Gila Meir

Stimulation of tumour growth by wound-derived growth factors.

SummaryThe goal of this work was to determine the molecular basis for the induction of tumour vascularization and progression by injury. Magnetic resonance imaging (MRI) studies demonstrated that administration of wound fluid derived from cutaneous injuries in pigs reduced the lag for vascularization and initiation of growth of C6 glioma spheroids, implanted in nude mice, and accelerated tumour doubling time. The former effect can be attributed to the angiogenic capacity of wound fluid as detected in vivo by MRI, and in vitro in promoting endothelial cell proliferation. The latter effect, namely the induced rate of tumour growth, is consistent with the angiogenic activity of wound fluid as well as with the finding that wound fluid was directly mitogenic to the tumour cells, and accelerated growth of C6 glioma in spheroid culture. Of the multiple growth factors present in wound fluid, two key factors, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) and platelet-derived growth factor (PDGF), were identified as the dominant mitogens for C6 glioma, and inhibition of their activity using specific neutralizing antibodies suppressed the mitogenic effect of wound fluid on DNA synthesis in C6 glioma. This study suggests that the stimulatory effect of injury on tumour progression can possibly be attenuated by therapeutic targeting directed against a limited number of specific growth factors.

The role of angiogenesis, vascular maturation, regression and stroma infiltration in dormancy and growth of implanted MLS ovarian carcinoma spheroids

MLS ovarian epithelial carcinoma multicellular spheroids xenografted subcutaneously in CD‐1 nude mice displayed growth delay, or dormancy, of up to 52 days. In the study reported here, implanted MLS spheroids were used for testing the role of angiogenesis and vascular maturation in triggering the initiation of tumor progression. The kinetics and impact of neovascular maturation and functionality, in dormancy, and growth of MLS spheroid xenografts were studied noninvasively by BOLD contrast MRI. MR data were supported by histologic staining for biotinylated albumin as a blood pool marker and alpha‐smooth muscle actin (alpha‐SMA) as marker for perivascular mural cells. Although the tumor periphery showed higher levels of total and mature vasculature than normal skin, the fraction of mature out of the total vessels as detected by MRI vascular maturation index (VMIMRI) was significantly lower in the tumor both before and after tumor exit from dormancy. The neovasculature induced by the implanted spheroid was unstable and showed cycles of vessel growth and regression. Surprisingly, this instability was not restricted to the immature vessels, but rather included also regression of mature vessels. During dormancy, neovasculature was predominantly peripheral with no infiltration into the implanted spheroid. Infiltration of alpha‐SMA positive stroma cells into the spheroid was associated with functional vascularization and tumor growth. Thus, stroma infiltration and vascular maturation are an important checkpoint linking the angiogenic switch with initiation of tumor progression.

Stimulation of tumour angiogenesis by proximal wounds: spatial and temporal analysis by MRI

We show here, using high-resolution magnetic resonance imaging, that injured tissue provides a favourable milieu for the neovascularization and growth of C6 glioma spheroids, implanted subcutaneously in nude mice. Moreover, the presence of micro-tumours in an injured tissue inhibited the healing process, leaving an open persistent wound. In correlation with the induced angiogenesis of implanted spheroids in the presence of proximal wounds, a shorter lag period was observed for initiation of tumour growth. This effect was restricted spatially and was observed only for wounds within 5 mm from the tumour. In such proximal wounds, angiogenesis was enhanced in the first days after injury, and vessel regression, which normally starts 4 days after injury, did not occur. Injury causing interference to tumour perfusion promoted tumour vascularization and growth even for more remote incisions, possibly by activating stress-induced angiogenesis. The kinetics of vascularization and growth of these wound-tumour systems sheds light on the clinical observations of increased probability of metastatic recurrence and stimulated regrowth of residual tumour in the site of surgical intervention. High-resolution magnetic resonance imaging could detect the aberrant angiogenic activity of these tumour-wound systems as early as 1 week after injury.

Functional and molecular mapping of uncoupling between vascular permeability and loss of vascular maturation in ovarian carcinoma xenografts: The role of stroma cells in tumor angiogenesis

Maintaining homogeneous perfusion in tissues undergoing remodeling and vascular expansion requires tight orchestration of the signals leading to endothelial sprouting and subsequent recruitment of perivascular contractile cells and vascular maturation. This regulation, however, is frequently disrupted in tumors. We previously demonstrated the role of tumor‐associated myofibroblasts in vascularization and exit from dormancy of human ovarian carcinoma xenografts in nude mice. The aim of this work was to determine the contribution of stroma‐ and tumor cell‐derived angiogenic growth factors to the heterogeneity of vascular permeability and maturation in MLS human ovarian carcinoma tumors. We show by RT‐PCR and by in situ hybridization that VEGF was expressed by the tumor cells, while angiopoietin‐1 and ‐2 were expressed only by the infiltrating host stroma cells. Vascular maturation was detected in vivo by vasoreactivity to hypercapnia, measured by BOLD contrast MRI and validated by immunostaining of histologic sections to α‐smooth muscle actin. Vascular permeability was measured in vivo by dynamic contrast‐enhanced MRI using albumin‐based contrast material and validated in histologic sections by fluorescent staining of the biotinylated contrast material. MRI as well as histologic correlation maps between vascular maturation and vascular permeability revealed a wide range of vascular phenotypes, in which the distribution of vascular maturation and vasoreactivity did not overlap spatially with reduced permeability. The large heterogeneity in the degree of vascular maturation and permeability is consistent with the differential expression pattern of VEGF and angiopoietins during tumor angiogenesis.

Gonadotropin-Regulated Lymphangiogenesis in Ovarian Cancer Is Mediated by LEDGF-Induced Expression of VEGF-C

The risk and severity of ovarian carcinoma, the leading cause of gynecologic malignancy death, are significantly elevated in postmenopausal women. Ovarian failure at menopause, associated with a reduction in estrogen secretion, results in an increase of the gonadotropic luteinizing hormone (LH) and follicle-stimulating hormone (FSH), suggesting a role for these hormones in facilitating the progression of ovarian carcinoma. The current study examined the influence of hormonal stimulation on lymphangiogenesis in ovarian cancer cells. In vitro stimulation of ES2 ovarian carcinoma cells with LH and FSH induced expression of vascular endothelial growth factor (VEGF)-C. In vivo, ovariectomy of mice resulted in activation of the VEGF-C promoter in ovarian carcinoma xenografts, increased VEGF-C mRNA level, and enhanced tumor lymphangiogenesis and angiogenesis. Seeking the molecular mechanism, we examined the role of lens epithelium-derived growth factor (LEDGF/p75) and the possible contribution of its putative target, a conserved stress-response element identified in silico in the VEGF-C promoter. Using chromatin immunoprecipitation, we showed that LEDGF/p75 indeed binds the VEGF-C promoter, and binding is augmented by FSH. A corresponding hormonally regulated increase in the LEDGF/p75 mRNA and protein levels was observed. Suppression of LEDGF/p75 expression using small interfering RNA, suppression of LH and FSH production using the gonadotropin-releasing hormone antagonist cetrorelix, or mutation of the conserved stress-response element suppressed the hormonally induced expression of VEGF-C. Overall, our data suggest a possible role for elevated gonadotropins in augmenting ovarian tumor lymphangiogenesis in postmenopausal women.

Ferritin as a reporter gene for MRI: Chronic liver over expression of h-Ferritin during dietary iron supplementation and aging

The iron storage protein, ferritin, provides an important endogenous MRI contrast that can be used to determine the level of tissue iron. In recent years the impact of modulating ferritin expression on MRI contrast and relaxation rates was evaluated by several groups, using genetically modified cells, viral gene transfer and transgenic animals. This paper reports the follow‐up of transgenic mice that chronically over‐expressed the heavy chain of ferritin (h‐ferritin) in liver hepatocytes (liver‐hfer mice) over a period of 2 years, with the aim of investigating the long‐term effects of elevated level of h‐ferritin on MR signal and on the well‐being of the mice. Analysis revealed that aging liver‐hfer mice, exposed to chronic elevated expression of h‐ferritin, have increased R2 values compared to WT. As expected for ferritin, R2 difference was strongly enhanced at high magnetic field. Histological analysis of these mice did not reveal liver changes with prolonged over expression of ferritin, and no differences could be detected in other organs. Furthermore, dietary iron supplementation significantly affected MRI contrast, without affecting animal wellbeing, for both wildtype and ferritin over expressing transgenic mice. These results suggest the safety of ferritin over‐expression, and support the use of h‐ferritin as a reporter gene for MRI. Copyright

Harnessing Competing Endocytic Pathways for Overcoming the Tumor-Blood Barrier: Magnetic Resonance Imaging and Near-Infrared Imaging of Bifunctional Contrast Media

Ovarian cancer is the most lethal gynecologic malignancy, often diagnosed at advanced stage leading to poor prognosis. In the study reported here, magnetic resonance imaging and near-infrared reflectance imaging were applied for in vivo analysis of two competing endocytic pathways affecting retention of bifunctional daidzein-bovine serum albumin (BSA)-based contrast media by human epithelial ovarian carcinoma cells. Suppression of caveolae-mediated uptake using nystatin or by BSA competition significantly enhanced daidzein-BSA-GdDTPA/CyTE777 uptake by tumor cells in vitro. In vivo, perivascular myofibroblasts generated an effective perivascular barrier excluding delivery of BSA-GdDTPA/CyTE777 to tumor cells. The ability to manipulate caveolae-mediated sequestration of albumin by perivascular tumor myofibroblasts allowed us to effectively overcome this tumor-stroma barrier, increasing delivery of daidzein-BSA-GdDTPA/CyTE777 to the tumor cells in tumor xenografts. Thus, both in vitro and in vivo, endocytosis of daidzein-BSA-GdDTPA/CyTE777 by ovarian carcinoma cells was augmented by albumin or by nystatin. In view of the cardinal role of albumin in affecting the availability and pharmacokinetics of drugs, this approach could potentially also facilitate the delivery of therapeutics and contrast media to tumor cells.

Fibroblast recruitment as a tool for ovarian cancer detection and targeted therapy

Metastatic ovarian cancer, the most lethal of gynecologic malignancies, is typically managed by debulking surgery, followed by chemotherapy. However, despite significant efforts, survival rate remains low. We have previously demonstrated, in mouse models, a specific systemic homing of labeled fibroblasts to solid ovarian tumors. Here, we demonstrate the feasibility of utilizing this specific homing of genetically modified fibroblasts for detection and targeted therapy of orthotopic metastatic ovarian carcinoma model in immune‐deficient mice. Using an in vivo metastatic mouse model for ovarian cancer, we demonstrated that fibroblasts expressing fluorescent reporters injected intra‐peritoneally, were specifically recruited to peritoneal tumor nodules (resulting in 93‐100% co‐localization). We further used fibroblasts over expressing the soluble receptor variant of VEGFR1 (s‐Flt1). Mice bearing tumors were injected weekly with either control or s‐Flt1 expressing fibroblasts. Injection of s‐Flt1 expressing fibroblasts resulted in a significant reduction in the ascites volume, reduced vascularization of adherent metastases, and improved overall survival. Using fluorescently labeled fibroblasts for tumor detection with readily available intra‐operative fluorescence imaging tools may be useful for tumor staging and directing biopsies or surgical efforts during exploratory or debulking surgery. Fibroblasts may serve as a beacon pointing to the otherwise invisible metastases in the peritoneal cavity of ovarian cancer patients. Utilizing the recruited fibroblasts also for targeted delivery of anti angiogenic or antitumor molecules may aid in controlling tumor progression. Thus, these results suggest a novel approach for targeting ovarian tumor metastases for both tumor detection and therapy.

Mapping Neovascularization and Antineovascularization Therapy

The switch of tumors from avascular to the vascular phase and the onset of tumor angiogenesis mark a critical checkpoint in tumor progression. Avascular tumor dormancy can sometimes extend over many years, while upon vascularization, tumors show increased invasiveness, elevated metastatic potential and significantly worse prognosis (Weidner and Folkman, 1996). Regulation of the transition to the vascular phase depends on the balance between the production of promoters and inhibitors of angiogenesis (Hanahan and Folkman, 1996). The goal of our work was to define physiological scenarios which perturb this balance and can thus drive angiogenesis to previously dormant tumors. Such mechanisms that promote angiogenesis may explain epidemiological observations regarding age specific probabilities of certain tumors, environmental effects and the effects of trauma on tumor growth. In order to follow angiogenesis quantitatively, we developed an experimental system that relies on detection of vessel density by magnetic resonance imaging (MRI).