Hagit Dafni

Noninvasive Detection of Target Modulation following Phosphatidylinositol 3-Kinase Inhibition Using Hyperpolarized 13C Magnetic Resonance Spectroscopy

Numerous mechanism-based anticancer drugs that target the phosphatidylinositol 3-kinase (PI3K) pathway are in clinical trials. However, it remains challenging to assess responses by traditional imaging methods. Here, we show for the first time the efficacy of hyperpolarized (13)C magnetic resonance spectroscopy (MRS) in detecting the effect of PI3K inhibition by monitoring hyperpolarized [1-(13)C]lactate levels produced from hyperpolarized [1-(13)C]pyruvate through lactate dehydrogenase (LDH) activity. In GS-2 glioblastoma cells, PI3K inhibition by LY294002 or everolimus caused hyperpolarized lactate to drop to 42 +/- 12% and to 76 +/- 5%, respectively. In MDA-MB-231 breast cancer cells, hyperpolarized lactate dropped to 71 +/- 15% after treatment with LY294002. These reductions were correlated with reductions in LDH activity to 48 +/- 4%, 63 +/- 4%, and 69 +/- 12%, respectively, and were associated with a drop in levels of LDHA mRNA and LDHA and hypoxia-inducible factor-1alpha proteins. Supporting these findings, tumor growth inhibition achieved by everolimus in murine GS-2 xenografts was associated with a drop in the hyperpolarized lactate-to-pyruvate ratio detected by in vivo MRS imaging, whereas an increase in this ratio occurred with tumor growth in control animals. Taken together, our findings illustrate the application of hyperpolarized (13)C MRS of pyruvate to monitor alterations in LDHA activity and expression caused by PI3K pathway inhibition, showing the potential of this method for noninvasive imaging of drug target modulation.

In vivo BOLD contrast MRI mapping of subcutaneous vascular function and maturation: Validation by intravital microscopy

Bold contrast MRI was applied for mapping vascular maturation in tumor‐ and wound‐induced skin angiogenesis using the response of mature vessels to hypercapnia (inhalation of air vs. air 5% CO2) and the response of all vessels to hyperoxia (air 5% CO2 vs. oxygen 5% CO2 (carbogen)). MRI signal enhancement with hypercapnia was reduced in centered vs. linear phase encoding, suggesting increased blood flow. However, intravital microscopy demonstrated constriction of arterioles and reduced flux and density of red blood cells in mature capillaries with hypercapnia, with no change in the diameter of wound‐induced neovasculature. The discrepancy in flow between MRI and intravital microscopy is consistent with increased plasma flow and reduced hematocrit. Hyperoxia resulted in increased blood oxygenation and constriction of all vessels. These results provide a hemodynamic explanation for the selective registration of MRI response to hypercapnia with mature vessels and the response to hyperoxia with total vascular function. Magn Reson Med 45:887–898, 2001.

Vascular Remodeling and Angiogenesis in Ectopic Ovarian Transplants: A Crucial Role of Pericytes and Vascular Smooth Muscle Cells in Maintenance of Ovarian Grafts

Abstract Cancer patients, treated by either chemo- or radiotherapy, frequently suffer from ovarian failure and infertility. One of the new emerging techniques to preserve reproductive potential of such patients is cryopreservation of ovarian fragments prior to treatment and their retransplantation after healing. A major obstacle in survival of the ovarian implants is vascular failure, which leads to tissue necrosis. In order to investigate the role of angiogenesis in implant preservation, we used a xenograft model in which rat ovaries were transplanted into immunodeficient mice. Graft reception and maintenance were monitored by magnetic resonance imaging (MRI) and histology. Two transplantation sites were explored, i.e., subcutaneous and intramuscular. Comparison between these two transplantation sites revealed the importance of vascular smooth muscle cells and pericytes in sustaining vascular and tissue integrity. Histological examination of the grafts, at different time points and sizes, revealed that loss of perivascular cells preceded damage to endothelial cells and was closely correlated with loss of follicular and oocyte integrity. Intramuscular implantation provided better maintenance of implant perivascular cells relative to subcutaneous implantation. Accordingly, follicular integrity was superior in the intramuscular implants and the number of damaged follicles was significantly lower compared with the subcutaneous transplantation site. These results suggest that improving ovarian implant maintenance should be directed toward preservation of perivascular support.

Hyperpolarized 13C Spectroscopic Imaging Informs on Hypoxia-Inducible Factor-1 and Myc Activity Downstream of Platelet-Derived Growth Factor Receptor

The recent development of hyperpolarized (13)C magnetic resonance spectroscopic imaging provides a novel method for in vivo metabolic imaging with potential applications for detection of cancer and response to treatment. Chemotherapy-induced apoptosis was shown to decrease the flux of hyperpolarized (13)C label from pyruvate to lactate due to depletion of NADH, the coenzyme of lactate dehydrogenase. In contrast, we show here that in PC-3MM2 tumors, inhibition of platelet-derived growth factor receptor with imatinib reduces the conversion of hyperpolarized pyruvate to lactate by lowering the expression of lactate dehydrogenase itself. This was accompanied by reduced expression of vascular endothelial growth factor and glutaminase, and is likely mediated by reduced expression of their transcriptional factors hypoxia-inducible factor-1 and c-Myc. Our results indicate that hyperpolarized (13)C MRSI could potentially detect the molecular effect of various cell signaling inhibitors, thus providing a radiation-free method to predict tumor response.

Angiogenesis in ectopic ovarian xenotransplantation: Multiparameter characterization of the neovasculature by dynamic contrast-enhanced MRI

It has been suggested that ovarian cryopreservation and xenotransplantation can be used to preserve oocytes from damage during anticancer treatments. The main obstacle to subsequent ovarian grafting is loss of oocytes due to impaired perfusion. The aim of this study was to characterize angiogenic events following ovary xenotransplantation. Rat ovaries were transplanted into or onto the muscle of immunocompromised CD1‐nude mice. Ovariectomy (OVX) of host mice prior to transplantation supported the resumption of follicular development, as manifested by the prevalence of antral follicles and corpora lutea. Two days after transplantation, the grafts were devoid of blood supply. Functional vessels within the graft were detected by MRI and histology from day 7 and on. By 2–3 weeks, both blood volume fraction and permeability in the graft, as measured with the use of albumin‐based MR contrast material, were significantly elevated relative to the adjacent muscle. Extravasation of contrast material from the graft neovasculature was followed by interstitial convection in the muscle surrounding the graft, and draining toward the proximal popliteal lymph node. Development of the vasculature was monitored noninvasively, providing a time scale for revascularization and recovery of ovarian function following xenotransplantation of ovarian grafts. Magn Reson Med 52:741–750, 2004.

Modulation of the pharmacokinetics of macromolecular contrast material by avidin chase: MRI, optical, and inductively coupled plasma mass spectrometry tracking of triply labeled albumin.

The goal of this work was to develop an MRI method for mapping the clearance of interstitial macromolecular plasma proteins after their extravasation from permeable blood vessels. To that end, a well‐defined window of exposure to elevated blood levels was generated by inducing rapid clearance of macromolecular contrast material from the blood. Experimental removal of the intravascular component allowed subsequent tracking of clearance from the interstitial compartment in the absence of further contrast extravasation. The contrast material was based on albumin triply labeled with biotin, fluorescent tag, and GdDTPA, allowing optical, inductively coupled plasma mass spectrometry (ICP‐MS) and MRI detection. The biotin tag was used here for in vivo chasing of the contrast material from the blood by intravenous administration of avidin. Upon administration of avidin the contrast material disappeared from the blood vessels and was cleared by the liver and spleen as detected by MRI, fluorescence of blood samples and histological sections, and by ICP‐MS. Nonbiotinylated fluorescent albumin was not affected by administration of avidin. Contrast material that extravasated from leaky blood vessels in a VEGF overexpressing tumor, prior to administration of avidin, was not cleared by the addition of avidin and showed continued interstitial convection. Thus, avidin‐chase provides an effective tool for in vivo manipulation of the arterial input function by providing experimental control over the rate of clearance of the contrast material from the circulation. Magn Reson Med 50:904–914, 2003.

Molecular imaging of angiogenesis

Angiogenesis (the growth of new blood vessels) is a complex multistep process that involves multiple cell types, numerous growth factors, and complex regulatory checks and balances. Tight control of vascular remodeling evolved to ensure stability of the vasculature while maintaining the bodys ability to rapidly mount an angiogenic response requiring a high degree of plasticity. Angiogenesis is critical not only for physiological development, but also for the progression of pathologies, and is thus a target for therapeutic intervention. The importance of the process coupled with the ease of access for delivery of contrast agents makes the vasculature at large, and angiogenesis in particular, a favorable target of functional and molecular imaging. Recent developments in molecular imaging tools have expanded our views to encompass many components of the process. Functional imaging of blood volume, vessel permeability, and vasoreactivity is complemented by novel contrast agents that reveal specific targets on endothelial cells. Methods have been developed to label vascular cells so as to track their recruitment to sites of angiogenesis, and new “smart” contrast agents have been designed to reveal the activity of enzymatic reactions in altering the extracellular matrix (ECM) during angiogenesis. J. Magn. Reson. Imaging 2007.

Magnetic Resonance Imaging Visualization of Hyaluronidase in Ovarian Carcinoma

Hyaluronan, a high molecular weight, negatively charged polysaccharide, is a major constituent of the extracellular matrix. High molecular weight hyaluronan is antiangiogenic, but its degradation by hyaluronidase generates proangiogenic breakdown products. Thus, by expression of hyaluronidase, cancer cells can tilt the angiogenic balance of their microenvironment. Indeed, hyaluronidase-mediated breakdown of hyaluronan correlates with aggressiveness and invasiveness of ovarian cancer metastasis and with tumor angiogenesis. The goal of this work was to develop a novel smart contrast material for detection of hyaluronidase activity by magnetic resonance imaging (MRI). Gadolinium-diethylenetriaminepentaacetic acid (GdDTPA) covalently linked to hyaluronan on the surface of agarose beads showed attenuated relaxivity. Hyaluronidase, either purified from bovine testes or secreted by ES-2 and OVCAR-3 human epithelial ovarian carcinoma cells, activated the hyaluronan-GdDTPA-beads by rapidly altering the R1 and R2 relaxation rates. The change in relaxation rates was consistent with the different levels of biologically active hyaluronidase secreted by those cells. Hyaluronan-GdDTPA-beads were further used for demonstration of MRI detection of hyaluronidase activity in the proximity of s.c. ES-2 ovarian carcinoma tumors in nude mice. Thus, hyaluronan-GdDTPA-beads could allow noninvasive molecular imaging of hyaluronidase-mediated tilt of the peritumor angiogenic balance.

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.

Macromolecular dynamic contrast-enhanced (DCE)-MRI detects reduced vascular permeability in a prostate cancer bone metastasis model following anti-platelet-derived growth factor receptor (PDGFR) therapy, indicating a drop in vascular endothelial growth factor receptor (VEGFR) activation.

The antivascular function of the platelet‐derived growth factor receptor (PDGFR) inhibitor imatinib combined with paclitaxel has been demonstrated by invasive immunohistochemistry. The purpose of this study was to 1) noninvasively monitor the response to anti‐PDGFR treatment, and 2) understand the underlying mechanism of this response. Thus, response to treatment was studied in a prostate cancer bone metastasis model using macromolecular (biotin–bovine serum albumin [BSA]–Gd‐diethylene triamine pentaacetic acid [GdDTPA]) dynamic contrast‐enhanced magnetic resonance imaging (DCE‐MRI). Human prostate cancer (PC‐3MM2) bone metastases that caused osteolysis and grew in neighboring muscle showed a high blood‐volume fraction (fBV) and vascular permeability (PS) at the tumor periphery compared to muscle tissue and intraosseous tumor. Imatinib alone or with paclitaxel significantly reduced PS by 35% (one‐tailed paired t‐test, P = 0.045) and 40% (P = 0.0003), respectively, whereas paclitaxel alone or no treatment had no effect. Based on changes in PS, we hypothesized that imatinib interferes with the signaling pathway of vascular endothelial growth factor (VEGF). This mechanism was verified by immunohistochemistry. It demonstrated reduced activation of both PDGFR‐β and VEGF receptor 2 (VEGFR2) in imatinib‐treated mice. Our study therefore demonstrates that macromolecular DCE‐MRI can be used to detect early vascular effects associated with response to therapy targeted to PDGFR, and provides insight into the role played by VEGF in anti‐PDGFR therapy. Magn Reson Med 60:822–833, 2008.