Arvind P. Pathak

Twist Overexpression Induces In vivo Angiogenesis and Correlates with Chromosomal Instability in Breast Cancer

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.

Utility of simultaneously acquired gradient-echo and spin-echo cerebral blood volume and morphology maps in brain tumor patients

An interleaved gradient‐echo (GE) / spin‐echo (SE) EPI sequence was used to acquire images during the first pass of a susceptibility contrast agent, in patients with brain tumors. Maps of 1) GE (total) rCBV (relative cerebral blood volume), 2) SE (microvascular) rCBV, both corrected for T1 leakage effects, and 3) (ΔR2*/ΔR2), a potential marker of averaged vessel diameter, were determined. Both GE rCBV and ΔR2*/ΔR2 correlated strongly with tumor grade (P = 0.01, P = 0.01, n = 15), while SE rCBV did not (P = 0.24, n = 15). When the GE rCBV data were not corrected for leakage effects, the correlation with tumor grade was no longer significant (P = 0.09, n = 15). These findings suggest that MRI measurements of total blood volume fraction (corrected for agent extravasation) and ΔR2*/ΔR2, as opposed to maps of microvascular volume, may prove to be the most appropriate markers for the evaluation of tumor angiogenesis (the induction of new blood vessels) and antiangiogenic therapies. Magn Reson Med 43:845–853, 2000.

MR-derived cerebral blood volume maps: Issues regarding histological validation and assessment of tumor angiogenesis

In an effort to develop MRI methods for the evaluation of tumor angiogenesis (new blood vessel formation), MRI‐derived cerebral blood volume (CBV) information has been compared to histologic measures of microvessel density (MVD). Although MVD is a standard marker of angiogenesis, it is not a direct correlate of the volume measurements made with MRI, and therefore inappropriate for the development and validation of the MR techniques. Therefore, the goal of this study was to develop an approach by which MR measurements of CBV can be directly correlated. To this end, dynamic susceptibility contrast (DSC) MRI experiments were performed in six Fisher rats implanted with 9L gliosarcoma brain tumors. Subsequently, the circulation was perfused with a latex compound (Microfil®), after which 50‐μm tissue sections were analyzed for vessel count, diameter, and the fraction of area comprised of vessels. The results demonstrate that while fractional area (FA) does not provide a good measure of CBV, FA corrected for section thickness effects does. Whereas the FA in normal brain was found to be 13.03 ± 1.83% the corrected FA, or fractional volume (FV), was 1.89 ± 0.39%, a value in agreement with those reported in the literature for normal brain. Furthermore, while no significant difference was found between normal brain and tumor FA (P = 0.55), the difference was significant for FV (P = 0.036), as would be expected. And only with FV does a correlation with the MRI‐derived CBV become apparent (rS = 0.74). There was strong correlation (rs = 0.886) between the tumor / normal blood volume ratios as estimated by each technique, although the MR‐ratio (1.56 ± 0.29) underestimated the histologic‐ratio (2.35 ± 0.75). Thus, the correlation of MRI CBV methods requires a measurement of fractional vessel area and correction of this area for section thickness effects. This new independent correlative measure should enable efficient and accurate progress in the development of MRI methods to evaluate tumor angiogenesis. Magn Reson Med 46:735–747, 2001.

Molecular and functional imaging of cancer: Advances in MRI and MRS

Publisher Summary This chapter presents an overview of the endogenous and exogenous magnetic resonance (MR) contrast mechanisms utilized in characterizing tumor vasculature. Every contrast mechanism for probing the tumor vasculature, including the use of exogenous MR contrast agents, is in some way a result of the changes in the MR signal intensity brought about by changes in tissue relaxation times. Noninvasive multinuclear magnetic resonance imaging (MRI) and MR spectroscopic imaging (MRSI) provide a wealth of spatial and temporal information on tumor vasculature, metabolism, and physiology. The most commonly used MR contrast agents (CA) are paramagnetic gadolinium chelates. These agents are tightly bound complexes of the rare earth element gadolinium and various chelating agents. A simple linear compartment model, describing uptake of the contrast agent from plasma, postulates a negligible reflux of the contrast agent from the interstitium back to the blood compartment. Blood concentrations of the CA can be approximated to be constant for the duration of the MR experiment, and under these conditions, contrast uptake is a linear function of time. The MR detection of cellular targets is also elaborated.

A novel technique for modeling susceptibility-based contrast mechanisms for arbitrary microvascular geometries: The finite perturber method

Recently, we demonstrated that vessel geometry is a significant determinant of susceptibility-induced contrast in MRI. This is especially relevant for susceptibility-contrast enhanced MRI of tumors with their characteristically abnormal vessel morphology. In order to better understand the biophysics of this contrast mechanism, it is of interest to model how various factors, including microvessel morphology contribute to the measured MR signal, and was the primary motivation for developing a novel computer modeling approach called the Finite Perturber Method (FPM). The FPM circumvents the limitations of traditional fixed-geometry approaches, and enables us to study susceptibility-induced contrast arising from arbitrary microvascular morphologies in 3D, such as those typically observed with brain tumor angiogenesis. Here we describe this new modeling methodology and some of its applications. The excellent agreement of the FPM with theory and the extant susceptibility modeling data, coupled with its computational efficiency demonstrates its potential to transform our understanding of the factors that engender susceptibility contrast in MRI.

Characterizing vascular parameters in hypoxic regions: A combined magnetic resonance and optical imaging study of a human prostate cancer model

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.

Characterizing Extravascular Fluid Transport of Macromolecules in the Tumor Interstitium by Magnetic Resonance Imaging

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.

Antiangiogenic effects of dexamethasone in 9L gliosarcoma assessed by MRI cerebral blood volume maps.

Depending on dose, dexamethasone has been shown to inhibit or stimulate growth of rat 9L gliosarcoma and decrease the expression of vascular endothelial growth factor (VEGF), an important mediator of tumor-associated angiogenesis. We demonstrate, by constructing relative cerebral blood volume (rCBV) maps with MRI, that dexamethasone also decreases total blood volume while increasing microvascular blood volume in Fischer rats bearing intracranial 9L gliosarcoma. Animals were inoculated with 1 x 10(5) 9L gliosarcoma tumor cells. On days 10-14 after tumor cell inoculation, animals were intra-peritoneally injected with dexamethasone (3 mg/kg) over 5 days. MRI-derived gradient echo (GE) and spin-echo (SE) rCBV maps were created to demonstrate total vasculature (GE) and microvasculature (SE). After MRI studies were performed, the rats vasculature was perfused with a latex compound. Total vessel volume and diameters were assessed by microscopy. Dexamethasone decreased the tumor-enhancing area of postcontrast T1-weighted images (P < 0.0001) and total tumor volume(P = 0.0085). In addition, there was a greater than 50% decrease in GE rCBV (total vasculature) (P = 0.007) as well as a significant decrease in total fractional blood volume, as validated by histology (P = 0.0007). Conversely, there was an increase in SE rCBV signal (microvasculature) in animals treated with dexamethasone (P = 0.05), which was consistent with microscopy (P < 0.0001). These data demonstrate that (1) dexamethasone selectively treats tumor vasculature, suggesting a vessel-size selective effect and (2) MRI-derived rCBV is a noninvasive technique that can be used to evaluate changes in blood volume and vascular morphology.

The effect of brain tumor angiogenesis on the in vivo relationship between the gradient-echo relaxation rate change (ΔR2*) and contrast agent (MION) dose

To determine in vivo if the susceptibility calibration factor for gradient‐echo imaging (kG), which characterizes the relationship between the tissue T2* relaxation rate change (ΔR2*) and tissue contrast agent concentration, is independent of tissue type and condition; in addition, to assess the consequences of such an assumption on the use of relative cerebral blood volume (rCBV) measurements as a direct index of tumor angiogenesis.

Molecular Imaging of Metastatic Potential

If molecular imaging is to prove clinically useful it will have to surpass current, primarily anatomic techniques in terms of sensitivity and the ability to detect minimal changes in tissue. One of the most important tests for molecular imaging is to determine whether it can image the metastatic potential of tumors. Like all predictive endeavors, the imaging of such “potential” is a daunting task, but one that only molecular imaging—rather than standard, anatomic techniques—is likely to solve. Although difficult, imaging of metastatic potential is also arguably the most important task for molecular imaging of cancer because it is generally the dissemination of malignant tissue, not its prolonged residence in an inopportune site, which kills the patient. Below are examples of uses of molecular imaging of metastases as well as of metastatic potential, the former being a far more developed area of clinical inquiry.