Biomedical subjects
Publications and source records attributed to Zaver M Bhujwalla.
The conjugate of bacterial cytosine deaminase (bCD) and poly-l-lysine (PLL) that was functionalized with biotin, rhodamine, and Gd3+-DOTA was synthesized and characterized. It demonstrated high relaxivity, improved enzymatic specificity to prodrug 5-fluorocytosine, low cytotoxicity, efficient cell uptake, and high enzymatic stability in fresh mouse serum and human breast cancer cell culture.
The integration of imaging technologies with the capabilities of genetic engineering has created novel opportunities for understanding and imaging cancer. Here, we have combined vascular magnetic resonance imaging (MRI) and optical imaging to understand the relationship between hypoxia and vascularization in a human prostate cancer model engineered to express enhanced green fluorescent protein (EGFP) under hypoxia. Characterization and validation of EGFP expression under hypoxic conditions was done in culture and in solid tumors in vivo. MRI measurements showed that vascular volume was significantly lower in fluorescing regions. These regions also frequently exhibited high permeability. These data were further supported by the detection of low vessel density in EGFP-positive regions, as determined by the distribution of intravascularly administered, fluorescence-labeled Lycopersicon esculentum lectin in frozen tumor sections. These observations are consistent with the possibility that regions of low vascular volumes are hypoxic, which induces increased expression of functionally active vascular endothelial growth factor, a potent vascular permeability factor.
[structure: see text] Two novel near-infrared (NIR) fluorescent probes have been synthesized by linking a carbocyanine fluorophore and glucosamine through different linkers. These probes demonstrated a high quantum yield, low cytotoxicity, reversible pH-dependent fluorescence in the physiological pH range, and a decreased aggregation tendency in aqueous solutions. In vitro NIR optical imaging studies revealed cellular uptake and strong intracellular NIR fluorescence of these two probes in four breast epithelial cell lines.
Interactions between the tumor stromal compartment and cancer cells play an important role in the spread of cancer. In this study, we have used noninvasive in vivo magnetic resonance imaging (MRI) of two human breast cancer models with significantly different invasiveness, to quantify and understand the role of interstitial fluid transport, lymphatic-convective drain, and vascularization in the regional spread of breast cancer to the axillary lymph nodes. Quantitative fluorescence microscopy was done to morphometrically characterize lymphatic vessels in these tumors. Significant differences in vascular and extravascular transport variables as well as in lymphatic vessel morphology were detected between the two breast cancer models, which also exhibited significant differences in lymph node and lung metastasis. These data are consistent with a role of lymphatic drain in lymph node metastasis and suggest that increased lymph node metastasis may occur due to a combination of increased invasiveness, and reduced extracellular matrix integrity allowing increased pathways of least resistance for the transport of extravascular fluid, as well as tumor cells. It is also possible that lymph node metastasis occurred via the cancer cell-bearing tumoral lymphatic vessels. The congestion of these tumoral lymphatics with cancer cells may have restricted the entry and transport of macromolecules.
Magnetic resonance studies from the last 10 years have conclusively demonstrated that choline metabolism is altered in a wide variety of cancers. In cancer, the choline metabolite profile is characterized by an elevation of phosphocholine and total choline-containing compounds. This elevation is increasingly being used as an endogenous biomarker of cancer. Importantly, the enzymes and pathways resulting in these distinct alterations in phosphocholine and total choline may provide novel molecular targets for specific, targeted anticancer therapies. In this article, we have summarized some of the magnetic resonance spectroscopy and positron emission tomography techniques that are currently available, or will be in the near future, for choline metabolism-based diagnosis, staging and therapy assessment in cancer patients. This review also outlines currently known molecular alterations that cause the aberrant choline metabolite profile in cancers and concludes with a summary of recent research findings that may, in the future, lead to novel anticancer therapies targeting choline metabolism.
We have developed a noninvasive magnetic resonance imaging (MRI) assay to characterize human umbilical vein endothelial cell (HUVEC) motility, invasion, and network formation in response to the presence of cancer cells. HUVECs were labeled with a superparamagnetic iron oxide T(2) contrast agent and cocultured with MDA-MB-231 breast cancer cells in the presence of an extracellular matrix (ECM) gel. Invasion into the ECM gel by HUVECs in response to paracrine factors secreted by MDA-MB-231 cancer cells, as well as network formation by HUVECs, was easily tracked with MRI. The invasive behavior of HUVECs was not observed in the absence of cancer cells. This noninvasive assay used to characterize the response of endothelial cells (ECs) can be used to understand the role of proangiogenic or antiangiogenic stimuli, and to study the interactions between ECs and other disease-specific cells in pathologies with aberrant angiogenesis, such as retinopathy and arthritis.
Human mammary epithelial cells (HMECs) exhibit an increase in phosphocholine (PC) and total choline-containing compounds, as well as a switch from high glycerophosphocholine (GPC)/low PC to low GPC/high PC, with progression to malignant phenotype. The treatment of human breast cancer cells with a nonsteroidal anti-inflammatory agent, indomethacin, reverted the high PC/low GPC pattern to a low PC/high GPC pattern indicative of a less malignant phenotype, supported by decreased invasion. Here, we have characterized mechanisms underlying indomethacin-induced alterations in choline membrane metabolism in malignant breast cancer cells and nonmalignant HMECs labeled with [1,2-13C]choline using 1H and 13C magnetic resonance spectroscopy. Microarray gene expression analysis was performed to understand the molecular mechanisms underlying these changes. In breast cancer cells, indomethacin treatment activated phospholipases that, combined with an increased choline phospholipid biosynthesis, led to increased GPC and decreased PC levels. However, in nonmalignant HMECs, activation of the anabolic pathway alone was detected following indomethacin treatment. Following indomethacin treatment in breast cancer cells, several candidate genes, such as interleukin 8, NGFB, CSF2, RHOB, EDN1, and JUNB, were differentially expressed, which may have contributed to changes in choline metabolism through secondary effects or signaling cascades leading to changes in enzyme activity.
Choline kinase is overexpressed in breast cancer cells and activated by oncogenes and mitogenic signals, making it a potential target for cancer therapy. Here, we have examined, for the first time, the effects of RNA interference (RNAi)-mediated down-regulation of choline kinase in nonmalignant and malignant human breast epithelial cell lines using magnetic resonance spectroscopy (MRS) as well as molecular analyses of proliferation and differentiation markers. RNAi knockdown of choline kinase reduced proliferation, as detected by proliferating cell nuclear antigen and Ki-67 expression, and promoted differentiation, as detected by cytosolic lipid droplet formation and expression of galectin-3. The functional importance of RNAi-mediated choline kinase down-regulation on choline phospholipid metabolism was confirmed by the significant reduction of phosphocholine detected by MRS. These results strongly support the targeting of choline kinase in breast cancer cells with RNAi and show the potential ability of noninvasive MRS to detect and evaluate future treatments incorporating such strategies.
Noninvasive imaging techniques to image and characterize delivery and transport of macromolecules through the extracellular matrix (ECM) and supporting stroma of a tumor are necessary to develop treatments that alter the porosity and integrity of the ECM for improved delivery of therapeutic agents and to understand factors which influence and control delivery, movement, and clearance of macromolecules. In this study, a noninvasive imaging technique was developed to characterize the delivery as well as interstitial transport of a macromolecular agent, albumin-GdDTPA, in the MCF-7 human breast cancer model in vivo, using magnetic resonance imaging. The transport parameters derived included vascular volume, permeability surface area product, macromolecular fluid exudate volume, and drainage and pooling rates. Immunohistochemical staining for the lymphatic endothelial marker LYVE-1 was done to determine the contribution of lymphatics to the macromolecular drainage. Distinct pooling and draining regions were detected in the tumors using magnetic resonance imaging. A few lymphatic vessels positively stained for LYVE-1 were also detected although these were primarily collapsed and tenuous suggesting that lymphatic drainage played a minimal role, and that the bulk of drainage was due to convective transport through the ECM in this tumor model.
PURPOSE: To investigate the feasibility of combined dynamic contrast enhanced (DCE) and magnetic resonance spectroscopy (MRS) in evaluating breast lesions. METHODS: Nine patients with positive mammograms scheduled for either biopsy or mastectomy were examined on a 1.5-T MR scanner. DCE was performed with administration of gadolinium-DTPA contrast using a two-dimensional spoiled gradient recall echo (SPGR) sequence. Proton spectroscopy (TR/TE = 2000/272 msec) was performed using PRESS single slice (10 mm). Lesion time intensity curves were classified as persistent (type 1), plateau (type 2), or washout (type 3) pattern enhancement. Choline (Cho) signal-to-noise ratios (SNRs) and enhancement patterns were compared between benign and malignant lesions as determined by histopathology. RESULTS: Five patients had breast carcinoma and four had benign lesions. Type 1 enhancement was found in two benign cases, type 2 enhancement in two of four benign and four of five malignant lesions, and one malignant case exhibited a type 3 pattern. Choline SNR was significantly different (P < 0.003) between benign and malignant lesions (2.0 +/- 0.3 vs. 5.7 +/- 1.4; P < 0.003). Choline SNR was less than 4.0 in all of the benign lesions, including the two lesions with type 2 enhancement. CONCLUSION: Proton MRS appears to be a promising technique for classification of breast lesions when DCE results are equivocal. A combination of DCE and MRS is feasible, and may have improved specificity compared to either modality alone.
This paper describes the construction of three dimensional (3D) encapsulation devices in large numbers, using a novel self-assembling strategy characterized by high mechanical stability, controlled porosity, extreme miniaturization, high reproducibility and the possibility of integrating sensing and actuating electromechanical modules. We demonstrated encapsulation of microbeads and cells within the containers, thereby demonstrating one possible application in cell encapsulation therapy. Magnetic resonance (MR) images of the containers in fluidic media suggest radio frequency (RF) shielding and a susceptibility effect, providing characteristic hypointensity within the container, thereby allowing the containers to be easily detected. This demonstration is the first step toward the design of 3D, micropatterned, non-invasively trackable, encapsulation devices.
Proton magnetic resonance spectroscopy ((1)H MRS) consistently detects significant differences in choline phospholipid metabolites of malignant versus benign breast lesions. It is critically important to understand the molecular causes underlying these metabolic differences, because this may identify novel targets for attack in cancer cells. In this study, differences in choline membrane metabolism were characterized in breast cancer cells and normal human mammary epithelial cells (HMECs) labeled with [1,2-(13)C]choline, using (1)H and (13)C magnetic resonance spectroscopy. Metabolic fluxes between membrane and water-soluble pool of choline-containing metabolites were assessed by exposing cells to [1,2-(13)C]choline for long and short periods of time to distinguish between catabolic and anabolic pathways in choline metabolism. Gene expression analysis using microarrays was performed to understand the molecular mechanisms underlying these changes. Breast cancer cells exhibited increased phosphocholine (PC; P < 0.001), total choline-containing metabolites (P < 0.01), and significantly decreased glycerophosphocholine (P < 0.05) compared with normal HMECs. Decreased (13)C-enrichment was detected in choline (P < 0.001) and phosphocholine (P < 0.05, P < 0.001) of breast cancer cells compared with HMECs, indicating a higher metabolic flux from membrane phosphatidylcholine to choline and phosphocholine in breast cancer cells. Choline kinase and phospholipase C were significantly overexpressed, and lysophospholipase 1, phospholipase A2, and phospholipase D were significantly underexpressed, in breast cancer cells compared with HMECs. The magnetic resonance spectroscopy data indicated that elevated phosphocholine in breast cancer cells was primarily attributable to increased choline kinase activity and increased catabolism mediated by increased phospholipase C activity. These observations were consistent with the overexpression of choline kinase and phospholipase C detected in the microarray analyses.
A method for measuring contrast agent concentration in blood was developed using a switchable RF tail coil to obtain MR signal from the mouse tail vein. The switchable RF coil was used to obtain quantitative high-resolution T1 maps of the tail in combination with a second coil used to obtain images from regions of interest in a mouse tumor model. The effect of flow rates on the T1 measurements was validated using a flow-phantom. Reliable estimates of blood T1 were obtained for flow rates up to 0.5 cm/sec in SCID mice. There was no significant difference between the in vivo tail coil measurements and in vitro blood sample relaxation rates of blood. The ability to measure the concentration of contrast agents in mouse blood in vivo is useful for several applications, such as obtaining quantitative values of tumor vascular volume and permeability, determining the effectiveness of drug delivery to tumors, or for detecting lymphatic drain.
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The recent surge of interest in the lymphatic system can be attributed to two factors: the discovery of cytokines that induce the growth of new lymphatic capillaries and the identification of lymphatic endothelial-specific markers. In contrast to the above, there is a paucity of techniques for studying lymphatic function in vivo. This article reviews imaging and other techniques that allow the assessment of lymphatic function, particularly in the tumor microenvironment, and proposes novel solutions for probing the same in vivo.
Mutations in the p53 gene are the most frequently observed genetic lesions in human cancers. Human cancers that contain a p53 mutation are more aggressive, more apt to metastasize, and more often fatal. p53 controls numerous downstream targets that can influence various outcomes such as apoptosis, growth arrest, and DNA repair. Based on previous observations using (1)H magnetic resonance spectroscopy (MRS), we have identified choline phospholipid metabolite intensities typical of increased malignancy. Here we have used (1)H MRS to characterize the choline phospholipid metabolite levels of p53(+/ +) and p53(-/-) cells, and demonstrated that loss of p53 function results in increased phosphocholine and total choline. These data suggest that the increased malignancy of cancer cells resulting from loss of p53 may be mediated, in part, through the choline phospholipid pathway.
The exploration of novel imaging methods that have the potential to improve specificity for the identification of malignancy is still critically needed in breast imaging. Changes in physiologic alterations of soft tissue water associated with breast cancer can be visualized by magnetic resonance (MR) imaging. However, it is unlikely that a single MR parameter can characterize the complexity of breast tissue. Techniques such as multiparametric MR imaging, proton magnetic resonance spectroscopic (MRSI) imaging, and 23Na sodium MR imaging when used in combination provide a comprehensive data set with potentially more power to diagnose breast disease than any single measure alone. A combination of MR, MRSI, and 23Na sodium MR parameters may be examined in a single MR imaging examination, potentially resulting in improved specificity for radiologic evaluation of malignancy.
Over the past decade MR (magnetic resonance) imaging has emerged as one of the major modalities for noninvasive functional imaging. Recent advances in the development of targeted MR contrast agents have added significantly to the capabilities of MR imaging. In particular, the use of targeted contrast agents to report on the expression of cell surface receptors, combined with the functional capabilities of MR imaging, together provide unique opportunities to understand receptor-mediated pathways. In this article we have reviewed current MRI strategies used to visualize receptor expression, the potential advantages and drawbacks of these strategies, and novel areas of focus for the future.