Magnetic resonance pharmacoangiography to detect and predict chemotherapy delivery to solid tumors.

Detection and prediction of drug delivery to the tumor interstitium are of critical importance in cancer chemotherapy. Prediction of drug delivery derived from standard pharmacokinetic models is frequently inadequate because of the complex nature of tumor blood flow and the microenvironment. Although drug concentrations can be directly sampled with microdialysis or in biopsy samples, we currently lack methods capable of detecting and/or predicting drug delivery to tumors noninvasively. In this study, we describe a novel magnetic resonance (MR) technique to directly detect the drug, and we present the correlation between delivery of drug and the delivery of MR contrast agents to the tumor. Experiments were performed with tumor xenografts in severe combined immunodeficient mice. Three-dimensional maps of the drug distribution within the tumors were obtained with 13C spectroscopic MR imaging with a spatial resolution of 2 x 2 x 2 mm, using signals of the 13C-labeled anticancer agent phenylacetate. Three-dimensional maps of uptake of gadolinium-diethylenetriaminepentaacetic acid (GdDTPA) contrast agent were obtained for the same tumors using dynamic MR imaging. Experimental data were analyzed for correlation between delivery of the drug and the contrast. Histological analysis was performed for excised tumors. Experimental data demonstrated a significant spatial correlation (r = 0.59 with P < 0.001) between the parameter representing delivery of the contrast to tumor interstitium, determined from the kinetic curves of GdDTPA, and integral tissue drug concentrations for two different tumor models. The method is designed to probe extravasation of the drug molecules from the bloodstream into the tumor interstitium. Although therapeutic efficiency of the drug will also depend upon drug retention in the tumor and the ability of the molecules to cross cellular membranes, inefficient drug transfer from plasma to tissue can be a major impediment in achieving effective tumor chemotherapy. The results of this study demonstrate that the uptake kinetics of a low molecular weight MR contrast agent can be used to predict delivery of drug molecules of similar size to the interstitium of solid tumors.

Intratumoral conversion of 5-fluorocytosine to 5-fluorouracil by monoclonal antibody-cytosine deaminase conjugates: noninvasive detection of prodrug activation by magnetic resonance spectroscopy and spectroscopic imaging.

The monitoring of antibody-directed enzyme-prodrug therapies requires evaluation of drug activation within the tissues of interest. We have demonstrated the feasibility of noninvasive magnetic resonance spectroscopy and spectroscopic imaging (chemical shift imaging) to detect activation of the prodrug 5-fluorocytosine (5-FCyt) to the cytotoxic species 5-fluorouracil (5-FU) by monoclonal antibody-cytosine deaminase (CD) conjugates. In vitro, L6-CD but not 1F5-CD selectively metabolized 5-FCyt to 5-FU on H2981 human lung adenocarcinoma cells because of the presence and absence of cell surface L6 and CD20 antigens, respectively. After pretreatment of H2981 tumor-bearing mice with L6-CD, in vivo metabolism of 5-FCyt to 5-FU within the tumors was detected by 19F magnetic resonance spectroscopy; the chemical shift separation between 5-FCyt and 5-FU resonances was approximately 1.2 ppm. 5-FU levels were 50-100% of 5-FCyt levels in tumors 10-60 min after 5-FCyt administration. Whole body 19F chemical shift imaging (6 x 6 mm in-plane resolution) of tumor-bearing mice demonstrated the highest signal intensity of 5-FU within the tumor region. This study supports further development of noninvasive magnetic resonance methods for preclinical and clinical monitoring of CD enzyme-prodrug therapies.

Vascular differences detected by MRI for metastatic versus nonmetastatic breast and prostate cancer xenografts.

Several studies have linked vascular density, identified in histologic sections, to "metastatic risk." Functional information of the vasculature, not readily available from histologic sections, can be obtained with contrast-enhanced MRI to exploit for therapy or metastasis prevention. Our aims were to determine if human breast and prostate cancer xenografts preselected for differences in invasive and metastatic characteristics established correspondingly different vascular volume and permeability, quantified here with noninvasive MRI of the intravascular contrast agent albumin-GdDTPA. Tumor vascular volume and permeability of human breast and prostate cancer xenografts were characterized using MRI. Parallel studies confirmed the invasive behavior of these cell lines. Vascular endothelial growth factor (VEGF) expression in the cell lines was measured using ELISA and Western blots. Metastasis to the lungs was evaluated with spontaneous as well as experimental assay. Metastatic tumors formed vasculature with significantly higher permeability or vascular volume (P<.05, two-sided unpaired t test). The permeability profile matched VEGF expression. Within tumors, regions of high vascular volume usually exhibited low permeability whereas regions of low vascular volume exhibited high permeability. We observed that although invasion was necessary, without adequate vascularization it was not sufficient for metastasis to occur.

Imaging prostate cancer invasion with multi-nuclear magnetic resonance methods: the Metabolic Boyden Chamber.

The physiological milieu within solid tumors can influence invasion and metastasis. To determine the impact of the physiological environment and cellular metabolism on cancer cell invasion, it is necessary to measure invasion during well-controlled modulation of the physiological environment. Recently, we demonstrated that magnetic resonance imaging can be used to monitor cancer cell invasion into a Matrigel layer [Artemov D, Pilatus U, Chou S, Mori N, Nelson JB, and Bhujwalla ZM (1999). Dynamics of prostate cancer cell invasion studied in vitro by NMR microscopy. Mag Res Med 42, 277-282.]. Here we have developed an invasion assay ("Metabolic Boyden Chamber") that combines this capability with the properties of our isolated cell perfusion system. Long-term experiments can be performed to determine invasion as well as cellular metabolism under controlled environmental conditions. To characterize the assay, we performed experiments with prostate cancer cell lines preselected for different invasive characteristics. The results showed invasion into, and degradation of the Matrigel layer, by the highly invasive/metastatic line (MatLyLu), whereas no significant changes were observed for the less invasive/metastatic cell line (DU-145). With this assay, invasion and metabolism was measured dynamically, together with oxygen tensions within the cellular environment and within the Matrigel layer. Such a system can be used to identify physiological and metabolic characteristics that promote invasion, and evaluate therapeutic interventions to inhibit invasion.

The physiological environment in cancer vascularization, invasion and metastasis.

One of the most lethal aspects of cancer arises from its ability to invade and metastasize. Determining the factors that promote cancer cell invasion and metastasis is therefore critically important in treating this disease. The tumour physiological environment is uniquely different from normal tissue, and exhibits hypoxia, acidic extracellular pH and high levels of lactate. This environment, dictated largely by abnormal tumour vasculature and metabolism, in turn also promotes angiogenesis. The physiological environment, tumour metabolism, angiogenesis and vascularization are therefore inextricably linked. We have developed and applied non-invasive magnetic resonance (MR) imaging (I) and spectroscopy (S) techniques to understand the role of vascular, physiological and metabolic properties in cancer invasion and metastasis. These MR studies are performed with human breast and prostate cancer cells maintained in culture or grown as solid tumours in immune-suppressed mice. We have detected significant differences in vascular, physiological and metabolic characteristics of metastatic and non-metastatic human breast and prostate cancer models with MRI and MRS. Using a combined MRI/MRS approach we are currently acquiring metabolic, extracellular pH and vascular images from the same localized regions within a solid tumour to further understand the dynamics between these parameters and their role in cancer invasion and metastasis.

'The metabolism of tumours': 70 years later.

Otto Warburg's classic treatise on the reprogramming of tumour metabolism from oxidative to glycolytic metabolism was published in London in 1930. Although the Warburg effect is one of the most universal characteristics of solid tumours, the molecular basis for this phenomenon has only recently been elucidated by studies indicating that increased expression of genes encoding glucose transporters and glycolytic enzymes in tumour cells is mediated by the transcription factors c-MYC and HIF-1. Whereas c-myc is a direct target for oncogenic mutations, expression of hypoxia-inducible factor 1 (HIF-1) is indirectly up-regulated via gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes that result increased HIF-1alpha protein expression and/or increased HIF-1 transcriptional activity in a cell-type-specific manner. As a result of genetic alterations and intratumoral hypoxia, HIF-1alpha is overexpressed in the majority of common human cancers relative to the surrounding normal tissue. In human breast cancer and brain tumours, HIF-1alpha overexpression is strongly correlated with tumour grade and vascularity.

Tumor angiogenesis, vascularization, and contrast-enhanced magnetic resonance imaging.

Angiogenesis, the process by which new blood vessels are generated, occurs during wound healing, in the female reproductive system during ovulation and gestation, and during embryonic development. The process is carefully controlled with positive and negative regulators, because several vital physiological functions require angiogenesis. The consequences of abnormal angiogenesis are either excessive or insufficient blood vessel growth. Ulcers, strokes, and heart attacks can result from the absence of angiogenesis normally required for natural healing, whereas excessive blood vessel proliferation may favor tumor growth and dissemination, blindness, and arthritis. In this review, the process of angiogenesis and the characteristics of the resulting tumor vasculature are outlined. Contrast-enhanced magnetic resonance imaging techniques that currently are available for basic research and clinical applications to study various aspects of tumor angiogenesis and neovascularization are discussed.

[Cellular factors of ischemia/reperfusion injury pathogenesis in the renal transplantation].

A review of literature reveales the current conception of Russian and foreign authors on the cellular and humoral pathogenetic mechanisms of ischemic and reperfusion injury of kidney transplant.

Twist contributes to hormone resistance in breast cancer by downregulating estrogen receptor-α.

The role of estrogen receptor-α (ER) in breast cancer development, and as a primary clinical marker for breast cancer prognosis, has been well documented. In this study, we identified the oncogenic protein, TWIST1 (Twist), which is overexpressed in high-grade breast cancers, as a potential negative regulator of ER expression. Functional characterization of ER regulation by Twist was performed using Twist low (MCF-7, T-47D) and Twist high (Hs 578T, MDA-MB-231, MCF-7/Twist) expressing cell lines. All Twist high expressing cell lines exhibited low ER transcript and protein levels. By chromatin immunoprecipitation and promoter assays, we demonstrated that Twist could directly bind to E-boxes in the ER promoter and significantly downregulate ER promoter activity in vitro. Functionally, Twist overexpression caused estrogen-independent proliferation of breast cells, and promoted hormone resistance to the selective estrogen receptor modulator tamoxifen and selective estrogen receptor down-regulator fulvestrant. Importantly, this effect was reversible on downregulating Twist. In addition, orthotopic tumors generated in mice using MCF-7/Twist cells were resistant to tamoxifen. These tumors had high vascular volume and permeability surface area, as determined by magnetic resonance imaging (MRI). Mechanistically, Twist recruited DNA methyltransferase 3B (DNMT3B) to the ER promoter, leading to a significantly higher degree of ER promoter methylation compared with parental cells. Furthermore, we demonstrated by co-immunoprecipitation that Twist interacted with histone deacetylase 1 (HDAC1) at the ER promoter, causing histone deacetylation and chromatin condensation, further reducing ER transcript levels. Functional re-expression of ER was achieved using the demethylating agent, 5-azacytidine, and the HDAC inhibitor, valproic acid. Finally, an inverse relationship was observed between Twist and ER expression in human breast tumors. In summary, the regulation of ER by Twist could be an underlying mechanism for the loss of ER activity observed in breast tumors, and may contribute to the generation of hormone-resistant, ER-negative breast cancer.

Imaging of cationic multifunctional liposome-mediated delivery of COX-2 siRNA.

Liposomes are a useful means of delivering molecular targeting agents such as small interfering RNA (siRNA) to downregulate specific pathways important in cancer growth and progression. The ability to non-invasively image these carriers is important to ascertain their delivery within the tumor. As cyclooxygenase-2 (COX-2) is an important therapeutic target in cancer, we investigated loading COX-2-specific siRNA into cationic liposomes containing MR contrast agents for imaging delivery in cancer cells and tumors. COX-2 and GAPDH siRNA, as well as Magnevist or Feridex, were loaded directly into the liposomes. These lipoplexes were used for cell transfection of the poorly differentiated and highly metastatic breast cancer cell line MDA-MB-231. PEGylated liposomes loaded with Feridex and fluorescently labeled COX-2 siRNA were used for in vivo delivery of lipoplexes in MDA-MB-231 breast cancer xenografts in female SCID mice. Transient transfection assays demonstrated potent and specific downregulation of the COX-2 protein in cells in culture. Tail vein injections of PEGylated COX-2 lipoplexes resulted in intratumoral delivery of siRNA. Biodistribution studies showed significant localization in the lung, liver and kidney at 24 h. These data demonstrate the feasibility of liposomal-mediated delivery of COX-2-specific siRNA to downregulate COX-2 in cancer cells, and multi-modality imaging of the delivery of specific siRNA in tumors.

Molecular and functional imaging of breast cancer.

Despite several major advances in breast cancer diagnosis and treatment, the American Cancer Society has estimated that in the US alone 43300 women and 400 men will die from breast cancer in 2007. Breast cancer typically is a multi-focal, multi-faceted disease, with the major cause of mortality being complications due to metastasis. Whereas a decade ago genetic alterations were the primary focus in cancer research, it is now apparent that the physiological tumor microenvironment, interactions between cancer cells and stromal cells such as endothelial cells, fibroblasts and macrophages, the extracellular matrix, and a multitude of secreted factors and cytokines influence progression, aggressiveness, and response of the disease to treatment. Prevention, early diagnosis, and treatment are the three broad challenges for MR molecular and functional imaging in reducing mortality from this disease. Multi-parametric molecular and functional MRI provides unprecedented opportunities for identifying novel targets for imaging and therapy at the bench, as well as for accurate diagnosis and monitoring response to therapy at the bedside. Here we provide an overview of the current status of molecular and functional MRI of breast cancer, outlining some key developments, as well as identifying some of the important challenges facing this field in the future.

In vivo selective measurement of (1-13C)-glucose metabolism in tumors by heteronuclear cross polarization.

Selective detection of (1-13C)-glucose and its glycolytic product, (3-13C)-lactate, was achieved by selective 13C NMR spectroscopy with 1H cross polarization. The total sensitivity of conventional broadband experiments was retained, and peak intensities were at least equivalent to those obtained with the inverse detection technique (i.e., 1H(13C)) for single proton resonances. A key advantage of the method is that it maintains the specific absorption rate (SAR) within FDA limits of 5 W/kg by reducing power deposition during decoupling. In this study we have monitored the kinetics of metabolism of 13C-labeled glucose to lactate following intravenous infusion of 0.55 ml of 0.18 M labeled glucose. Physiological effects were minimized by a) maintaining total plasma glucose concentrations below 20 mM throughout the course of NMR experiment and b) by avoiding significant heating of the tumor.

Characterization of a major permeability barrier in the zebrafish embryo.

Fish embryos represent a class of multicompartmental biological systems that have not been successfully cryopreserved, primarily because of the lack of understanding of how water and cryoprotectants permeate the compartments. We are using the zebrafish embryo as a model to understand these kinetics. Zebrafish embryos have two major compartments, the blastoderm and the yolk, which is surrounded by the multinucleated yolk syncytial layer (YSL). We determined the water and cryoprotectant permeability in these compartments using two methods. First, we measured shrink/swell dynamics in optical volumetric experiments. Zebrafish embryos shrank over time and did not re-expand while immersed in dimethyl sulfoxide (DMSO) or propylene glycol. Second, we measured DMSO uptake with diffusion-weighted nuclear magnetic resonance spectroscopy. DMSO uptake was rapid during the first few minutes, then gradual thereafter. We used one- and two-compartment models to analyze the data and to determine the permeability parameters. We found that the two-compartment model provided a better fit to the data. On the basis of this model and in the presence of DMSO, the yolk and blastoderm had very similar water permeabilities (i.e., 0.01 and 0. 005 micron x min-1atm-1, respectively), but they had different DMSO permeabilities separated by three orders of magnitude (i.e.,

Two-compartment model for determination of glycolytic rates of solid tumors by in vivo 13C NMR spectroscopy.

Carbon-13 NMR spectroscopy of 13C enriched substrates is useful for non-invasively determining metabolic fluxes of cells and tissues. Our study demonstrates that for RIF-1 tumor cells, examined under monolayer culture with continuous perfusion and also grown as solid subcutaneously (sc) implanted tumors in vivo, the levels of intracellular glucose and intermediates of the glycolytic pathway are below the level of detection by NMR spectroscopy. For these tumors, glucose transport into the cell is the most probable rate limiting step of the glycolytic pathway. Under these limiting conditions a simple two-compartment model of glycolysis applies. This model yields two parameters: the average rate of glycolysis and the rate of lactate clearance through the vasculature. For the RIF-1 tumor these parameters were 0.022 +/- 0.01 and 0.034 +/- 0.006 min(-1), respectively.

Combined vascular and extracellular pH imaging of solid tumors.

The unique physiological environment of solid tumors, frequently characterized by areas of poor flow, hypoxia, high lactate and low extracellular pH (pHe), influences vascularization, invasion and metastasis. Thus, vascularization and the physiological and metabolic environment play permissive (and conversely preventive) roles in invasion and metastasis. By using a multi-parametric approach of combined vascular and spectroscopic imaging, we can begin to evaluate which combinations of vascular, metabolic and physiological regions in a solid tumor represent the highest 'metastatic threat'. Here, we present measurements of pHe, vascular volume and permeability from co-localized regions within a solid tumor. These studies were performed for a group of metastatic (MDA-MB-231) and non-metastatic (MCF-7) human breast cancer xenografts. In this study, we have demonstrated the feasibility of such an approach, and presented methods of analyses to detect differences in patterns of combined parameters obtained from spatially co-registered regions in a solid tumor.

Real-time measurements of cellular oxygen consumption, pH, and energy metabolism using nuclear magnetic resonance spectroscopy.

Changes in molecular expression or apoptotic behavior, induced by malignant transformation or anticancer treatment, are frequently reflected in cellular metabolism and oxygen consumption. A technique to monitor oxygen consumption, cell physiology, and metabolism noninvasively would provide a better understanding of interactions between molecular changes and metabolism in malignant transformation and following cancer treatment. Such a system was developed in this study by adapting multinuclear MRI and spectroscopic techniques to an isolated cell perfusion system. The system was evaluated by studying the effects of two agents, carbonyl cyanide m-chlorophenylhydrazone (CCCP) which is an uncoupler of oxidative phosphorylation, and antimycin, an inhibitor of oxidative phosphorylation, on the oxygen consumption and metabolism of MCF-7 and MatLyLu cancer cell lines.

Dynamics of prostate cancer cell invasion studied in vitro by NMR microscopy.

Understanding the dynamics and pathogenesis of invasion is vital for developing strategies to prevent cancer metastasis. Conventional invasion assays provide information for a single time point. NMR microscopic imaging as used in the current study to measure cell invasion in vitro provides a nondestructive method for scoring cell invasion thus offering a unique possibility to study this process dynamically. An additional advantage is that cells can be retrieved for metabolic and physiological characterization. Two prostate cancer cell lines, DU-145 and Mat-Ly-Lu, preselected for differences in invasive behavior, were studied. Cells were seeded in 12-mm culture plate inserts containing a 15-microm-thick porous membrane with 3.0 microm pore size that was coated with a 100 microm Matrigel layer. Cell invasion in the Matrigel layer was obtained from the profile of intracellular water measured with diffusion-weighted 1D imaging. Additional experiments were also performed with confocal microscopy to validate the NMR results. Significant differences were detected between the invasive behavior of DU-145 and Mat-Ly-Lu cells. The obtained results show that NMR microscopy can be used to dynamically study invasion by cancer cells. The noninvasive nature of NMR microscopy permits determination of cell migration dynamically for any given sample, which is especially important if cell availability is limited to the unique sample, such as for biopsy specimens. Magn Reson Med 42:277-282, 1999.

Intracellular volume and apparent diffusion constants of perfused cancer cell cultures, as measured by NMR.

Diffusion NMR spectroscopy was used to study intracellular volume and apparent water diffusion constants in different cell lines (DU145, human prostate cancer; AT3, rat prostate cancer; MCF-7, human breast cancer; RIF-1, mouse fibrosacroma). The cells were grown on various matrices (collagen sponge, collagen beads, polystyrene beads) which enabled continuous growth in perfused high density cell culture suitable for NMR studies. In perfused cell systems, the attenuation of the water signal versus the squared gradient strength was fitted by the sum of two decaying exponentials. For the slowly decaying component the apparent water diffusion constant at 37 degrees C was 0.22 (+/-0.02) x 10(-9) s/m2 for all cell lines at diffusion times > 100 ms. It continuously increased up to 0.47 (+/-0.05) x 10(-9) s/m2 when the diffusion time was decreased to 8 ms, indicating restricted diffusion. No significant effect of the matrices was observed. The fractional volume of the slow component as determined from the biexponential diffusion curve correlated with the relative intracellular volume, as obtained from the cell density in the sample and the cell size as measured by light microscopy. Therefore, this simple NMR approach can be used to determine intracellular volume in perfused cell cultures suitable for NMR studies. Using this information in combination with spectroscopic data, changes in intracellular metabolite concentration can be detected even when the cellular volume is changing during the experiment. The apparent diffusion constant for the fast diffusing component varied with growth matrix, cell density and cell type and also showed the typical characteristics of restricted diffusion (increase of apparent diffusion constant with time).

Broadband proton decoupling for in vivo brain spectroscopy in humans.

A new decoupling sequence, PBAR, is described for broadband heteronuclear decoupling in vivo in humans at 1.5T. The sequence uses non-adiabatic, frequency- and amplitude-modulated inversion pulses designed to minimize decoupling sidebands at low applied gammaB(2) RF field levels and to cover only the narrow range of resonance offsets encountered in practice. The offset dependence of the decoupling efficiency of PBAR is demonstrated and compared to the conventional WALTZ-4 sequence. At the same average power levels, PBAR had slightly reduced bandwidth but significantly less intense decoupling sidebands. Applications of PBAR are shown in vivo in the human brain both for (31)P and natural abundance (13)C spectroscopy using volume decoupling coils. The PBAR sequence allows whole brain [(1)H]-[13]C decoupling to be performed at 1.5T with a standard head coil within FDA guidelines for RF power deposition. Magn Reson Med 45:226-232, 2001.