Joan E. Carpenter

Imaging Tumor Angiogenesis With Contrast Ultrasound and Microbubbles Targeted to αvβ3

Background Angiogenesis is a critical determinant of tumor growth and metastasis. We hypothesized that contrastenhanced ultrasound (CEU) with microbubbles targeted to &agr;v‐integrins expressed on the neovascular endothelium could be used to image angiogenesis. Methods and Results Malignant gliomas were produced in 14 athymic rats by intracerebral implantation of U87MG human glioma cells. On day 14 or day 28 after implantation, CEU was performed with microbubbles targeted to &agr;v&bgr;33 by surface conjugation of echistatin. CEU perfusion imaging with nontargeted microbubbles was used to derive tumor microvascular blood volume and blood velocity. Vascular &agr;v‐integrin expression was assessed by immunohistochemistry, and microbubble adhesion was characterized by confocal microscopy. Mean tumor size increased markedly from 14 to 28 days (2±1 versus 35±14 mm2, P<0.001). Tumor blood volume increased by ≈35% from day 14 to day 28, whereas microvascular blood velocity decreased, especially at the central portions of the tumors. On confocal microscopy, &agr;v&bgr;3‐targeted but not control microbubbles were retained preferentially within the tumor microcirculation. CEU signal from &agr;v&bgr;3‐targeted microbubbles in tumors increased significantly from 14 to 28 days (1.7±0.4 versus 3.3±1.0 relative units, P<0.05). CEU signal from &agr;v&bgr;3‐targeted microbubbles was greatest at the periphery of tumors, where &agr;v‐integrin expression was most prominent, and correlated well with tumor microvascular blood volume (r=0.86). Conclusions CEU with microbubbles targeted to &agr;v&bgr;3 can noninvasively detect early tumor angiogenesis. This technique, when coupled with changes in blood volume and velocity, may provide insights into the biology of tumor angiogenesis and be used for diagnostic applications. (Circulation. 2003;108:336‐341.)

Insulin-like growth factor-1 content and pattern of expression correlates with histopathologic grade in diffusely infiltrating astrocytomas.

Studies of experimental tumorigenesis have strongly implicated signaling of the insulin-like growth factor 1 (IGF-1) as a key component in astrocytic neoplasia; however, its role in the growth of low-grade and malignant human tumors is not well understood. Correlative analyses of IGF-1, p53, and Ki-67 (MIB-1) immunohistochemistry and IGF-1 receptor (IGF-1R) mRNA expression were performed to examine the cellular pattern of IGF-1 signaling in 39 cases of astrocytoma (World Health Organization grades II-IV). Tumor cells expressing IGF-1 and IGF-1R were present in all tumor grades. The proportion of tumor cells that expressed IGF-1 correlated with both histopathologic grade and Ki-67 labeling indices, while expression of IGF-1R mRNA correlated with Ki-67 indices. In cases where stereotactic tissue sampling could be identified with a specific tumor area by neuroimaging features, the numbers of IGF-1 immunoreactive cells correlated with the tumor zones of highest cellularity and Ki-67 labeling. In glioblastomas, the localization of IGF-1 immunoreactivity was notable for several features: frequent accentuation in the perivascular tumor cells surrounding microvascular hyperplasia; increased levels in reactive astrocytes at the margins of tumor infiltration; and selective expression in microvascular cells exhibiting endothelial/pericytic hyperplasia. IGF-1R expression was particularly prominent in tumor cells adjacent to both microvascular hyperplasia and palisading necrosis. These data suggest that IGF-1 signaling occurs early in astroglial tumorigenesis in the setting of cell proliferation. The distinctive correlative patterns of IGF-1 and IGF-1R expression in glioblastomas also suggest that IGF-1 signaling has an association with the development of malignant phenotypes related to aberrant angiogenesis and invasive tumor interactions with reactive brain.

Phorbol 12-Myristate 13-Acetate Induces Protein Kinase Cη-specific Proliferative Response in Astrocytic Tumor Cells

Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-η expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (α, βI, βII, and γ) and novel (θ and ε) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-η, was only expressed by U-251 MG cells. In contrast, PKC-δ was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-η was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-η cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 μm) and the MEK inhibitor, PD 98059 (50 μm). Transient transfection of wild type U-251 with PKC-η antisense oligonucleotide (1 μm) also blocked the PMA-induced increase in [3H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-η plays a determining role in the different proliferative capacity.

An Extensive Invasive Intracranial Human Glioblastoma Xenograft Model : Role of High Level Matrix Metalloproteinase 9

The lack of an intracranial human glioma model that recapitulates the extensive invasive and hypervascular features of glioblastoma (GBM) is a major hurdle for testing novel therapeutic approaches against GBM and studying the mechanism of GBM invasive growth. We characterized a high matrix metalloproteinase-9 (MMP-9) expressing U1242 MG intracranial xenograft mouse model that exhibited extensive individual cells and cell clusters in a perivascular and subpial cellular infiltrative pattern, geographic necrosis and infiltrating tumor-induced vascular proliferation closely resembling the human GBM phenotype. MMP-9 silencing cells with short hairpin RNA dramatically blocked the cellular infiltrative pattern, hypervascularity, and cell proliferation in vivo, and decreased cell invasion, colony formation, and cell motility in vitro, indicating that a high level of MMP-9 plays an essential role in extensive infiltration and hypervascularity in the xenograft model. Moreover, epidermal growth factor (EGF) failed to stimulate MMP-9 expression, cell invasion, and colony formation in MMP-9-silenced clones. An EGF receptor (EGFR) kinase inhibitor, a RasN17 dominant-negative construct, MEK and PI3K inhibitors significantly blocked EGF/EGFR-stimulated MMP-9, cell invasion, and colony formation in U1242 MG cells, suggesting that MMP-9 is involved in EGFR/Ras/MEK and PI3K/AKT signaling pathway-mediated cell invasion and anchorage-independent growth in U1242 MG cells. Our data indicate that the U1242 MG xenograft model is valuable for studying GBM extensive invasion and angiogenesis as well as testing anti-invasive and anti-angiogenic therapeutic approaches.

Protein kinase C-η regulates resistance to UV- and γ-irradiation-induced apoptosis in glioblastoma cells by preventing caspase-9 activation

Both increased cell proliferation and apoptosis play important roles in the malignant growth of glioblastomas. We have demonstrated recently that the differential expression of protein kinase C (PKC)-eta increases the proliferative capacity of glioblastoma cells in culture; however, specific functions for this novel PKC isozyme in the regulation of apoptosis in these tumors has not been defined. In the present study of several glioblastoma cell lines, we investigated the role of PKC-eta in preventing UV- and gamma-irradiation-induced apoptosis and in caspase-dependent signaling pathways that mediate cell death. Exposure to UV or gamma irradiation killed 80% to 100% of PKC-eta-deficient nonneoplastic human astrocytes and U-1242 MG cells, but had little effect on the PKC-eta-expressing U-251 MG and U-373 MG cells. PKC-eta appears to mediate resistance to irradiation specifically such that when PKC-eta was stably expressed in U-1242 MG cells, more than 80% of these cells developed resistance to irradiation-induced apoptosis. Reducing PKC-eta expression by transient and stable expression of antisense PKC-eta in wild-type U-251 MG cells results in increased sensitivity to UV irradiation in a fashion similar to U-1242 MG cells and nonneoplastic astrocytes. Irradiation of PKC-eta-deficient glioblastoma cells resulted in the activation of caspase-9 and caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and a substantial increase in subdiploid DNA content that did not occur in PKC-eta-expressing tumor cells. A specific inhibitor (Ac-DEVD-CHO) of caspase-3 blocked apoptosis in PKC-eta-deficient U-1242 MG cells. The data demonstrate that resistance to UV and gamma irradiation in glioblastoma cell lines is modified significantly by PKC-eta expression and that PKC-eta appears to block the apoptotic cascade at caspase-9 activation.

Protein Kinase C-α–Mediated Regulation of Low-Density Lipoprotein Receptor–Related Protein and Urokinase Increases Astrocytoma Invasion

Aggressive and infiltrative invasion is one of the hallmarks of glioblastoma. Low-density lipoprotein receptor-related protein (LRP) is expressed by glioblastoma, but the role of this receptor in astrocytic tumor invasion remains poorly understood. We show that activation of protein kinase C-alpha (PKC-alpha) phosphorylated and down-regulated LRP expression. Pretreatment of tumor cells with PKC inhibitors, phosphoinositide 3-kinase (PI3K) inhibitor, PKC-alpha small interfering RNA (siRNA), and short hairpin RNA abrogated phorbol 12-myristate 13-acetate-induced down-regulation of LRP and inhibited astrocytic tumor invasion in vitro. In xenograft glioblastoma mouse model and in vitro transmembrane invasion assay, LRP-deficient cells, which secreted high levels of urokinase-type plasminogen activator (uPA), invaded extensively the surrounding normal brain tissue, whereas the LRP-overexpressing and uPA-deficient cells did not invade into the surrounding normal brain. siRNA, targeted against uPA in LRP-deficient clones, attenuated their invasive potential. Taken together, our results strongly suggest the involvement of PKC-alpha/PI3K signaling pathways in the regulation of LRP-mediated astrocytoma invasion. Thus, a strategy of combining small molecule inhibitors of PKC-alpha and PI3K could provide a new treatment paradigm for glioblastomas.

Epidermal growth factor differentially regulates low density lipoprotein receptor-related protein gene expression in neoplastic and fetal human astrocytes.

Low density lipoprotein receptor–related protein (LRP) is a multi‐functional endocytotic receptor that may modify the biological activity of reactive astrocytes in neuroplasticity and neurodegeneration and of malignant astrocytes in brain invasion. In this study, the regulation of LRP by epidermal growth factor receptor (EGFR) ligands in both cultured human fetal astrocytes and astrocytic tumor cell lines (U‐251 MG and U‐1242 MG) was investigated. All astrocytic cell types expressed LRP, as determined by the binding of activated α2‐macroglobulin (α2M*) on intact cells and by Western and Northern blot analyses of cell extracts. Primary cultured astrocytes expressed the highest levels of α2M*‐binding capacity (Bmax = 30 fmol/mg protein). This was twofold higher than for the U‐1242 MG astrocytoma cells (Bmax = 15 fmol/mg protein) and fourfold greater than for the glioblastoma U‐251 MG cells (7.0 fmol/mg protein). Receptor affinity (KD) ranged from 0.25 to 0.6 nM in all the astroglial cell types. Functional LRP at the surface was down‐regulated by EGF, compared with controls, as indicated by a reduction of both Bmax and LRP‐mediated endocytosis by approximately 50% and 60%, respectively. In comparison, EGF treatment of primary astrocytes did not down‐regulate LRP expression or LRP‐mediated endocytosis. Treatment of the tumor cells with EGF or TGFα (25 ng/ml) significantly down‐regulated total cellular LRP. Receptor‐associated protein (RAP) mRNA expression was not affected by EGF in either tumor cells or primary astrocytes. The reduction of LRP in the tumor cells resulted from a specific decrease in LRP mRNA transcription, as determined by Northern blot and nuclear run‐on experiments. These data suggest that EGF mediates a functional down‐regulation of LRP endocytotic activity in astrocytic tumor cells and that LRP expression is differentially regulated in neoplastic and non‐neoplastic astrocytes. GLIA 25:71–84, 1999.

Epidermal Growth Factor Receptor-Mediated Regulation of Urokinase Plasminogen Activator Expression and Glioblastoma Invasion via C-SRC/MAPK/AP-1 Signaling Pathways

One of the major pathophysiological features of malignant astrocytomas is their ability to infiltrate surrounding brain tissue. The epidermal growth factor receptor (EGFR) and proteases are known to be overexpressed in glioblastomas (GBMs), but the interaction between the activation of the EGFR and urokinase plasminogen activator (uPA) in promoting astrocytic tumor invasion has not been fully elucidated. Here, we characterized the signal transduction pathway(s) by which EGF regulates uPA expression and promotes astrocytoma invasion. We show that EGFR activation and constitutively active EGFR vIII in GBM cell lines upregulate uPA expression. Small-molecule inhibitors of mitogen-activated protein kinase, tyrosine kinase, and small interfering RNA targeting c-Src blocked uPA upregulation. Similarly, mutations in the activator protein 1 binding site of the uPA promoter reduced EGF-induced increases in uPA promoter activity. Treatment of GBM cells with EGF increased in vitro cell invasion, and the invasive phenotype was attenuated by gene silencing of uPA using small interfering RNA and short hairpin RNA. In addition, uPA knockdown clones formed smaller well-circumscribed tumors than nontarget U1242 control cells in a xenograft GBM mouse model in vivo. In summary, these results suggest that c-Src, mitogen-activated protein kinase, and a composite activator protein 1 on the uPA promoter are responsible for EGF-induced uPA expression and GBM invasion.

Site-specific imaging of tumor angiogenesis using contrast-enhanced ultrasound imaging with microbubbles targeted to alpha-V beta-3

flow, was mildly increased at 1 hr (2.0+2.3), then increased over the first week peaking at day 4 (5.6+2.6), then declined rapidly (0.950.7 at day 26). The MB, Signal peak preceded increases in normalized blood flow and blood volume, were not detected until after day 14 (0.52+0.11 and 0.87*0.09, for blood flow and volume at 28 days). MB, Signal peak also preceded an increase in ischemic muscle tissue PO, (normalized values Of 0.24+0.06 and 0.47+0.10, at 1 hr and 28 days, respectively). We conclude that CEU with microbubbles targeted for endothelial a,-in&grins can be used to non-invasively assess anglogenic responses in skeletal muscle. These results suggest that targeted CEU imaging of endothelial markers of angiogenesis may potentially be used for assessing intrinsic and therapeutic angiogenesis prior to changes tn perfusion. IO:00 a.m.