive cells and accumulated at the front of invasion plays a significant role in the first step of invasion.

Formation and propagation of brain oedema fluid around human brain metastases. A CT Study.

Computerized tomography (CT) was used to examine the time-course of the propagation of extravasated contrast medium from small brain metastases into the peritumoural oedematous white matter, following infusion of 200 ml of meglumine amidtrizoate for 3 hours. Four patients with a metastatic brain tumour were examined. CT scans at identical levels were taken 1.5, 3, 6, 9, and 12 hours after start of contrast infusion. Following 4-7 days of dexamethasone treatment (8-12 mg/day i.v.) the examination was repeated. A contrast-enhanced area was observed surrounding the clearly delineated tumours, expanding gradually in a circular fashion into the peritumoural white matter oedema. The expanding circular enhancement was measured planimetrically on the various scans. From these values, the increase in radius/hr respectively in volume/hr was calculated, assuming a spherical geometry. This enabled a determination of the rate of oedema fluid formation and of the speed of oedema fluid propagation. The formation rate of oedema fluid amounted to 0.5-3.2 ml/hour and the speed of oedema fluid spreading to 1.9 mm/hour. Following treatment with dexamethasone the formation rate of oedema fluid is reduced by 30-50%. The important clinical implications of these new findings are discussed.