Establishing a Mouse Genotyping Core Facility Based on Automation, High-resolution Melting Analysis, and Purpose-developed MATLAB Applications for Sample Tracking, Data Analysis, and Report Generation.

An in-house genotyping facility should aim to be more cost-effective than outsourced service and more reliable than genotyping performed by short-term employees or students of individual research groups. Reliable genotyping allows efficient and economical management of mice colonies and promotes accurate and reproducible research results. Here we provide a detailed description of our approach to establishing a genotyping core facility, relying on automated PCR assembly and high-resolution melting (HRM) analysis (first derivative). The workflow we devised was tightly managed by purpose-designed applications developed using MATLAB App Designer that allowed straightforward work planning, ensured sample tracking throughout the process, and provided a platform for reliable data analysis and generation of genotyping reports. We successfully transitioned PCR product analysis of more than 250 different target genes from standard gel electrophoresis to the more advanced HRM analysis. About 23% of the target genes required a redesign of primers to adapt to our protocol. The process was highly universal, and only 2% of the target genes required deviation from the standard PCR method to a more restricted protocol that reduces the amplification of nonspecific products. We currently run more than 1,000 PCR reactions weekly, of samples taken at weaning or experimental endpoint, and assemble a large variety of target genes in every PCR plate. We also showed that genotyping of blastocytes instead of embryos can serve as quality control of cryopreservation. Thus, our genotyping protocol promotes the 3Rs (Replacement, Reduction, and Refinement) principles. Our refined genotyping process facilitates cost-effective colony management, replaces tissue types as well as traditional methods with advanced ones, and provides reliable results in a timely manner. MATLAB codes and related data are available in supplementary materials and online.

Competitive fungal commensalism mitigates candidiasis pathology.

The mycobiota are a critical part of the gut microbiome, but host-fungal interactions and specific functional contributions of commensal fungi to host fitness remain incompletely understood. Here, we report the identification of a new fungal commensal, Kazachstania heterogenica var. weizmannii, isolated from murine intestines. K. weizmannii exposure prevented Candida albicans colonization and significantly reduced the commensal C. albicans burden in colonized animals. Following immunosuppression of C. albicans colonized mice, competitive fungal commensalism thereby mitigated fatal candidiasis. Metagenome analysis revealed K. heterogenica or K. weizmannii presence among human commensals. Our results reveal competitive fungal commensalism within the intestinal microbiota, independent of bacteria and immune responses, that could bear potential therapeutic value for the management of C. albicans-mediated diseases.

Prediction of Ovarian Follicular Dominance by MRI Phenotyping of Hormonally Induced Vascular Remodeling.

In the mammalian female, only a small subset of ovarian follicles, known as the dominant follicles (DFs), are selected for ovulation in each reproductive cycle, while the majority of the follicles and their resident oocytes are destined for elimination. This study aimed at characterizing early changes in blood vessel properties upon the establishment of dominance in the mouse ovary and application of this vascular phenotype for prediction of the follicles destined to ovulate. Sexually immature mice, hormonally treated for induction of ovulation, were imaged at three different stages by dynamic contrast-enhanced (DCE) MRI: prior to hormonal administration, at the time of DF selection, and upon formation of the corpus luteum (CL). Macromolecular biotin-bovine serum albumin conjugated with gadolinium-diethylenetriaminepentaacetic acid (b-BSA-GdDTPA) was intravenously injected, and the dynamics of its extravasation from permeable vessels as well as its accumulation in the antral cavity of the ovarian follicles was followed by consecutive T1-weighted MRI. Permeability surface area product (permeability) and fractional blood volume (blood volume) were calculated from b-BSA-GdDTPA accumulation. We found that the neo-vasculature during the time of DF selection was characterized by low blood volume and low permeability values as compared to unstimulated animals. Interestingly, while the vasculature of the CL showed higher blood volume compared to the DF, it exhibited a similar permeability. Taking advantage of immobilized ovarian imaging, we combined DCE-MRI and intravital light microscopy, to reveal the vascular properties of follicles destined for dominance from the non-ovulating subordinate follicles (SFs). Immediately after their selection, permeability of the vasculature of DF was attenuated compared to SF while the blood volume remained similar. Furthermore, DFs were characterized by delayed contrast enhancement in the avascular follicular antrum, reflecting interstitial convection, whereas SFs were not. In this study, we showed that although DF selection is accompanied by blood vessel growth, the new vasculature remained relatively impermeable compared to the vasculature in control animal and compared to SF. Additionally, DFs show late signal enhancement in their antrum. These two properties may aid in clinical prediction of follicular dominance at an early stage of development and help in their diagnosis for possible treatment of infertility.

The Likelihood of Misidentifying Rodent Pasteurellaceae by Using Results from a Single PCR Assay.

The precise identification of rodent Pasteurellaceae is known to be highly challenging. An unknown strain of Pasteurellaceae appeared and rapidly spread throughout our animal facilities. Standard microbiology, combined with biochemical analysis, suggested that the bacteria strain was Rodentibacter pneumotropicus or R. heylii. We submitted samples of the unknown bacteria and known isolates of R. pneumotropicus, R. heylii, and Muribacter muris, to 2 service laboratories that provide animal health monitoring. Results of microbiology tests performed by both laboratories, species-specific PCR analysis performed by one laboratory, and independent 16S rRNA gene sequencing yielded identical identification of the unknown bacteria as Pasteurellaceae (Pasteurella spp.) and not R. pneumotropicus or R. heylii. In contrast, the similarly intended PCR assay performed by the other laboratory identified the bacteria as R. heylii. Careful evaluation of all of the results led us to conclude that the correct identification of the bacteria is Pasteurellaceae. From our experience, we recommend that a combination of several methods should be used to achieve correct identification of rodent Pasteurellaceae. Specifically, we advise that all primer sets used should be disclosed when reporting PCR test results, including in health reports provided by service laboratories and animal vendors. Careful, correct, and informative health monitoring reports are most beneficial to animal researchers and caretakers who might encounter the presence and effects of rodent Pasteurellaceae.

Whole Organ Blood and Lymphatic Vessels Imaging (WOBLI).

Thin section histology is limited in providing 3D structural information, particularly of the intricate morphology of the vasculature. Availability of high spatial resolution imaging for thick samples, would overcome the restriction dictated by low light penetration. Our study aimed at optimizing the procedure for efficient and affordable tissue clearing, along with an appropriate immunofluorescence labeling that will be applicable for high resolution imaging of blood and lymphatic vessels. The new procedure, termed whole organ blood and lymphatic vessels imaging (WOBLI), is based on two previously reported methods, CLARITY and ScaleA2. We used this procedure for the analysis of isolated whole ovary, uterus, lung and liver. These organs were subjected to passive clearing, following fixation, immunolabeling and embedding in hydrogel. Cleared specimens were immersed in ScaleA2 solution until transparency was achieved and imaged using light sheet microscopy. We demonstrate that WOBLI allows detailed analysis and generation of structural information of the lymphatic and blood vasculature from thick slices and more importantly, from whole organs. We conclude that WOBLI offers the advantages of morphology and fluorescence preservation with efficient clearing. Furthermore, WOBLI provides a robust, cost-effective method for generation of transparent specimens, allowing high resolution, 3D-imaging of blood and lymphatic vessels networks.

Noninvasive mapping of endothelial dysfunction in myocardial ischemia by magnetic resonance imaging using an albumin-based contrast agent.

Noninvasive preclinical methods for the characterization of myocardial vascular function are crucial to an understanding of the dynamics of ischemic cardiac disease. Ischemic heart disease is associated with myocardial endothelial dysfunction, resulting in leakage of plasma albumin into the extravascular space. These features can be harnessed in a novel noninvasive three-dimensional magnetic resonance imaging method to measure fractional blood volume (fBV) and vascular permeability (permeability-surface area product, PS) using labeled albumin as a blood pool contrast agent. C57BL/6 mice were imaged before and 3 days after myocardial infarction (MI). Following the quantification of endogenous myocardial R1 , the dynamics of intravenously injected albumin-based contrast agent, extravasating from permeable myocardial blood vessels, were tracked on short-axis magnetic resonance images of the entire heart. This study successfully discriminated between infarcted and remote regions at 3 days post-infarct, based on a reduced fBV and increased PS in the infarcted region. These findings were confirmed using ex vivo fluorescence imaging and histology. We have demonstrated a novel method to quantify blood volume and permeability in the infarcted myocardium, providing an imaging biomarker for the assessment of endothelial dysfunction. This method has the potential to three-dimensionally visualize subtle changes in myocardial permeability and to track endothelial function for longitudinal cardiac studies determining pathophysiological processes during infarct healing.

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.

Multimodal Correlative Preclinical Whole Body Imaging and Segmentation.

Segmentation of anatomical structures and particularly abdominal organs is a fundamental problem for quantitative image analysis in preclinical research. This paper presents a novel approach for whole body segmentation of small animals in a multimodal setting of MR, CT and optical imaging. The algorithm integrates multiple imaging sequences into a machine learning framework, which generates supervoxels by an efficient hierarchical agglomerative strategy and utilizes multiple SVM-kNN classifiers each constrained by a heatmap prior region to compose the segmentation. We demonstrate results showing segmentation of mice images into several structures including the heart, lungs, liver, kidneys, stomach, vena cava, bladder, tumor, and skeleton structures. Experimental validation on a large set of mice and organs, indicated that our system outperforms alternative state of the art approaches. The system proposed can be generalized to various tissues and imaging modalities to produce automatic atlas-free segmentation, thereby enabling a wide range of applications in preclinical studies of small animal imaging.

Genetic and Pharmacological Modulation of Akt1 for Improving Ovarian Graft Revascularization in a Mouse Model.

Ovarian tissue cryopreservation and transplantation is one of a few available treatments for fertility preservation in women diagnosed with cancer. Rapid revascularization is essential for reducing hypoxic damage after grafting and protecting the primordial follicles reserve. Using a mouse model of heterotopic ovarian graft transplantation, we have delineated the role of endothelial Akt1 expression using longitudinal magnetic resonance imaging follow-up to quantify angiogenic response. Endothelial Akt1 activation in ovarian grafts promoted angiogenesis to support the graft during posttransplantation hypoxic period. Similarly, simvastatin therapy activated Akt1 at the transplantation site and improved the revascularization and vascular support of ovarian grafts. These results serve as an important first step toward pharmacological intervention to improve revascularization of ovarian grafts and restoration of fertility in cancer survivors. The pro-angiogenic effects reported here may extend beyond improving ovarian graft reception in fertility preservation and could potentially be used for different organ or tissue transplantation.

Using bimodal MRI/fluorescence imaging to identify host angiogenic response to implants.

Therapies that promote angiogenesis have been successfully applied using various combinations of proangiogenic factors together with a biodegradable delivery vehicle. In this study we used bimodal noninvasive monitoring to show that the host response to a proangiogenic biomaterial can be drastically affected by the mode of implantation and the surface area-to-volume ratio of the implant material. Fluorescence/MRI probes were covalently conjugated to VEGF-bearing biodegradable PEG-fibrinogen hydrogel implants and used to document the in vivo degradation and liberation of bioactive constituents in an s.c. rat implantation model. The hydrogel biodegradation and angiogenic host response with three types of VEGF-bearing implant configurations were compared: preformed cylindrical plugs, preformed injectable microbeads, and hydrogel precursor, injected and polymerized in situ. Although all three were made with identical amounts of precursor constituents, the MRI data revealed that in situ polymerized hydrogels were fully degraded within 2 wk; microbead degradation was more moderate, and plugs degraded significantly more slowly than the other configurations. The presence of hydrogel degradation products containing the fluorescent label in the surrounding tissues revealed a distinct biphasic release profile for each type of implant configuration. The purported in vivo VEGF release profile from the microbeads resulted in highly vascularized s.c. tissue containing up to 16-fold more capillaries in comparison with controls. These findings demonstrate that the configuration of an implant can play an important role not only in the degradation and resorption properties of the materials, but also in consequent host angiogenic response.

In search of signaling pathways critical for ovarian graft reception: Akt1 is essential for long-term survival of ovarian grafts.

OBJECTIVE: To explore the role of Akt1, a principle modulator of angiogenesis, in ovarian graft reception and to investigate whether Akt1 deficiency can alter ovarian graft reception. DESIGN: Experimental mouse model. SETTING: Research institute. ANIMAL(S): Donors: Akt1 knockout (Akt1(-/-)) and wild types (Akt1(+/+)) mice. Recipients: CD-1 nude immune deficient female mice. INTERVENTION(S): Ovaries from Akt1(-/-) and Akt1(+/+) mice transplanted in the biceps femoris muscle of immunocompromised CD-1 mice, and ovarian graft viability, perfusion, and revascularization explored in vivo by magnetic resonance imaging (MRI). MAIN OUTCOME MEASURE(S): Vascular density and permeability of newly formed graft blood vessels quantified by dynamic contrast-enhanced MRI 7, 14, 30, and 60 days after grafting as indicators for angiogenesis and reestablishment of blood perfusion. RESULT(S): The Akt1(-/-) ovarian grafts showed a gradual decrease in angiogenic response with time after transplantation, ultimately leading to complete or near-complete graft destruction coinciding with massive follicular loss. Sixty days after transplantation, the mean blood volume fraction (fBV) and vessel permeability (PS) were statistically significantly lower in Akt1(-/-) transplants compared with Akt1(+/+). CONCLUSION(S): Akt1 is essential for ovarian graft reception. However, surprisingly the impact of Akt1 deficiency was most profound not in the early stages of angiogenesis but rather in long-term survival of the graft.

Vascular patterning and permeability in prostate cancer models with differing osteogenic properties.

Bone metastasis is a major cause of morbidity and mortality in prostate cancer. However, the lack of clinically relevant models hinders our understanding of the disease as well as development of effective therapies and imaging approaches. We used noninvasive MRI, histology and micro CT to further characterize the newly established prostate cancer bone metastases-derived model MDA-PCa-118b, and to compare it to the well-established PC-3MM2 model with regard to bone structure and vascular patterning. The PC-3MM2 model is highly osteolytic whereas the MDA-PCa-118b model shows a robust osteoblastic reaction, as often seen in clinical cases. Macromolecular contrast enhanced MRI revealed differences in vascular permeability patterns, which appeared peripheral for PC-3MM2 and nodular for MDA-PCa-118b, matching the microscopic cellular composition of each model: PC-3MM2 exclusively recruits endothelial cells to form thin tumor-core blood vessels and enlarged, leaky peripheral vessels, whereas MDA-PCa-118b also recruits bone-forming cells and pericytes such that small tumor nests are encircled with leaky vessels and embedded in bone-like tissue dotted with pericyte-covered vessels. Despite these structural differences, vascular permeability was reduced in both tumor models by either imatinib or SU10944 treatment. This study highlights the importance of clinically relevant osteogenic models of human prostate cancer and the value of such models not only in enhancing our understanding of tumorigenesis, metastasis and response to therapy, but also for development of appropriate methods for noninvasive imaging of these processes.

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.

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.

Dynamic nuclear polarization in metabolic imaging of metastasis: common sense, hypersense and compressed sensing.

Tumorigenesis and metastasis are associated with metabolic reprogramming, including enhanced glycolysis and glutaminolysis. Recent developments in dynamic nuclear polarization (DNP) dramatically enhance the sensitivity of magnetic resonance spectroscopy, providing a novel method for metabolic imaging in real-time through 'hypersensing' of polarized metabolites. In parallel, advanced signal acquisition sequences, such as compressed sensing, improve spatial and temporal resolution providing better depiction of rapid metabolic events in small tumors and metastases. Here we review the potential of hyperpolarized magnetic resonance spectroscopy for studying the crosstalk between oncogenic cell signaling and metabolism, and for probing diagnostic biomarkers and metabolic indicators of therapeutic response in primary tumors and metastases.

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-beta 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.

Monitoring histone deacetylase inhibition in vivo: noninvasive magnetic resonance spectroscopy method.

Histone deacetylase inhibitors (HDACis) are emerging as promising and selective antitumor agents. However, HDACis can lead to tumor stasis rather than shrinkage, in which case, traditional imaging methods are not adequate to monitor response. Consequently, novel approaches are needed. We have shown in cells that (19)F magnetic resonance spectroscopy (MRS)-detectable levels of the HDAC substrate Boc-Lys-TFA-OH (BLT) are inversely correlated with HDAC activity. We extended our investigations to a tumor xenograft model. Following intraperitoneal injection of BLT, its accumulation within the tumor was monitored by in vivo (19)F MRS. In animals treated with the HDACi suberoylanilide hydroxamic acid (SAHA), tumoral BLT levels were higher by 77% and 132% on days 2 and 7 of treatment compared with pretreatment levels (n = 6; p < .05). In contrast, tumoral BLT levels remained unchanged in control animals and in normal tissue. Thus, (19)F MRS of BLT detected the effect of HDACi treatment as early as day 2 of treatment. Importantly, tumor size confirmed that SAHA treatment leads to inhibition of tumor growth. However, difference in tumor size reached significance only on day 6 of treatment. Thus, this work identifies BLT as a potential molecular imaging agent for the early noninvasive MRS detection of HDAC inhibition in vivo.

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 body's 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.

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 alpha-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.