A triple suicide gene strategy that improves therapeutic effects and incorporates multimodality molecular imaging for monitoring gene functions.

Gene-directed enzyme prodrug therapy (GDEPT), or suicide gene therapy, has shown promise in clinical trials. In this preclinical study using stable cell lines and xenograft tumor models, we show that a triple-suicide-gene GDEPT approach produce enhanced therapeutic efficacy over previous methods. Importantly, all the three genes (thymidine kinase, cytosine deaminase and uracil phosphoribosyltransferase) function simultaneously as effectors for GDEPT and markers for multimodality molecular imaging (MMI), using positron emission tomography, magnetic resonance spectroscopy and optical (fluorescent and bioluminescent) techniques. It was demonstrated that MMI can evaluate the distribution and function/activity of the triple suicide gene. The concomitant expression of these genes significantly enhances prodrug cytotoxicity and radiosensitivity in vitro and in vivo.

Gaussian mixture model-based classification of dynamic contrast enhanced MRI data for identifying diverse tumor microenvironments: preliminary results.

Tumor hypoxia develops heterogeneously, affects radiation sensitivity and the development of metastases. Prognostic information derived from the in vivo characterization of the spatial distribution of hypoxic areas in solid tumors can be of value for radiation therapy planning and for monitoring the early treatment response. Tumor hypoxia is caused by an imbalance between the supply and consumption of oxygen. The tumor oxygen supply is inherently linked to its vasculature and perfusion which can be evaluated by dynamic contrast enhanced (DCE-) MRI using the contrast agent Gd-DTPA. Thus, we hypothesize that DCE-MRI data may provide surrogate information regarding tumor hypoxia. In this study, DCE-MRI data from a rat prostate tumor model were analysed with a Gaussian mixture model (GMM)-based classification to identify perfused, hypoxic and necrotic areas for a total of ten tumor slices from six rats, of which one slice was used as training data for GMM classifications. The results of pattern recognition analyzes were validated by comparison to corresponding Akep maps defining the perfused area (0.84 ± 0.09 overlap), hematoxylin and eosin (H&E)-stained tissue sections defining necrosis (0.64 ± 0.15 overlap) and pimonidazole-stained sections defining hypoxia (0.72 ± 0.17 overlap), respectively. Our preliminary data indicate the feasibility of a GMM-based classification to identify tumor hypoxia, necrosis and perfusion/permeability from non-invasively acquired, in vivo DCE-MRI data alone, possibly obviating the need for invasive procedures, such as biopsies, or exposure to radioactivity, such as positron emission tomography (PET) exams.

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.

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.

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.

Detection of increased choline compounds with proton nuclear magnetic resonance spectroscopy subsequent to malignant transformation of human prostatic epithelial cells.

In this study, a panel of normal human prostate cells (HPCs) and tumor cells derived from metastases were studied by (1)H NMR spectroscopy to determine whether the malignant transformation of HPCs results in the elevation of choline compounds. Although an elevated choline signal has been observed previously in clinical studies, the contribution of the different Cho compounds to this elevation, as well as their quantification, has not been established until now. Here we have shown that HPCs derived from metastases exhibit significantly higher phosphocholine as well as glycerophosphocholine levels compared with normal prostate epithelial and stromal cells. Thus the elevation of the choline peak observed clinically in prostate cancer is attributable to an alteration of phospholipid metabolism and not simply to increased cell density, doubling time, or other nonspecific effects. Androgen deprivation of the androgen receptor-positive cell lines resulted in a significant increase of choline compounds after chronic androgen deprivation of the LNCaP cell line and in a decrease of choline compounds after a more acute androgen deprivation of the LAPC-4 cell line. These data strongly support the use of proton magnetic resonance spectroscopic imaging to detect the presence of prostate cancer for diagnosis, to detect response subsequent to androgen ablation therapy, and to detect recurrence.

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.