Cornelia Matei

Metabolic Signatures Associated with a NAD Synthesis Inhibitor–Induced Tumor Apoptosis Identified by 1H-Decoupled-31P Magnetic Resonance Spectroscopy

Purpose: Attempts to selectively initiate tumor cell death through inducible apoptotic pathways are increasingly being exploited as a potential anticancer strategy. Inhibition of NAD+ synthesis by a novel agent FK866 has been recently reported to induce apoptosis in human leukemia, hepatocarcinoma cells in vitro, and various types of tumor xenografts in vivo. In the present study, we used 1H-decoupled phosphorus (31P) magnetic resonance spectroscopy (MRS) to examine the metabolic changes associated with FK866 induced tumor cell death in a mouse mammary carcinoma. Experimental Design: Induction of apoptosis in FK866-treated tumors was confirmed by histology and cytofluorometric analysis. FK866-induced changes in mammary carcinoma tumor metabolism in vivo were investigated using 1H-decoupled 31P MRS. To discern further the changes in metabolic profiles of tumors observed in vivo, high-resolution in vitro 1H-decoupled 31P MRS studies were carried out with perchloric acid extracts of mammary carcinoma tumors excised after similar treatments. In addition, the effects of FK866 on mammary carcinoma tumor growth and radiation sensitivity were studied. Results: Treatment with FK866 induced a tumor growth delay and enhanced radiation sensitivity in mammary carcinoma tumors that was associated with significant increases in the 31P MR signal in the phosphomonoester region and a decrease in NAD+ levels, pH, and bioenergetic status. The 31P MRS of perchloric acid extracts of treated tumors identified the large unresolved signal in the phosphomonoester region as the resultant of resonances originating from intermediates of tumor glycolysis and guanylate synthesis in addition to alterations in pyridine nucleotide pools and phospholipid metabolism. Conclusion: The present results suggest that FK866 interferes with multiple biochemical pathways that contribute to the increased cell death (apoptosis) and subsequent radiation sensitivity observed in the mammary carcinoma that could be serially monitored by 31P MRS.

In vivo multiple-mouse imaging at 1.5 T

A multiple‐mouse solenoidal MR coil was developed for in vivo imaging of up to 13 mice simultaneously to screen for tumors on a 1.5 T clinical scanner. For the coil to be effective as a screening tool, it should permit acquisition of MRIs in which orthotopic tumors with diameters >2 mm are detectable in a reasonable period of time (<1 hr magnet time) and their sizes accurately measured. Using a spin echo sequence, we demonstrated that this coil provides sufficient sensitivity for moderately high resolution images (156–176 μm in plane‐resolution, 1.5 mm slice thickness). This spatial resolution permitted detection of primary brain tumors in transgenic/knockout mice and orthotopic xenografts. Brain tumor size as measured by MRI was correlated with size measured by histopathology (P < 0.001). Metastatic tumors in the mouse lung were also successfully imaged in a screening setting. The multiple mouse coil is simple in construction and may be implemented without any significant modification to the hardware or software on a clinical scanner. Magn Reson Med 49:551–557, 2003.

Characterization of reactive versus tumor–bearing lymph nodes with interstitial magnetic resonance lymphography in an animal model

RATIONALE AND OBJECTIVESTo determine if magnetic resonance lymphography performed with subcutaneously administered AMI-227, a nanoparticulate iron oxide contrast agent, can distinguish reactive from tumor-bearing lymph nodes. MATERIALS AND METHODSMature male Copenhagen rats were inoculated with cell suspensions of R3327-MAT-LyLu rat prostate carcinoma (n = 21) or Freunds complete adjuvant (n = 15) in the left footpad to generate ipsilateral popliteal lymph node metastases or lymphadenitis. At 12 to 14 days after inoculation, T1- and T2-weighted magnetic resonance images of bilateral popliteal areas were obtained before and 24 hours after subcutaneous administration of AMI-227. Contrast-to-noise ratios were calculated in precontrast and postcontrast images. Bilateral popliteal nodes were excised for pathologic assessment. RESULTSAMI-227 resulted in decreased contrast-to-noise ratios in reactive (Tl − W = −7.01 ± 1.13, T2 − W = −31.64 ± 5.35) and normal (T1 − W = −13.56 ± 1.97, T2 − W = −21.62 ± 2.51) nodes. Contrast-to-noise ratios were unchanged (T1 − W = −0.22 ± 1.71, T2 − W = −2.20 ± 4.19) in tumor-containing nodes. These differences in contrast-to-noise ratio changes between tumor-bearing versus nontumor-bearing nodes were statistically significant (P < 0.05). Histologic analysis showed similar distribution of AMI-227 within normal and reactive nodes, but not in tumor-bearing nodes. CONCLUSIONSDifferences in AMI-227-uptake between tumor- and nontumor-bearing nodes detected with magnetic resonance imaging are helpful for distinguishing the two entities.

In vivo 19F magnetic resonance spectroscopy and chemical shift imaging of tri-fluoro-nitroimidazole as a potential hypoxia reporter in solid tumors

Purpose: 2-Nitro-α-[(2,2,2-trifluoroethoxy)methyl]-imidazole-1-ethanol (TF-MISO) was investigated as a potential noninvasive marker of tissue oxygen levels in tumors using 19F magnetic resonance spectroscopy (MRS) and 19F chemical shift imaging. Experimental Designs:In vitro data were obtained using high-performance liquid chromatography on tumor cells incubated under varying oxygen conditions to determine the oxygen-binding characteristics. In vivo data were obtained using a well-characterized hypoxic murine breast tumor (MCa), in addition to studies on a rat prostate tumor model (R3327-AT) implanted in nude mice. Detection of intratumor 19F signal from TF-MISO was done using MRS for up to 10 h following a 75 mg/kg i.v. injection. Localized distribution of the compound in the implanted MCa tumor has been imaged using slice-selective two-dimensional chemical shift imaging 6 h after injection. Results: The in vitro results showed that TF-MISO preferentially accumulates in cells incubated under anoxic conditions. The in vivo 19F MR spectral features (line width and chemical shift) were recorded as a function of time after injection, and the results indicate that the fluorine atoms are indeed sensitive to changes in the local environment while still providing a detectable MR signal. Ex vivo spectra were collected and established the visibility of the 19F signal under conditions of maximum hypoxia. Late time point (>6 h) tumor tissue concentrations, as obtained from 19F MRS, suggest that TF-MISO is reduced and retained in hypoxic tumor. The feasibility of obtaining TF-MISO tumor distribution maps in a reasonable time frame was established. Conclusions: Based on the results presented herein, it is suggested that TF-MISO has the potential to be a valid magnetic resonance hypoxia imaging reporter for both preclinical hypoxia studies and hypoxia-directed clinical therapy.

Preclinical evaluation of tumor microvascular response to a novel antiangiogenic/antitumor agent RO0281501 by dynamic contrast-enhanced MRI at 1.5 T

Inhibition of tumor angiogenesis is a promising approach in cancer treatment. The purpose of this study was to evaluate the vascular response of human lung tumor xenografts in vivo to RO0281501, an inhibitor of tyrosine kinase receptors, including vascular endothelial growth factor receptor 2, fibroblast growth factor receptor, and platelet-derived growth factor receptor, using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Human non–small cell lung carcinoma (H460a) xenografts grown s.c. in athymic nu/nu mice were treated p.o. with the antiangiogenic agent RO0281501. Treatment-induced changes in tumor volume, epiphyseal growth plate thickness, and microvessel density assessed by CD31 immunohistochemistry were analyzed. Tumor vascular permeability and perfusion were measured in tumors using DCE-MRI with gadopentetate dimeglumine on a 1.5 T clinical scanner to assess vascular function. Treatment with RO0281501 resulted in significant growth retardation of H460a tumors. RO0281501-treated tumors showed histologic evidence of growth plate thickening and relatively lower microvessel density compared with the controls. Regarding DCE-MRI variables, the initial slope of contrast uptake and Akep were significantly decreased on day 7 of treatment. RO0281501 is a novel antiangiogenic/antitumor agent, which is active in the H460a xenograft model. Its effects on tumor vasculature can be monitored and assessed by DCE-MRI on a 1.5 T human MR scanner with clinically available gadopentetate dimeglumine contrast, which will facilitate clinical trials with this or similar agents. [Mol Cancer Ther 2006;5(8):1950–7]

Lactate MRSI and DCE MRI as surrogate markers of prostate tumor aggressiveness

Longitudinal studies of lactate MRSI and dynamic contrast‐enhanced MRI were performed at 4.7 T in two prostate tumor models grown in rats, Dunning R3327‐AT (AT) and Dunning R3327‐H (H), to determine the potential of lactate and the perfusion/permeability parameter Akep as markers of tumor aggressiveness. Subcutaneous AT (n = 12) and H (n = 6) tumors were studied at different volumes between 100 and 2900 mm3 (Groups 1–5). Lactate concentration was determined using selective multiple quantum coherence MRSI with the phantom substitution method. Tumor enhancement after the administration of gadolinium diethylenetriaminepenta‐acetic acid was analyzed using the Brix–Hoffmann model and the Akep parameter was used as a measure of tumor perfusion/permeability. Lactate was not detected in the smallest AT tumors (Group 1; 100–270 mm3). In larger AT tumors, the lactate concentration increased from 2.8 ± 1.0 mm (Group 2; 290–700 mm3) to 8.4 ± 2.9 mm (Group 3; 1000–1340 mm3) and 8.2 ± 2.2 mm (Group 4; 1380–1750 mm3), and then decreased to 5.0 ± 1.7 mm (Group 5; 1900–2500 mm3), and was consistently higher in the tumor core than in the rim. Lactate was not detected in any of the H tumors. The mean tumor Akep values decreased with increasing volume in both tumor types, but were significantly higher in H tumors. In AT tumors, the Akep values were significantly higher in the rim than in the core. Histological hypoxic and necrotic fractions in AT tumors increased with volume from 0% in Group 1 to about 20% and 30%, respectively, in Group 5. Minimal amounts of hypoxia and necrosis were found in H tumors of all sizes. Thus, the presence of lactate and heterogeneous perfusion/permeability are signatures of aggressive, metabolically deprived tumors. Copyright

A quantitative assessment of liver metabolites during jaundice using three dimensional phosphorus chemical shift imaging

Phosphorus metabolites in the jaundiced rat liver were studied by three-dimensional phosphorus chemical shift imaging (CSI). Animals were studied at 1, 2, and 3 weeks post-ligation of the common bile duct. Quantitation of metabolites was performed using an external standard. Metabolite T(1) values were assessed in CSI experiments on normal untreated animals. High-performance liquid chromatography (HPLC) was used to measure adenine nucleotides in a separate group of jaundiced rats. 3D-CSI did not detect significant changes in NTP in jaundiced animals relative to baseline controls. At two and three weeks post bile duct ligation, pH was significantly elevated. HPLC data comparing ATP levels to baseline controls also detected no change except for elevated ATP detected on Day 21. (31)P NMR chemical shift imaging may be used to assess liver metabolites under conditions of stress such as jaundice. However, absolute quantitation requires careful attention to many factors including point spread function, correct T(1) values, and adequate signal-to-noise ratio.

In Vivo Detection by 31P NMR of Pentose Phosphate Pathway Block Secondary to Biochemical Modulation

The chemotherapeutic regimen of N‐(phosphonacetyl)‐lsc‐aspartate (PALA) followed 17 h later by 6‐methylmercaptopurine riboside (MMPR) and 6‐aminonicotanamide (6AN) has been shown to be a potent sensitizer of anti‐neoplastic therapy. We undertook this study to compare the therapeutic and metabolic effects of this triple drug combination vs one of its components, 6AN, in a murine mammary carcinoma. After treatment with PALA, MMPR and 6AN, a new peak was detected which was assigned to 6‐phosphogluconate (6PG), which is a marker of inhibition of the pentose phosphate pathway at the 6‐phosphogluconate dehydrogenase step. Treatment with PALA, MMPR and 6AN also induced a decrease in the ratios of nucleoside triphosphate/inorganic phosphate (NTP/Pi) and phosphocreatine/inorganic phosphate (PCr/Pi) similar to previous results with a different tumor model. These effects were most pronounced at 6 and 10 h. In addition, an increase in PME′/phosphocholine (PME′=downfield peak in the phosphomonoester region) was detected, which was expected because of the cytotoxic effect of this regimen. Treatment with 6AN alone also resulted in the detection of 6PG with a maximum intensity at 6 h post‐6AN. Treatment with 6AN alone induced a smaller change in PME′/PC and failed to cause a decrease in PCr/Pi or NTP/Pi at 6 and 10 h. The enhanced response to the combination of PALA, MMPR and 6AN vs 6AN alone, both with regard to cytotoxicity and radiosensitization, may be due to energy depletion.

Evaluation of chemotherapy and radiation enhancement and 31P NMR spectral changes induced by biochemical modulation

The combination of N-(phosphonacetyl)-L-aspartate (PALA), 6-methylmercaptopurine riboside (MMPR), and 6-aminonicotinamide (6AN) has been shown to be an effective antineoplastic regimen and also to enhance the effects of other antineoplastic agents (1-4). To further enhance the effect of this combination, we investigated the effects of adding adriamycin, at its maximally tolerated dose, to this regimen. The response rate (complete regression+partial regression) for the four-drug regimen was higher than for the three-drug regimen, and the tumor growth delay was also significantly higher than for treatment with PALA, MMPR, 6AN, or after treatment with maximally tolerated doses of adriamycin alone (11 mg/kg). The addition of adriamycin to PALA, MMPR, 6AN did not result in enhancement of the effect of radiation, as measured by tumor growth delay studies and tumor control (complete and partial regression rate). The mechanism of action of the combination of PALA, MMPR, and 6AN is not known definitively, but a possible mechanism previously suggested is biochemical modulation of energy metabolism and inhibition of production of tumor ATP. Treatment with PALA, MMPR, 6AN, and adriamycin (at 2.5 hr post MMPR, 6AN) resulted in a nadir NTP/Pi value, as determined by 31P NMR spectroscopy, at approximately 10 hr post MMPR + 6AN (7.5 hr post adriamycin), which was not significantly different from the NTP/Pi value determined after treatment with the three-drug combination.