Biomedical subjects
Publications and source records attributed to Dmitri Artemov.
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.
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.
Among the primary reasons for failure of anticancer chemotherapy are insufficient drug delivery to the tumor because of inadequate tumor vascularization and/or the antivascular effects of chemotherapy. Thus, determining the spatial intratumoral distribution of anticancer agents by noninvasive methods such as MRI/MRSI is important for monitoring cancer chemotherapy. We therefore studied the distribution of the 13C-labeled anticancer agent temozolomide ([13C]TMZ) in MCF-7 tumor-bearing mice using 1H/13C MRSI. In phantom studies inverse 13C detection with heteronuclear multiple quantum coherence (HMQC) provided a 2.3-fold gain in signal-to-noise ratio (SNR) over direct nuclear overhauser effect (NOE)-enhanced 13C-MRS. This enabled detection of [13C]TMZ in the micromolar range. Three-dimensional (3D) maps of drug distribution with a nominal 2.5-mm isotropic resolution were obtained following intraperitoneal administration of [13C]TMZ, for a total dose of 200 mg/kg. The status of the blood supply of tumors was assessed by gadolinium (Gd)-enhanced dynamic MRI. Nonuniform distributions of the drug and the contrast agent were detected in the tumors. Although carbon-13 MRSI has an inherently low sensitivity for detection, the novel technique described here demonstrates the feasibility of studying the delivery of 13C-labeled drugs and contrast uptake during the course of chemotherapy.
Aggressive cancer phenotypes are a manifestation of many different genetic alterations that promote rapid proliferation and metastasis. In this study, we show that stable overexpression of Twist in a breast cancer cell line, MCF-7, altered its morphology to a fibroblastic-like phenotype, which exhibited protein markers representative of a mesenchymal transformation. In addition, it was observed that MCF-7/Twist cells had increased vascular endothelial growth factor (VEGF) synthesis when compared with empty vector control cells. The functional changes induced by VEGF in vivo were analyzed by functional magnetic resonance imaging (MRI) of MCF-7/Twist-xenografted tumors. MRI showed that MCF-7/Twist tumors exhibited higher vascular volume and vascular permeability in vivo than the MCF-7/vector control xenografts. Moreover, elevated expression of Twist in breast tumor samples obtained from patients correlated strongly with high-grade invasive carcinomas and with chromosome instability, particularly gains of chromosomes 1 and 7. Taken together, these results show that Twist overexpression in breast cancer cells can induce angiogenesis, correlates with chromosomal instability, and promotes an epithelial-mesenchymal-like transition that is pivotal for the transformation into an aggressive breast cancer phenotype.
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.
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.
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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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.
Magnetic resonance imaging (MRI) produces high-resolution three-dimensional maps delineating morphological features of the specimen. Differential contrast in soft tissues depends on endogenous differences in water content, relaxation times, and/or diffusion characteristics of the tissue of interest. The specificity of MRI can be further increased by exogenous contrast agents (CA) such as gadolinium chelates, which have been successfully used for imaging of hemodynamic parameters including blood perfusion and vascular permeability. Development of targeted MR CA directed to specific molecular entities could dramatically expand the range of MR applications by combining the noninvasiveness and high spatial resolution of MRI with specific localization of molecular targets. However, due to the intrinsically low sensitivity of MRI (in comparison with nuclear imaging), high local concentrations of the CA at the target site are required to generate detectable MR contrast. To meet these requirements, the MR targeted CA should recognize targeted cells with high affinity and specificity. They should also be characterized by high relaxivity, which for a wide variety of CA depends on the number of contrast-generating groups per single molecule of the agent. We will review different designs and applications of targeted MR CA and will discuss feasibility of these approaches for in vivo MRI.
The HER-2/neu receptor is a member of the epidermal growth factor family and is amplified in multiple cancers. It is under intense investigation both as a prognostic marker and for therapy, using monoclonal antibodies targeted against the receptor. We have developed a novel two-component gadolinium-based MR contrast agent to image the HER-2/neu receptor. Positive T1 contrast in MR images was generated by the specific binding of avidin-gadolinium complexes to tumor cells prelabeled with a biotinylated anti-HER-2/neu antibody. Significant intensity enhancement was observed in HER-2/neu-expressing cell lines and in vivo in a breast cancer model. Potential applications of this approach may include determination of the HER-2/neu status for prognosis and for selecting tumors for monoclonal antibody therapy.
MR molecular imaging is an exciting new frontier in the biomedical applications of MR. One of the clinically relevant targets is the tyrosine kinase Her-2/neu receptor, which has a significant role in staging and treating breast cancer. In this study Her-2/neu receptors were imaged in a panel of breast cancer cells expressing different numbers of the receptors on the cell membrane. Commercially available streptavidin-conjugated superparamagnetic nanoparticles were used as targeted MR contrast agent. The nanoparticles were directed to receptors prelabeled with a biotinylated monoclonal antibody and generated strong T(2) MR contrast in Her-2/neu-expressing cells. The contrast observed in MR images was proportional to the expression level of Her-2/neu receptors determined independently with FACS analysis. In these experiments, iron oxide nanoparticles were attached to the cell surface and were not internalized into the cells, which is a major advantage for in vivo applications of the method.
Magnetic resonance studies have previously shown that solid tumors and cancer cells in culture typically exhibit high phosphocholine and total choline. Treatment of cancer cells with the anti-inflammatory agent, indomethacin (INDO), reverted the phenotype of choline phospholipid metabolites in cancer cells towards a less malignant phenotype. Since endothelial cells form a key component of tumor vasculature, in this study, we used MR spectroscopy to characterize the phenotype of choline phospholipid metabolites in human umbilical vein endothelial cells (HUVECs). We determined the effect of growth factors, the anti-inflammatory agent INDO, and conditioned media obtained from a malignant cell line, on choline phospholipid metabolites. Growth factor depletion or treatment with INDO induced similar changes in the choline phospholipid metabolites of HUVECs. Treatment with conditioned medium obtained from MDA-MB-231 cancer cells induced changes similar to the presence of growth factor supplements. These results suggest that cancer cells secrete growth factors and/or other molecules that influence the choline phospholipid metabolism of HUVECs. The ability of INDO to alter choline phospholipid metabolism in the presence of growth factor supplements suggests that the inflammatory response pathways of HUVECs may play a role in cancer cell-HUVEC interaction and in the response of HUVECs to growth factors.
PURPOSE: The availability of noninvasive techniques to detect the effects of antiangiogenic agents is critically important for optimizing treatment of cancer with these agents. Magnetic resonance imaging (MRI) is one such noninvasive technique that is routinely used clinically. EXPERIMENTAL DESIGN: In this study, we have evaluated the use of MRI of the intravascular contrast agent albumin-GdDTPA to detect the effects of the antiangiogenic agent TNP-470 on the vascular volume and permeability of the MatLyLu prostate cancer model. RESULTS: TNP-470-treated tumors demonstrated a significant decrease of vascular volume, as well as a significant reduction in vascular and permeable regions, compared with volume-matched control tumors. Although the fractional volume of permeable regions in the tumor decreased, the average value of tumor permeability did not decrease significantly. This was attributable to increase in permeability in some regions of the tumor. These regions were mostly associated with low vascular volume. ELISA assays of control and treated MatLyLu tumors also detected a significant increase of vascular endothelial growth factor in the TNP-470-treated tumors. CONCLUSION: MRI detected significant changes in tumor vascular characteristics after treatment with TNP-470.
Choline metabolites in malignant human mammary epithelial cells (HMECs) are significantly altered compared to normal HMECs. (1)H NMR studies of cell extracts have shown that treatment of malignant HMECs with a nonsteroidal anti-inflammatory agent, indomethacin, results in a distribution of choline compounds more typical of nonmalignant HMECs. To follow the time course of these changes, in this study real-time monitoring of choline compounds of malignant MDA-MB-231 cells was performed during treatment with indomethacin. The contribution of changes in intra- and extracellular pH to changes in choline compounds was also examined. Changes in water-soluble choline phospholipid metabolites, such as phosphocholine (PC), glycerophosphocholine (GPC), and total choline, as well as intracellular pH, were monitored by (31)P and diffusion-weighted (1)H NMR spectroscopy of living cells using an NMR-compatible perfusion system. An accumulation of GPC and a decrease of PC, resulting in an increased [GPC]/[PC] ratio, were detected within 2 hr of treatment with 200 microM indomethacin. Since a decreased [GPC]/[PC] ratio is associated with increased malignancy, these data demonstrate that nonspecific cyclooxygenase inhibition by indomethacin alters the choline metabolite profile of malignant cells towards a less malignant phenotype. These changes were not related to alterations of intra- or extracellular pH.
We previously observed that changes in choline phospholipids of two malignant human mammary epithelial cells (HMECs) following treatment with a high dose of the cyclooxygenase (COX) inhibitor, indomethacin, mimicked changes following transfection with a metastasis suppressor gene, nm23. The similarity between response to indomethacin and nm23 transfection led us to 1) expand our (1)H NMR spectroscopy study of indomethacin treatment by determining the response at two doses for two nonmalignant and three malignant HMECs, 2) investigate COX-1 and COX-2 levels in HMECs and their relationship with choline phosholipid metabolites, and 3) determine changes in Nm23 expression following treatment with indomethacin. All HMECs exhibited a significant change in choline phospholipids following treatment with 300 microM indomethacin. At the lower dose of 50 microM, only nonmalignant HMECs and the estrogen-dependent malignant cell line, MCF-7, responded. COX-1 levels were significantly higher in malignant HMECs than in nonmalignant HMECs. A significant increase in Nm23 expression following 300 microM indomethacin was detected in MCF-12A and MCF-7 cells but not in MDA-MB-231 and MDA-MB-435 cells. These results suggest that COX-1 expression and its inhibition play a role in the choline phospholipid metabolism of HMECs, and the effect of indomethacin on HMECs may be mediated, in part, through upregulation of nm23.