Immunohistochemical study of tight junction-related protein in neovasculature in astrocytic tumor

To clarify whether the neovasculature of brain tumors preserves blood-brain barrier (BBB) functions, we studied the expression of a tight junction—related protein, Zo-1, using immunohistochemistry. Twenty-six astrocytic tumors were examined using an anti-Zo-1 Mab, and Zo-1 expression was compared with the expression of vasicular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR) (flt-1), and antiproliferative cell nuclear antigen (PCNA). A positive reaction for Zo-1 was seen in the endothelial cells in micro-blood vessels in all astrocytic tumors. The reactions for Zo-1 in the endothelial cells forming glomeruloid proliferations in newly formed micro-blood vessels in high-grade tumors were weaker than those in the endothelial cells of normal cerebral capillaries. Although there is a negative correlation between positive immunoreactions for BBB-related proteins and the expression of VEGF of the endothelial cells in micro-blood vessels, the proliferative activity of tumor cells, and histological grades, the present findings suggest that the endothelial cells of the neovasculature of high-grade tumors preserve partial BBB function at the cellular level. Because of the ease of immunohistochemical procedures compared with electron microscopic examination, the immunohistochemical detection of Zo-1 should provide a useful marker for tight junctions.     

Glomeruloid Structures as Vascular Reaction in Human Gastrointestinal Carcinoma

Malignant tumors induce angiogenesis and modulation of microvasculature. Based on histologic and immunohistochemical analysis of human surgical material, we describe here the occurrence of glomeruloid structures in gastrointestinal carcinomas, and compare them with the microvasculature in inflammatory granulation tissue. The glomeruloid structures were composed of clusters of mutually fused capillaries with prominent swelling of endothelial cells and pericytes. They were thought to be specific for glioblastoma of the brain. The glomeruloid structures were observed juxtaposed to carcinoma nests in one-third of gastric carcinoma of intestinal type and colorectal carcinoma in the area of invasive growth beyond the muscularis mucosae. They were not observed in gastric carcinoma of diffuse type, intramucosal carcinoma, or inflammatory granulation tissue. The glomeruloid structures can be regarded as an extreme example of endothelial hyperplastic changes observed in cancer stroma. Our results suggested that glomeruloid structures can occur in carcinomas as vascular reaction, a mechanism different from that in inflammatory granulation tissues.     

Glomeruloid structures as vascular reaction in human gastrointestinal carcinoma.

Malignant tumors induce angiogenesis and modulation of microvasculature. Based on histologic and immunohistochemical analysis of human surgical material, we describe here the occurrence of glomeruloid structures in gastrointestinal carcinomas, and compare them with the microvasculature in inflammatory granulation tissue. The glomeruloid structures were composed of clusters of mutually fused capillaries with prominent swelling of endothelial cells and pericytes. They were thought to be specific for glioblastoma of the brain. The glomeruloid structures were observed juxtaposed to carcinoma nests in one-third of gastric carcinoma of intestinal type and colorectal carcinoma in the area of invasive growth beyond the muscularis mucosae. They were not observed in gastric carcinoma of diffuse type, intramucosal carcinoma, or inflammatory granulation tissue. The glomeruloid structures can be regarded as an extreme example of endothelial hyperplastic changes observed in cancer stroma. Our results suggested that glomeruloid structures can occur in carcinomas as vascular reaction, a mechanism different from that in inflammatory granulation tissues.     

Distribution of collagen Type IV in brain tumors: An immunohistochemical study

Summary One hundred-twenty seven human brain tumors were examined by an immunoperoxidase technique for the expression of collagen Type IV, a major constituent of basement membrane. The parenchymal components were negative for the marker protein in all tumors except for neurilemmomas which were positively stained. In every case, the antibody to collagen Type IV showed distinct staining of the vascular pattern. In gliomas, capillaries increased in number and the vascular staining increased in intensity. Fine branching capillaries and endothelial glomeruloid proliferations characteristic each of oligodendrogliomas and glioblastomas could be distinctly illustrated. In two ependymomas, marked capillary proliferation was noted in periventricular areas. Fibrillar staining was observed between the tumor cells in seven of 34 meningiomas. Pericapillary lamellar deposition of collagen Type IV suggests a vascular origin of psammoma bodies. In some malignant tumors, pial-glial membranes were disrupted and the Virchow-Robin spaces were filled with malignant cells. Collagen Type IV was absent around the stromal cells of hemangioblastomas, suggesting that these stromal cells were unrelated histogenetically with endothelial cells. Collage Type IV may be useful in the differential diagnosis between meningiomas and neurilemmonas.     

The significance of immuno-stain with HLC3-AB in cerebral metastatic tumor

By using the immunohistochemical technique (ABC method), 186 cases of cerebral metastatic tumors labeled with McAb against human lung carcinoma are reported. They were divided into two groups: primary site determined group and undetermined group. In determined group (59 cases), 33/36 cases (91.6%) of lung carcinomas showed HLC₃ positive reactions, but among 23 cases of other cancers, only two cases showed weak positive reactions. This result indicated that the HLC₃ have strong specific reaction to the lung carcinomas. In undetermined group (127 cases), 68 cases of metastatic tumors reacted to HLC₃ positively. In all 186 cases of cerebral metastatic tumor, there were 101 cases (53.5%) showing positive reaction to HLC₃−AB. Among 125 cases of cerebral metastatic adenocarcinoma, 76 cases (61%) showed positive reactions with HLC₃-AB. The result in this paper proved that the lung carcinoma was the important primary site of the cerebral metastatic tumor operated neurosurggically. The specifity of McAb HLC₃- AB, the incidance of the cerebral metastatic lung carcinoma and its metastatic course were discaussed.     

Expression of aquaporine-4 in central nervous system tumors

Cerebral edema is associated with common brain tumors. Aquaporine-4 (AQP4) is a member of the water channel protein family, which is thought to be a major factor regulating cerebral edema. To elucidate the characterization of the expression of AQP4 and the relationship of the expression of VEGF, we investigated the expression of AQP4 in tumors of the central nervous system immunohistochemically. Brain tumors and nontumorous cerebral tissue for control were evaluated by immunohistochemical staining using anti-AQP4, VEGF, CD34, and MIB-1. In tumor cells, only glial tumor cells showed a positive reaction for AQP4. The reactivities for immunostaining increased according to WHO grades. Reactive glial cells in edematous tissue also showed positive reactions. Although endothelial cells were negative and/or weakly positive for AQP4, the positive relationship suggested the expression of VEGF in endothelial cells in neovasculature and that of AQP 4 in tumor cells. APQ4 expression increased in human astrocytic tumors and edematous cerebral tissue. Upregulation of APQ4 by tumor cells and reactive astroglia were major factors of cerebral edema.     

Immunohistochemical demonstration of alpha-1-proteinase inhibitor in brain tumors

Using light microscopy and immunoperoxidase methods (PAP), the presence of alpha-1-proteinase inhibitor (API) was studied in seventeen brain tumors and four normal brain samples. The brain tumors included four glioblastomas, five low-grade gliomas, two metastatic lung carcinomas, two acoustic schwannomas, and four meningiomas. Normal brain displayed a finely granular intracytoplasmic staining confined to neuronal cells. Glial cells were negative for API. Fifteen of the 17 brain tumors were positive for API. Only two of five low-grade gliomas were negative for API. Glioblastoma and metastatic tumors exhibited the strongest positivity followed by acoustic neuroma, meningioma, and low-grade glioma. All positive samples exhibited finely granular intracytoplasmic API, and 50% exhibited extracellular API positivity. Metastatic and glioblastoma tumors demonstrated prominent extracellular API staining. Our results support the concept of a local production of API by brain tumors.     

ATP-binding cassette transporters in primary central nervous system lymphoma: decreased expression of MDR1 P-glycoprotein and breast cancer resistance protein in tumor capillary endothelial cells

Most tumors in the central nervous system are drug resistant partly because of the presence of the blood-brain barrier (BBB) between circulating blood and tumor tissues. Primary central nervous system lymphoma (PCNSL) is one of the exceptions, as it is highly sensitive to high-dose methotrexate (MTX)-based chemotherapy. The aim of this study was to evaluate the BBB function of tumor capillary endothelial cells in PCNSL. Expression of three major drug efflux transporters that belong to the ATP-binding cassette (ABC) superfamily, P-glycoprotein encoded by the human multidrug resistance gene (MDR1 Pgp; ABCB1), breast cancer resistance protein (BCRP; ABCG2), and multidrug resistance-associated protein 1 (MRP1; ABCC1), was evaluated. Immunohistochemistry was performed in capillary endothelial cells of 30 tumor areas from 22 PCNSL cases and compared with that of 30 gliomas. The microenvironment around tumor capillaries was assessed by examining the distribution of astrocytes and by counting the number of macrophages and T-cells, the principal cytokine producers. In PCNSL, expression of MDR1 Pgp and BCRP in tumor capillary endothelial cells was decreased in 63 and 93% of tumor areas examined, respectively, and these reduction levels differed significantly from those of gliomas (P<0.05). When the PCNSLs were further segregated by way of infiltration of tumor cells into three patterns (dense, perivascular and sparse), decreased MDR1 Pgp and BCRP in tumor capillary endothelial cells were much more prominent in dense and perivascular patterns. In all tumors and non-tumor areas of the brain, MRP1 was undetected on capillary endothelial cells. Assessment of the microenvironment around the tumor capillaries suggested that dissociation from astrocytes and infiltration of macrophages and T-cells was involved in the down-regulation of MDR1 Pgp and BCRP in capillary endothelial cells. In conclusion, we report that expression of the major ABC transporters of the BBB, MDR1 Pgp and BCRP, decreases in tumor capillary endothelial cells of PCNSL. Thus, decreased expression may permit delivery of chemotherapeutic agents to the tumor tissues.     

THE SIGNIFICANCE OF IMMUNO-STAIN WITH HLC_3-AB IN CEREBRAL METASTATIC TUMOR

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Prognostic relevance of P-glycoprotein expression in breast cancer

BACKGROUND: P-glycoprotein (Pgp) expression has been reported to be associatedwith a poor prognosis in some malignancies such as neuroblastoma,soft tissue sarcoma and acute myeloid leukemia. The prognosticrole of Pgp expression in breast cancer is still unclear. Weinvestigated the expression of Pgp in primary and metastaticbreast cancer tissues in relation to patient characteristicsand treatment outcome. PATIENTS AND METHODS: Pgp expression was evaluated in 92 primary and 12 metastaticbreast cancers by the use of immunohisto/cytochemistry withthree monoclonal antibodies (MAbs) (JSB-1, C219, MRK16), andan RNAse protection assay. Follow-up information was availablefor 77 primary breast cancer patients (median follow-up 42 months;range 2–63 months). RESULTS: Concordance among the anti-Pgp MAbs varied, the highest beingbetween JSB-1 and MRK16 (71%; p = 0.002). Pgp expression wasmore frequent in metastatic disease (58%) than in primary breastcancer (29%) (JSB-1; p     

Endothelial Cell Proliferation and Vascular Endothelial Growth Factor Expression in Primary Colorectal Cancer and Corresponding Liver Metastases

Background: . Colorectal carcinoma (CRC) is one of the major causes of cancer death worldwide. Data fromthe literature indicate differences between the proliferation rate of endothelial cells relative to the morphologygrowth type, possibly due to origin of specimens (autopsy material, surgery fragments) or quantification methods.Vascular endothelial growth factor (VEGF) is a factor that stimulates the proliferation of endothelial cells. It isexpressed in more than 90% of cases of metastatic CRC. Aim: The aim of this study was to evaluate the endothelialcell proliferation and VEGF expression in primary tumors and corresponding liver metastases. Materials andMethods: Our study included 24 recent biopsies of primary tumors and corresponding liver metastases of CRCcases. CD34/ Ki67 double immunostaining and RNA scope assay for VEGF were performed. Results: In theprimary tumors analysis of VEGFmRNA expression indicated no significant correlation with differentiationgrade, proliferative and non-proliferative vessels in the intratumoral and peritumoral areas. In contrast, in thecorresponding liver metastases, VEGFmRNA expression significantly correlated with the total number of nonproliferativevessels and total number of vessels. CD34/ Ki67 double immunostaining in the cases with poorlydifferentiated carcinoma indicated a high number of proliferating endothelial cells in the peritumoral area anda low number in the intratumoral area for the primary tumor. Moderately differentiated carcinomas of colonshowed no proliferating endothelial cells in the intratumoral area in half of the cases included in the study, forboth, primary tumor and liver metastasis. In well differentiated CRCs, in primary tumors, a high proliferationrate of endothelial cells in the intratumoral area and a lower proliferation rate in the peritumoral area were found.A low value was found in corresponding liver metastasis. Conclusions: The absence of proliferative endothelialcells in half of the cases for the primary tumors and liver metastases in moderately differentiated carcinomasuggest a vascular mimicry phenomenon. The mismatch between the total number of vessels and endothelialproliferation in primary tumors indicate that a functional vascular network is already formed or the existenceof some mechanisms influenced by other angiogenic factors.     

A monoclonal antibody reacting with endothelial cells of budding vessels in tumors and inflammatory tissues, and non-reactive with normal adult tissues

This report describes a monoclonal antibody (MAb) generated by immunization of mice with cell suspensions of capillary-rich fragments of mammary carcinomas. The antibody (EN 7/44) belongs to the IgM class and detects a 30.5 kDa antigen which is found in the cytoplasm of human placental and umbilical vein-endothelial cells (HUVEC) and on the surface of tumor endothelium. Using indirect immunofluorescence and immunoperoxidase techniques, we did not find the MAb in peripheral blood cells or in any human cell lines tested, nor in endothelial cells from normal, nonproliferating adult tissues. Positively staining endothelium was found in the placenta, the umbilical vein and in proliferating normal tissues (intestine). Positive endothelial cells were found in acute inflammatory reactions and in tumors. In the latter, the strongest reactions were seen in newly formed budding capillaries, which were identified by their reactivity with Ulex europaeus I-lectin and antibodies against F VIII-RAG. In tumor tissues, large vessels could be positively stained with the EN 7/44 antigen, in contrast to inflammatory tissues. It is concluded that endothelial cells at the tip of a budding capillary express distinct phenotypic characteristics.     

Expression of mediated P-glycoprotein multidrug resistance related to Tc-99m MIBI scintimammography results

We prospectively studied a total of 24 patients with breast cancer to evaluate the relationship between the degree of accumulation of technetium-99m sestamibi (Tc-99m MIBI) and p-glycoprotein (Pgp) expression in tumor tissues. All 24 patients underwent Tc-99m MIBI scintimammography before surgery or biopsy. Immunohistochemical studies were performed on multiple non-consecutive sections of the same tumor using a Pgp specific monoclonal antibody, JSB-1. Planar images were started 10 min after injection of Tc-99m MIBI. Tumor to background (T/B) ratios calculated from the planar images were correlated with Pgp expression as determined by immunohistochemical studies. The T/B ratios were significantly lower for tumors in eight patients with positive Pgp expression (Group 1) than in 16 patients with negative expression (Group 2) (1.40+/-0.11 and 2.76+/-0.60, P<0. 05). Our data confirmed that Tc-99m MIBI scintimammography is useful for determination of the presence of multidrug resistance due to Pgp expression in patients with breast cancer.     

Localization of Acidic and Basic Fibroblast Growth Factor mRNA in Human Brain Tumors

Acidic and basic fibroblast growth factors (aFGF and bFGF) are closely related peptide mitogens acting on both mesoderm- and neuroectoderm-derived cells, including fibroblasts, endothelial cells and glial cells. In order to identify the expression of mRNAs for these growth factors, in situ hybridization using human aFGF and bFGF RNA probes was performed in 24 human brain tumors. The mRNAs for aFGF and bFGF were expressed in the cells of various tumors (1/1 and 1/1 astrocytoma, 2/2 and 2/2 anaplastic astrocytomas, 6/6 and 6/6 glioblastomas, 4/4 and 4/4 meningiomas, 3/3 and 3/3 schwannomas, 1/2 and 1/2 pituitary adenomas, 4/4 and 4/4 metastatic carcinomas, 0/1 and 0/1 hemangioblastoma, 0/1 and 0/1 craniopharyngioma) and were also detected in endothelial cells and surrounding neuronal cells of brain tumors. These results suggest the possibilities that aFGF and bFGF contribute to the uncontrolled growth of tumor cells and the proliferation of endothelial cells in autocrine and paracrine manners, and that the expression of mRNAs for these growth factors in the surrounding neuronal cells results in enhancement of tumor growth.     

Localization of acidic and basic fibroblast growth factor mRNA in human brain tumors

Acidic and basic fibroblast growth factors (aFGF and bFGF) are closely related peptide mitogens acting on both mesoderm- and neuroectoderm-derived cells, including fibroblasts, endothelial cells and glial cells. In order to identify the expression of mRNAs for these growth factors, in situ hybridization using human aFGF and bFGF RNA probes was performed in 24 human brain tumors. The mRNAs for aFGF and bFGF were expressed in the cells of various tumors (1/1 and 1/1 astrocytoma, 2/2 and 2/2 anaplastic astrocytomas, 6/6 and 6/6 glioblastomas, 4/4 and 4/4 meningiomas, 3/3 and 3/3 schwannomas, 1/2 and 1/2 pituitary adenomas, 4/4 and 4/4 metastatic carcinomas, 0/1 an 0/1 hemangioblastoma, 0/1 and 0/1 craniopharyngioma) and were also detected in endothelial cells and surrounding neuronal cells of brain tumors. These results suggest the possibilities that aFGF and bFGF contribute to the uncontrolled growth of tumor cells and the proliferation of endothelial cells in autocrine and paracrine manners, and that the expression of mRNAs for these growth factors in the surrounding neuronal cells results in enhancement of tumor growth.     

Clinical and cell line specific expression profiles of a human gene identified in experimental central nervous system metastases

Some cancers, particularly malignant melanomas and carcinomas of the breast and lung, metastasize to the central nervous system (CNS) in advanced stages. In order to develop into clinically manifest metastases, hematogenously disseminated tumor cells must respond to trophic factors within the CNS microenvironment. We have previously identified a nuclearfactor, com1, expressed in human breast carcinoma cells upon formation of experimental metastatic tumors in the CNS. In the present study distinct com1 mRNA expression was detected in cerebral metastases from patients with lung carcinomas, whereas the expression level was generally much lower in glioblastomas (primary brain tumors). In tissue specimens from normal brain and lung, as well as in glioma and lung carcinoma cell lines, com1 expression was barely detectable. One potential mechanism involved in the induction of com1 expression was indicated in the metastatic MCF7/LCC2 breast carcinoma cells. Significant increases in the level of com1 mRNA were observed upon activation of receptor tyrosine kinase signaling, which is known to operate during metastatic tumor cell proliferation within the CNS. The observations in this study strengthen the assumption that com1 may be involved in the tumor cell response to regulatory signals upon metastasis formation.     

Variable p-glycoprotein immunoreactivity unrelated to cytotoxic drug-resistance in-vitro of human adrenocortical carcinoma

Human adrenocortical tissue was immunohistochemically investigated with monoclonal antibodies C219 and JSB-1 for expression of P-glycoprotein (Pgp) associated with resistance to multiple cytotoxic drugs. All normal (n=7) and adenomatous (n=3) glands strongly expressed the protein, while the cancers (n=15) demonstrated variable Pgp reactivity. Cytotoxic drug sensitivity testing in vitro on freshly dispersed cells from one normal adrenal gland and from 6 carcinomas with no to extensive Pgp reactivity demonstrated universally poor sensitivity to 9 standard drugs including those with Pgp mediated resistance. The findings indicate that immunohistochemical staining for Pgp cannot be used to predict cytotoxic drug resistance and that other mechanisms mediate cytotoxic drug resistance in adrenocortical carcinoma.     

The effect of P-glycoprotein on paclitaxel brain and brain tumor distribution in mice

It may be inferred from the presence of P-glycoprotein (Pgp) in brain capillaries that this drug efflux pump is a factor in limiting the penetration of certain agents into brain tumors. However, by contrast with normal brain capillaries which constitute the blood-brain barrier, brain tumor capillaries are compromised or "leaky," and the extent to which Pgp expression in brain tumor neovasculature retains its capacity to limit drug penetration has not been determined. To address this question, we studied the normal brain and brain tumor distribution of paclitaxel (PAC), a known Pgp substrate, using steady-state PAC dosing regimens in wild-type and Pgp knockout (mdr1a -/- and mdr1b -/-) mice bearing an intracerebral B-16 melanoma. At comparable steady-state PAC plasma concentrations of approximately 5 microg/ml, steady-state PAC brain concentrations in Pgp knockout mice were approximately 3-, 1.8-, and 1.7-fold greater in left brain, right brain, and brain tumor, respectively, than in wild-type mice and statistically different (P < 0.05) in each brain region. Determination of the steady-state brain/plasma concentration ratios or partition coefficients, which take into account any differences in plasma concentrations between each group, indicated a similar pattern as did the absolute brain concentrations. It is concluded that even in the neovasculature of brain tumors, Pgp has the facility to limit drug penetration, although somewhat less so than in normal brain.     

Expression of p-glycoprotein in the normal colorectal epithelium and in colorectal-carcinoma

Expression of P-glycoprotein (pgp) was analyzed by immunohistochemical staining with monoclonal antibody JSB-1 in 145 frozen specimens (67 were samples of normal colorectal mucosa from sites adjacent to the tumor, 66 were colorectal carcinomas, 5 colorectal polyps, 5 metastatic lymph nodes, and 2 samples of metastatic liver tumors) of 67 patients with colorectal carcinoma and polyps. All 72 specimens of normal colorectal mucosa and adenomatous polyps expressed pgp to various degrees. By contrast, 18 of 39 (46.2%) samples from cases of well differentiated adenocarcinoma were positive for pgp but only 3 of 21 (14.3%) samples from cases of moderately differentiated adenocarcinoma and none of the 4 samples from cases of poorly differentiated adenocarcinoma were positive for pgp. There was no correlation between the clinicopathological stage of colorectal carcinoma and the expression of pgp. These findings indicate that the expression of P-glycoprotein is closely related to the differentiation of cells. In normal colorectal epithelium, pgp was expressed normally and in well differentiated adenocarcinomas, pgp was still expressed. However, expression of pgp was no longer detectable in carcinomas with moderate or poor differentiation.     

Fibulin-7 is overexpressed in glioblastomas and modulates glioblastoma neovascularization through interaction with angiopoietin-1

Glioblastoma (GBM) is pathologically characterized by highly malignant neoplastic cells, focal necrosis and aberrant blood vessels composed of disorganized endothelial cells and pericytes. The recent cancer microarray database revealed upregulation of fibulin-7 (Fbln7), a member of the fibulin family, but provided no information on the tissue localization or biological function. In the present study, we demonstrated that Fbln7 is markedly overexpressed by the GBM tissue among astrocytic tumors, and immunolocalized mainly to endothelial cells and pericytes of the glomeruloid and hypertrophied microvessels. The production of Fbln7 by endothelial cells and pericytes was confirmed in cultured human umbilical vein endothelial cells (HUVEC) and human brain vascular pericytes (HBVP) and vascular endothelial growth factor (VEGF) stimulated the Fbln7 expression in HUVEC. Fbln7 bound to angiopoietin-1, but not angiopoietin-2 or Tie2 receptor, through interaction between the N-terminal portions of Fbln7 and angiopoietin-1, and it blocked phosphorylation of Tie2 receptor in HUVEC. In a coculture assay using HUVEC and HBVP, multilayered and irregular-shaped tube-like structures of HUVEC were induced by treatment with a high concentration of VEGF. This was accompanied by Fbln7 overproduction by HUVEC and angiopoietin-1 expression by HBVP. The production of aberrant VEGF-induced tube-like structures was attenuated by treatment with antibody or synthetic peptides specific to the Fbln7 N-terminal domain or knockdown of Fbln7. These data demonstrate that Fbln7 is overexpressed by endothelial cells and pericytes of the abnormal microvessels in GBM, and suggest that Fbln7 may contribute to the aberrant vessel formation by modulation of the angiopoietin-1/angiopoietin-2-Tie2 axis.