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(1)H magnetic resonance spectroscopy (MRS) with the multiple quantum coherence (MQC) technique allows for the detection of lactate, an end product of glycolysis, in the environment of lipids. The method can also be used to detect alanine, a byproduct of glutaminolysis. An issue is that when both lactate and alanine are detected together by the MQC technique, a phase mismatch arises between lactate and alanine signals due to off-resonance rotations and the difference in double quantum coherence frequencies between the two molecules. Such phase mismatch can cause errors in spectral fitting and metabolite quantification. In this study, we designed two pulse sequences that eliminate such phase differences of lactate and alanine while suppressing lipid signals by modifications of the Selective Multiple Quantum Coherence (Sel-MQC) sequence, a well-known MQC technique. Using the product operator formalism and the off-resonance rotation matrices, the phase evolutions of lactate and alanine during the spectrally selective pulses and the free precession times of the sequence at the single quantum, double quantum and zero quantum coherence states of these molecules were calculated. The multiple quantum (MQ) evolution time t(1) that can remove the phase difference of lactate and alanine at the echo was calculated and fine-tuned with experiments. The lactate and alanine signal intensities and the editing efficiencies from the two modified Sel-MQC sequences were theoretically predicted by using the product operator evolutions and compared with the experimental data. The J-coupled lipid signals were successfully suppressed by both sequences. One of the two developed sequences was applied to a human body with a phantom of lactate and alanine, which resulted in successful in-phase editing of lactate and alanine and suppression of the lipid signals from the body. The study sets an important foundation for the noninvasive detection of lactate and alanine from tumors of cancer patients.

Objective. The selective multiple quantum coherence (Sel-MQC) sequence is a magnetic resonance spectroscopy (MRS) technique used to detect lactate and suppress co-resonant lipid signals in vivo. The coherence pathways of J-coupled lipids upon the sequence, however, have not been studied, hindering a logical design of the sequence to fully attenuate lipid signals. The objective of this study is to elucidate the coherence pathways of J-coupled lipids upon the Sel-MQC sequence and find a strategy to effectively suppress lipid signals from these pathways while keeping the lactate signal. Approach. The product operator formalism was used to express the evolutions of the J-coupled spins of lipids and lactate. The transformations of the product operators by the spectrally selective pulses of the sequence were calculated by using the off-resonance rotation matrices. The coherence pathways and the conversion rates of the individual pathways were derived from them. Experiments were performed on phantoms and two human subjects at 3 T. Main results. The coherence pathways contributing to the various lipid resonance signals by the Sel-MQC sequence depending on the gradient ratios and RF pulse lengths were identified. Theoretical calculations of the signals from the determined coherence pathways and signal attenuations by gradients matched the experimental data very well. Lipid signals from fatty tissues of the subjects were successfully suppressed to the noise level by using the gradient ratio −0.8:−1:2 or 1:0.8:2. The new gradient ratios kept the lactate signal the same as with the previously used gradient ratio 0:−1:2. Significance. The study has elucidated the coherence pathways of J-coupled lipids upon the Sel-MQC sequence and demonstrated how lipid signals can be effectively suppressed while keeping lactate signals by using information from the coherence pathway analysis. The findings enable applying the Sel-MQC sequence to lactate detection in an environment of high concentrations of lipids.

Background Glioblastoma Multiforme is the deadliest type of brain tumor and is characterized by very poor prognosis with a limited overall survival. Current optimal therapeutic approach has essentially remained unchanged for more than a decade, consisting in maximal surgical resection followed by radiotherapy plus temozolomide. Main body Such a dismal patient outcome represents a compelling need for innovative and effective therapeutic approaches. Given the development of new drugs is a process presently characterized by an immense increase in costs and development time, drug repositioning, finding new uses for existing approved drugs or drug repurposing, re-use of old drugs when novel molecular findings make them attractive again, are gaining significance in clinical pharmacology, since it allows faster and less expensive delivery of potentially useful drugs from the bench to the bedside. This is quite evident in glioblastoma, where a number of old drugs is now considered for clinical use, often in association with the first-line therapeutic intervention. Interestingly, most of these medications are, or have been, widely employed for decades in non-neoplastic pathologies without relevant side effects. Now, the refinement of their molecular mechanism(s) of action through up-to-date technologies is paving the way for their use in the therapeutic approach of glioblastoma as well as other cancer types. Short conclusion The spiraling costs of new antineoplastic drugs and the long time required for them to reach the market demands a profoundly different approach to keep lifesaving therapies affordable for cancer patients. In this context, repurposing can represent a relatively inexpensive, safe and fast approach to glioblastoma treatment. To this end, pros and cons must be accurately considered.

While studies have identified a number of mutations in mantle cell lymphoma (MCL), the list may still be incomplete and contribution to the pathogenesis remains unclear. We analyzed the mutational landscape of four mantle cell lymphoma biopsies obtained during an 8-year period from the same patient with his normal cells serving as control; we also established a cell line from the final stage of the disease. Numerous mutations with high allelic burden have been identified in all four biopsies. While a large subset of mutations was seen only in individual biopsies, the core of 21 mutations persisted throughout the disease. This mutational core is also maintained in the cell line that also displays DNA-methylation and cytokine secretion profiles of the primary mantle cell lymphoma cells. This cell line is uniquely sensitive to clinically relevant inhibitors of Bruton's Tyrosine Kinase. The response to Bruton Tyrosine Kinase's inhibition is enhanced by inhibitors of CDK4/6 and mTOR. Among the mutations seen in the primary and cultured MCL cells, mutations of three genes are involved in the control of H3K4 methylation: demethylase KDM5C, present already in the early disease, and methyltransferase KMT2D and cofactor BCOR, both of which are seen late in the disease and are novel and predicted to be pathogenic. The presence of these mutations was associated with hypermethylation of H3K4. Restoration of KDM5C expression affected expression of numerous genes involved in cell proliferation, adherence/movement, and invasiveness.

Rationale Mass spectrometric (MS) analysis of low molecular weight polar metabolites can be challenging because of poor chromatographic resolution of isomers and insufficient ionization efficiency. These metabolites include intermediates in key metabolic pathways, such as glycolysis, the pentose phosphate pathway, and the Krebs cycle. Therefore, sensitive, specific, and comprehensive quantitative analysis of these metabolites in biological fluids or cell culture models can provide insight into multiple disease states where perturbed metabolism plays a role. Methods An ion-pairing reversed-phase ultra-high-performance liquid chromatography (IP-RP-UHPLC)/MS approach to separate and analyze biochemically relevant phosphate- and carboxylic acid-containing metabolites was developed. Diisopropylethylamine (DIPEA) was used as an IP reagent in combination with reversed-phase liquid chromatography (RP-LC) and a triple quadrupole mass spectrometer using selected reaction monitoring (SRM) and negative electrospray ionization (NESI). An additional reagent, hexafluoroisopropanol (HFIP), which has been previously used to improve sensitivity of nucleotide analysis by UHPLC/MS, was used to enhance sensitivity. Results HFIP versus acetic acid, when added with the IP base, increased the sensitivity of IP-RP-UHPLC/NESI-MS up to 10-fold for certain analytes including fructose-1,6-bisphosphate, phosphoenolpyruvate, and 6-phosphogluconate. It also improved the retention of the metabolites on a C18 reversed-phase column, and allowed the chromatographic separation of important isomeric metabolites. This methodology was amenable to quantification of key metabolites in cell culture experiments. The applicability of the method was demonstrated by monitoring the metabolic adaptations resulting from rapamycin treatment of DB-1 human melanoma cells. Conclusions A rapid, sensitive, and specific IP-RP-UHPLC/NESI-MS method was used to quantify metabolites from several biochemical pathways. IP with DIPEA and HFIP increased the sensitivity and improved chromatographic separation when used with reversed-phase UHPLC.

Lonidamine (LND), a metabolic modulator, sensitizes DB-1 human melanoma to doxorubicin (DOX) chemotherapy by acidifying and de-energizing the tumor. This report compares the effects of LND on two human melanoma lines, DB-1 and WM983B, which exhibit different metabolic properties. Using liquid chromatography mass spectrometry and Seahorse analysis, we show that DB-1 was more glycolytic than WM983B in vitro. 31P magnetic resonance spectroscopy (MRS) indicates that LND (100 mg/kg, i.p.) induces similar selective acidification and de-energization of WM983B xenografts in immunosuppressed mice. Over three hours, intracellular pH (pHi) of WM983B decreased from 6.91 ± 0.03 to 6.59 ± 0.10 (p = 0.03), whereas extracellular pH (pHe) of this tumor changed from 7.03 ± 0.05 to 6.89 ± 0.06 (p = 0.19). A decline in bioenergetics (β-NTP/Pi) of 55 ± 5.0% (p = 0.03) accompanied the decline in pHi of WM983B. Using 1H MRS with a selective multiquantum pulse sequence and Hadamard localization, we show that LND induced a significant increase in tumor lactate levels (p

We seek to exploit the natural tendency of melanomas and other tumors to convert glucose to lactate as a method for the selective intracellular acidification of cancer cells and for the potentiation of the activity of nitrogen-mustard antineoplastic agents. We performed this study to evaluate whether the induction of hyperglycemia (26 mM) could enhance the effects of lonidamine (LND, 100 mg/kg; intraperitoneally) on the induction of intracellular acidification, bioenergetic decline and potentiation of the activity of melphalan (LPAM) against DB-1 melanoma xenografts in mice. Intracellular pH (pHi ), extracellular pH (pHe ) and bioenergetics (β-nucleoside triphosphate to inorganic phosphate ratio, β-NTP/Pi) were reduced by 0.7 units (p 0.05) and 51.4% (p < 0.05), respectively. The therapeutic response to LPAM (7.5 mg/kg; intravenously) + LND (100 mg/kg; intraperitoneally) was reduced by about a factor of three under hyperglycemic conditions relative to normoglycemia, producing a growth delay of 7.76 days (tumor doubling time, 5.31 days; cell kill, 64%) compared with LND alone of 1.70 days and LPAM alone of 0.29 days. Under normoglycemic conditions, LND plus LPAM produced a growth delay of 17.75 days, corresponding to a cell kill of 90% at the same dose for each of these agents. The decrease in tumor cell kill under hyperglycemic conditions correlates with an increase in tumor ATP levels resulting from increased glycolytic activity. However, hyperglycemia substantially increases lactic acid production in tumors by a factor of approximately six (p < 0.05), but hyperglycemia did not increase the effects of LND on acidification of the tumor, most probably because of the strong buffering action of carbon dioxide (the pKa of carbonic acid is 6.4). Therefore, this study demonstrates that the addition of glucose during treatment with LND diminishes the activity of this agent.

BACKGROUND/AIM: Since temozolomide (TMZ) is activated under alkaline conditions, we expected lonidamine (LND) to have no effect or perhaps diminish its activity, but initial results suggest it may actually enhance either or both short- and long-term activity of TMZ in melanoma xenografts. MATERIALS AND METHODS: Cohorts of 5 mice with subcutaneous xenografts ~5 mm in diameter were treated with saline (control (CTRL)), LND only, TMZ only or LND followed by TMZ at t=40 min (time required for maximal tumor acidification). RESULTS: Mean tumor volume for LND+TMZ for the period between 6 and 26 days was reduced compared to TMZ alone (repeated measures ANOVA F (1, 8), p=0.006), suggesting a pronounced impact of LND on this phenomenon. TMZ and LND+TMZ produced median growth delays of 82 and 106 days, respectively. CONCLUSION: The use of TMZ alone and in combination with LND deserves further investigation in treatment of melanoma and other malignancies.

We demonstrate that the effects of lonidamine (LND, 100 mg/kg, i.p.) are similar for a number of xenograft models of human cancer including DB-1 melanoma and HCC1806 breast, BT-474 breast, LNCaP prostate and A2870 ovarian carcinomas. Following treatment with LND, each of these tumors exhibits a rapid decrease in intracellular pH, a small decrease in extracellular pH, a concomitant monotonic decrease in nucleoside triphosphate and increase in inorganic phosphate over a 2–3 hr period. We previously demonstrated that selective intracellular tumor acidification potentiates response of this melanoma model to melphalan (7.5 mg/kg, i.v.), producing an estimated 89% cell kill based on tumor growth delay analysis. We now show that in both DB-1 melanoma and HCC1806 breast carcinoma, LND potentiates response to doxorubicin producing 95% cell kill in DB-1 melanoma at 7.5 mg/kg, i.v. doxorubicin and 98% cell kill at 10.0 mg/kg doxorubicin, and in HCC1806 breast carcinoma producing a 95% cell kill at 12.0 mg/kg doxorubicin. Potentiation of doxorubicin can result from cation trapping of the weakly basic anthracycline. Recent experience with the clinical treatment of melanoma and other forms of human cancer suggests that these diseases will probably not be cured by a single therapeutic procedure other than surgery. A multimodality therapeutic approach will be required. As a potent modulator of tumor response to N-mustards and anthracyclines as well as tumor thermo- and radiosensitivity, LND promises to play an important clinical role in the management and possible complete local control of a number of prevalent forms of human cancer.

Hepatocellular carcinoma (HCC), the primary form of human adult liver malignancy, is a highly aggressive tumor with average survival rates that are currently less than a year following diagnosis. Although bioinformatic analyses have indicated differentially expressed genes and cancer-related mutations in hepatocellular carcinoma (HCC), integrated genetic and metabolic pathway analyses remain to be investigated. Herein, gene (i.e. mRNA) enrichment analysis was performed to delineate significant alterations of metabolic pathways in HCC. The objective of this study was to investigate the pathway of aspartate metabolism in HCC of humans. Coupled with transcriptomic (i.e. mRNA) and NMR-based metabolomics of human tissue extracts, we utilized liquid chromatography mass spectrometry (LC-MS/MS)-based metabolomics analysis of stable [U-13C6]glucose metabolism or [U-13C5,15N2]glutamine metabolism of HCC cell culture. Our results indicated that aspartate metabolism is a significant and differentiable metabolic pathway of HCC compare to non-tumor liver (p-value < 0.0001). In addition, branched-chain amino acid metabolism (p-value< 0.0001) and tricarboxylic acid metabolism (p-value = < 0.0001) are significant and differentiable. Statistical analysis of measurable NMR metabolites indicated that at least two of the group means were significantly different for the metabolites alanine (p-value = 0.0013), succinate (p-value = 0.0001), lactate (p-value = 0.0114), glycerophosphoethanolamine (p-value = 0.015), and inorganic phosphate (p-value = 0.0001). However, 13C isotopic enrichment analysis of these metabolites revealed less than 50% isotopic enrichment with either stable [U-13C6]glucose metabolism or [U-13C5,15N2]glutamine. This may indicate the differential account of total metabolite pool versus de novo metabolites from a 13C labeled substrate. The ultimate translation of these findings will be to determine putative enzyme activity via 13C labeling, to investigate targeted therapeutics against these enzymes, and to optimize the in vivo performance of 13C magnetic resonance imaging techniques.

Lonidamine (LND) was initially introduced as an antispermatogenic agent. It was later found to have anticancer activity sensitizing tumors to chemo-, radio-, photodynamic-therapy and hyperthermia. Although the mechanism of action remained unclear, LND treatment has been known to target metabolic pathways in cancer cells. It has been reported to alter the bioenergetics of tumor cells by inhibiting glycolysis and mitochondrial respiration, while indirect evidence suggested that it also inhibited L-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). Recent studies have demonstrated that LND potently inhibits MPC activity in isolated rat liver mitochondria (Ki 2.5 μM) and cooperatively inhibits L-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevis oocytes with K0.5 and Hill Coefficient values of 36–40 μM and 1.65–1.85, respectively. In rat heart mitochondria LND inhibited the MPC with similar potency and uncoupled oxidation of pyruvate was inhibited more effectively (IC50 ~ 7 μM) than other substrates including glutamate (IC50 ~ 20 μM). LND inhibits the succinate-ubiquinone reductase activity of respiratory Complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species through Complex II and has been reported to promote cell death by suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. We conclude that MPC inhibition is the most sensitive anti-tumour target for LND, with additional inhibitory effects on MCT-mediated L-lactic acid efflux, Complex II and glutamine/glutamate oxidation.

We present the first validated metabolic network model for analysis of flux through key pathways of tumor intermediary metabolism, including glycolysis, the oxidative and non-oxidative arms of the pentose pyrophosphate shunt, the TCA cycle as well as its anaplerotic pathways, pyruvate-malate shuttling, glutaminolysis, and fatty acid biosynthesis and oxidation. The model that is called Bonded Cumomer Analysis for application to (13)C magnetic resonance spectroscopy ((13)C MRS) data and Fragmented Cumomer Analysis for mass spectrometric data is a refined and efficient form of isotopomer analysis that can readily be expanded to incorporate glycogen, phospholipid, and other pathways thereby encompassing all the key pathways of tumor intermediary metabolism. Validation was achieved by demonstrating agreement of experimental measurements of the metabolic rates of oxygen consumption, glucose consumption, lactate production, and glutamate pool size with independent measurements of these parameters in cultured human DB-1 melanoma cells. These cumomer models have been applied to studies of DB-1 melanoma and DLCL2 human diffuse large B-cell lymphoma cells in culture and as xenografts in nude mice at 9.4 T. The latter studies demonstrate the potential translation of these methods to in situ studies of human tumor metabolism by MRS with stable (13)C isotopically labeled substrates on instruments operating at high magnetic fields (≥7 T). The melanoma studies indicate that this tumor line obtains 51% of its ATP by mitochondrial metabolism and 49% by glycolytic metabolism under both euglycemic (5 mM glucose) and hyperglycemic conditions (26 mM glucose). While a high level of glutamine uptake is detected corresponding to ~50% of TCA cycle flux under hyperglycemic conditions, and ~100% of TCA cycle flux under euglycemic conditions, glutaminolysis flux and its contributions to ATP synthesis were very small. Studies of human lymphoma cells demonstrated that inhibition of mammalian target of rapamycin (mTOR) signaling produced changes in flux through the glycolytic, pentose shunt, and TCA cycle pathways that were evident within 8 h of treatment and increased at 24 and 48 h. Lactate was demonstrated to be a suitable biomarker of mTOR inhibition that could readily be monitored by (1)H MRS and perhaps also by FDG-PET and hyperpolarized (13)C MRS methods.

This study compared the steady state concentration of lactate in an inducible Her2/nue transgenic breast cancer mouse model and in tumours from the same Her2/neu model grown orthotopically. In vivo lactate was detected by MRS using the Hadamard encoded selective multiple quantum coherence pulse sequence (HadSelMQC) recently developed by our laboratory. A lower lactate signal was observed in the inducible tumours compared to orthotopic tumours in vivo, while ex vivo analysis of perchloric acid extracts revealed similar amounts of this metabolite in both models. Histological staining of mammary tumour specimens showed a much higher level of fat tissue in inducible tumours compared to the orthotopic model. Phantom studies with [3-C-13] lactate indicated that a lipid environment could significantly reduce the T2 of lactate and impede its detection. The transgenic inducible model for breast cancer not only better recapitulated the biological aspects of the human disease but also provided additional characteristics related to in vivo detection of lactate that are not available in orthotopic or xenograft models. This study suggests that the level of lactate measured by the HadSelMQC pulse sequence may be underestimated in human patients in the presence of high lipid levels that are typically encountered in the breast. Copyright (C) 2012 John Wiley & Sons, Ltd.

In vivo (31) P MRS demonstrates that human melanoma xenografts in immunosuppressed mice treated with lonidamine (LND, 100 mg/kg intraperitoneally) exhibit a decrease in intracellular pH (pH(i) ) from 6.90 ± 0.05 to 6.33 ± 0.10 (p 0.05) and a monotonic decline in bioenergetics (nucleoside triphosphate/inorganic phosphate) of 66.8 ± 5.7% (p 0.05) at 20 min post-LND, with no significant change in pH(e) and a small transient decrease in bioenergetics (32.9 ± 10.6%, p > 0.05) at 40 min post-LND. No changes in pH(i) or adenosine triphosphate/inorganic phosphate were detected in the brain (pH(i) , bioenergetics; p > 0.1) or skeletal muscle (pH(i) , pH(e) , bioenergetics; p > 0.1) for at least 120 min post-LND. Steady-state tumor lactate monitored by (1) H MRS with a selective multiquantum pulse sequence with Hadamard localization increased approximately three-fold (p = 0.009). Treatment with LND increased the systemic melanoma response to melphalan (LPAM; 7.5 mg/kg intravenously), producing a growth delay of 19.9 ± 2.0 days (tumor doubling time, 6.15 ± 0.31 days; log(10) cell kill, 0.975 ± 0.110; cell kill, 89.4 ± 2.2%) compared with LND alone of 1.1 ± 0.1 days and LPAM alone of 4.0 ± 0.0 days. The study demonstrates that the effects of LND on tumor pH(i) and bioenergetics may sensitize melanoma to pH-dependent therapeutics, such as chemotherapy with alkylating agents or hyperthermia.

Application of the kinase inhibitors in cancer treatment is rapidly growing. However, the methods for monitoring the effectiveness of the inhibitors are still poorly developed and currently rely mainly on tracking changes in the tumor volume, a rather late and relatively insensitive marker of therapeutic response. In contrast, magnetic resonance spectroscopy (MRS) can detect changes in cell metabolism, which has potential for providing early and patient-specific markers of drug activity. Using human B-cell lymphoma models and MRS, we demonstrate that inhibition of the mTOR signaling pathway can be detected in malignant cells in vitro and noninvasively in vivo by measuring lactate levels. An mTOR inhibitor, rapamycin, suppressed lactic acid production in the lymphoma cell line cultures and also diminished steady state lactate levels in xenotransplants. The inhibition was time dependent and first detectable 8 hours after drug administration in cell cultures. In the xenotransplants, two days of rapamycin treatment produced significant changes in lactic acid concentration in the tumor measured in vivo that were followed by tumor growth arrest and tumor volume regression. The rapamycin-induced changes in lactate production strongly correlated with inhibition of expression of hexokinase II, the key enzyme in the glycolytic pathway. These studies suggest that MRS or FDG PET detection of changes in glucose metabolism may be effective noninvasive methods for monitoring mTOR targeting therapy in lymphomas and other malignancies. Furthermore, measuring glucose metabolic inhibition by MRS or by FDG PET imaging may also prove effective in monitoring the efficacy of other kinase inhibitors, given that the rapamycin-sensitive mTOR is down-stream of many oncogenic signaling pathways.

Slowly cycling cells are believed to play a critical role in tumor progression and metastatic dissemination. The goal of this study was to develop a method for in vivo detection of slowly cycling cells. To distinguish these cells from more rapidly proliferating cells that constitute the vast majority of cells in tumors, we used the well-known effect of label dilution due to division of cells with normal cycle and retention of contrast agent in slowly dividing cells. To detect slowly cycling cells, melanoma cells were labeled with iron oxide particles. After labeling, we observed dilution of contrast agent in parallel with cell proliferation in the vast majority of normally cycling cells. A small and distinct subpopulation of iron-retaining cells was detected by flow cytometry after 20 days of in vitro proliferation. These iron-retaining cells exhibited high expression of a biological marker of slowly cycling cells, JARID1B. After implantation of labeled cells as xenografts into immunocompromised mice, iron-retaining cells were detected in vivo and ex vivo by magnetic resonance imaging that was confirmed by Prussian Blue staining. Magnetic resonance imaging detects not only iron retaining melanoma cells but also iron positive macrophages. Proposed method opens up opportunities to image subpopulation of melanoma cells, which is critical for continuous tumor growth. Magn Reson Med, 2011. © 2011 Wiley Periodicals, Inc.

The purpose of the study was to investigate the capability of (1)H MRS and MRI methods for detecting early response to radiation therapy in non-Hodgkin's lymphoma (NHL). Studies were performed on the WSU-DLCL2 xenograft model in nude mice of human diffuse large B-cell lymphoma, the most common form of NHL. Radiation treatment was applied as a single 15 Gy dose to the tumor. Tumor lactate, lipids, total choline, T(2) and apparent diffusion coefficients (ADC) were measured before treatment and at 24 h and 72 h after radiation. A Hadamard-encoded slice-selective multiple quantum coherence spectroscopy sequence was used for detecting lactate (Lac) while a stimulated echo acquisition mode sequence was used for detection of total choline (tCho) and lipids. T(2)- and diffusion-weighted imaging sequences were used for measuring T(2) and ADC. Within 24 h after radiation, significant changes were observed in the normalized integrated resonance intensities of Lac and the methylenes of lipids. Lac/H(2)O decreased by 38 +/- 15% (p = 0.03), and lipid (1.3 ppm, CH(2))/H(2)O increased by 57 +/- 14% (p = 0.01). At 72 h after radiation, tCho/H(2)O decreased by 45 +/- 14% (p = 0.01), and lipid (2.8 ppm, polyunsaturated fatty acid)/H(2)O increased by 970 +/- 36% (p = 0.001). ADC increased by 14 +/- 2% (p = 0.003), and T(2) did not change significantly. Tumor growth delay and regression were observed thereafter. This study enabled comparison of the relative sensitivities of various (1)H MRS and MRI indices to radiation and suggests that (1)H MRS/MRI measurements detect early responses to radiation that precede tumor volume changes.

Lactate is an important metabolite in normal and malignant tissues detectable by NMR spectroscopy; however, it has been difficult to clinically detect the lactate methyl resonance because it is obscured by lipid resonances. The selective homonuclear multiple quantum coherence transfer technique offers a method for distinguishing lipid and lactate resonances. We implemented a three-dimensional selective homonuclear multiple quantum coherence transfer version with Hadamard slice selection and two-dimensional phase encoding (Hadamard encoded-selective homonuclear multiple quantum coherence transfer-chemical shift imaging) on a conventional clinical MR scanner. Hadamard slice selection is explained and demonstrated in vivo. This is followed by 1-cm(3) resolution lactate imaging with detection to 5-mM concentration in 20 min on a 3-T clinical scanner. An analysis of QSel gradient duration and amplitude effects on lactate and lipid signal is presented. To demonstrate clinical feasibility, a 5-min lactate scan of a patient with a non-Hodgkin's lymphoma in the superficial thigh is reported. The elevated lactate signal coincides with the T(2)-weighted image of this tumor. As a test of selective homonuclear multiple quantum coherence transfer sensitivity, a thigh tourniquet was applied to a normal volunteer and an increase in lactate was detected immediately after tourniquet flow constriction. In conclusion, the Hadamard encoded-selective homonuclear multiple quantum coherence transfer-chemical shift imaging sequence is demonstrated on a phantom and in two lipid-rich, clinically relevant, in vivo conditions.

At 400 MHz the 1H chemical shifts, peptide NH-C alpha H coupling constants (JN alpha) and peptide hydrogen exchange rates of [Glu4] oxytocin in aqueous solution closely resemble those previously reported for oxytocin under comparable conditions, indicating that both the parent hormone and its analogue have similar conformations in this solvent. The hydrogen exchange data suggest a dynamic equilibrium between conformation(s) in which the peptide NH's of Asn5 and Cys6 are internally hydrogen bonded and conformation(s) in which these hydrogens are bonded to the solvent. [Glu4] oxytocin forms 1:1 complexes with lanthanide metal ions. The diamagnetic La3+ complex exhibits values of JN alpha very similar to those of the metal free hormone analogue, suggesting that coordination of the metal is accompanied by minimal perturbation of the peptide backbone. Specific average proton-metal distances estimated from Gd3+ induced paramagnetic relaxation effects indicate that the metal is probably coordinated to the Glu4 carboxyl group and the sidechain carbonyl of Asn5. Limiting shifts induced by binding of paramagnetic Yb3+ are also reported.

An effective method for in vivo detection of early therapeutic response of patients with non-Hodgkin's lymphoma would enable personalized clinical management of cancer therapy and facilitate the design of optimal treatment regimens. This study evaluates the feasibility of T2-weighted MRI (T2WI) and diffusion-weighted MRI (DWI) for in vivo detection of response of human diffuse large B-cell lymphoma xenografts in severe combined immunodeficient mice to chemotherapy. Each cycle of combination chemotherapy with cyclophosphamide, hydroxydoxorubicin, Oncovin, prednisone, and bryostatin 1 (CHOPB) was administered to tumor-carrying mice weekly for up to four cycles. T2WI and DWI were performed before the initiation of CHOPB and after each cycle of CHOPB. In order to corroborate the MRI results, histological analyses were carried out on control tumors and treated tumors after completion of all MRI studies. DWI revealed a significant (P

In order to identify early (1)H MRS metabolic markers of response to rituximab immunotherapy and to rituximab plus CHOP (cyclophosphamide, hydroxydoxorubicin, vincristine, and prednisone) combination therapy, we performed an in vivo MRS investigation of a non-Hodgkin's lymphoma (NHL) xenograft model. Human WSU-DLCL2 NHL cells were subcutaneously implanted into flanks of female severe combined immunodeficient mice. When tumor volumes reached approximately 600 mm(3), rituximab was administered for three weekly cycles at a dose of 25 mg/kg per cycle with or without CHOP. Before and after treatment, tumor lactate (Lac) and total choline (tCho) were detected using the selective multiple quantum coherence sequence and the stimulated echo acquisition mode sequence, respectively. Rituximab produced a small tumor growth delay ( approximately 5 days), whereas treatment with rituximab plus CHOP (RCHOP) led to approximately 20% tumor regression after three cycles of therapy. After one cycle of rituximab, the tCho/H(2)O ratio had decreased significantly (5%, P = 0.003), whereas the Lac/H(2)O ratio had not changed (P = 0.58). Both Lac/H(2)O and tCho/H(2)O had decreased after one cycle of RCHOP treatment (26%, P = 0.001; 10%, P = 0.016, respectively). After two cycles of RCHOP, Ki67 assay of histological tumor specimens indicated approximately 40% decrease in proliferation (P < 0.001) in the RCHOP-treated tumors; no change was detected after treatment with rituximab alone. This study suggests that decreases in tCho/H(2)O are more sensitive indices of response to rituximab, whereas decreases in Lac/H(2)O are more sensitive to response to CHOP combination therapy.

To identify 1H-MRS molecular biomarkers of early clinical therapeutic response in non-Hodgkin's lymphoma, an in vivo longitudinal study was performed on human non-Hodgkin's diffuse large B-cell lymphoma xenografts (WSU-DLCL2) grown in the flanks of female SCID mice. 31P-MRS measurements, which have been demonstrated to be prognostic clinical indices of response (Arias-Mendoza et al. Acad. Radiol. 2004; 11: 368-376) but which provide lower spatial resolution, were included for comparison. The animals received CHOP (cyclophosphamide, hydroxydoxorubicin, oncovin and prednisone) chemotherapy for three 1-week cycles, resulting in stable disease based on tumor volume. Localization of total choline and phosphorus metabolites in vivo was achieved with stimulated echo acquisition mode and image selected in vivo spectroscopy sequences, respectively. Significant decreases in lactate were detected by the selective multiple quantum coherence spectral editing technique after the first cycle of CHOP, whereas total choline and the phosphomonoester/nucleoside triphosphate ratio did not change until the third cycle. Ex vivo extract MRS of tumors corroborated the in vivo results. Histological staining with antibodies to Ki67 revealed a decrease in proliferation rate in CHOP-treated tumors that coincided with the decrease in lactate. This study demonstrates the utility of lactate as an early proliferation-sensitive indicator of therapeutic response in a mouse model of non-Hodgkin's lymphoma and serves as a basis for future clinical implementation of these methods.

A network model for the determination of tumor metabolic fluxes from 13C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mm [1,6-13C2]glucose under normoxic conditions at 37 °C and monitored by 13C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ∼50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ∼6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mm) and euglycemic (5 mm) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism.

Lonidamine (LND) is an anti-tumour drug particularly effective at selectively sensitizing tumours to chemotherapy, hyperthermia and radiotherapy, although its precise mode of action remains unclear. It has been reported to perturb the bioenergetics of cells by inhibiting glycolysis and mitochondrial respiration, whereas indirect evidence suggests it may also inhibit L-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). In the present study, we test these possibilities directly. We demonstrate that LND potently inhibits MPC activity in isolated rat liver mitochondria (Ki 2.5 μM) and co-operatively inhibits L-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevis oocytes with K0.5 and Hill coefficient values of 36–40 μM and 1.65–1.85 respectively. In rat heart mitochondria LND inhibited the MPC with similar potency and uncoupled oxidation of pyruvate was inhibited more effectively (IC50 ∼ 7 μM) than other substrates including glutamate (IC50 ∼ 20 μM). In isolated DB-1 melanoma cells 1–10 μM LND increased L-lactate output, consistent with MPC inhibition, but higher concentrations (150 μM) decreased L-lactate output whereas increasing intracellular [L-lactate] > 5-fold, consistent with MCT inhibition. We conclude that MPC inhibition is the most sensitive anti-tumour target for LND, with additional inhibitory effects on MCT-mediated L-lactic acid efflux and glutamine/glutamate oxidation. Together these actions can account for published data on the selective tumour effects of LND on L-lactate, intracellular pH (pHi) and ATP levels that can be partially mimicked by the established MPC and MCT inhibitor α-cyano-4-hydroxycinnamate (CHC).

The antitumor agent lonidamine (LND; 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid) is known to interfere with energy-yielding processes in cancer cells. However, the effect of LND on central energy metabolism has never been fully characterized. In this study, we report that a significant amount of succinate is accumulated in LND-treated cells. LND inhibits the formation of fumarate and malate and suppresses succinate-induced respiration of isolated mitochondria. Utilizing biochemical assays, we determined that LND inhibits the succinate-ubiquinone reductase activity of respiratory complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species through complex II, which reduced the viability of the DB-1 melanoma cell line. The ability of LND to promote cell death was potentiated by its suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. Using stable isotope tracers in combination with isotopologue analysis, we showed that LND increased glutaminolysis but decreased reductive carboxylation of glutamine-derived α-ketoglutarate. Our findings on the previously uncharacterized effects of LND may provide potential combinational therapeutic approaches for targeting cancer metabolism.

Low-density lipoproteins (LDLs) are a naturally occurring endogenous nanoplatform in mammalian systems. These nanoparticles (22 nm) specifically transport cholesterol to cells expressing the LDL receptor (LDLR). Several tumors overexpress LDLRs presumably to provide cholesterol to sustain a high rate of membrane synthesis. Amphiphilic gadolinium (Gd)-diethylenetriaminepentaacetic acid chelates have been incorporated into the LDL to produce a novel LDLR-targeted magnetic resonance imaging (MRI) contrast agent. The number of Gd chelates per LDL particle ranged between 150 and 496 Gd(III). In vitro studies demonstrated that Gd-labeled LDL retained a similar diameter and surface charge as the native LDL particle. In addition, Gd-labeled LDL retained selective cellular binding and uptake through LDLR-mediated endocytosis. Finally, Gd-labeled LDLs exhibited significant contrast enhancement 24 hours after administration in nude mice with human hepatoblastoma G2 xenografts. Thus, Gd-labeled LDL demonstrates potential use as a targeted MRI contrast agent for in vivo tumor detection.

Contains fulltext : 51298.pdf (Publisher’s version ) (Closed access) The standardization and reproducibility of techniques required to acquire anatomically localized 31P MR spectra non-invasively while studying tumors in cancer patients in a multi-institutional group at 1.5 T are reported. This initial group of patients was studied from 1995 to 2000 to test the feasibility of acquiring in vivo localized 31P MRS in clinical MR spectrometers. The cancers tested were non-Hodgkin's lymphomas, sarcomas of soft tissue and bone, breast carcinomas and head and neck carcinomas. The best accrual and spectral quality were achieved with the non-Hodgkin's lymphomas. The initial analysis of the spectral values of the sum of phosphoethanolamine plus phosphocholine normalized by the content of nucleotide triphosphates in a homogeneous sample of 32 NHL patients studied by in vivo (31)P MRS showed good reproducibility among different institutions. No statistical differences were found between the institution with the largest number of cases accrued and the rest of the multi-institutional NHL data (2.28 +/- 0.64, mean +/- standard error; n = 17, vs 2.08 +/- 0.14, n = 15). The preliminary data reported demonstrate that the institutions involved in this trial are obtaining reproducible 31P MR spectroscopic data non-invasively from human tumors. This is a fundamental prerequisite for the international cooperative group to be able to demonstrate the clinical value of the normalized determination of phosphoethanolamine plus phosphocholine by 31P MRS as predictor for treatment response in cancer patients.

We reported previously the in vivo detection of ectopic and transient expression of creatine kinase gene (ck) in the liver by phosphorus-31 magnetic resonance spectroscopy ((31)P MRS). Here we demonstrate the feasibility of using ck as a reporter gene to monitor the transfer of low-density lipoprotein receptor (LDLr) gene to LDLr(/) mice, a preclinical model for familial hypercholesterolemia. A recombinant adenovirus was generated that carries the creatine kinase gene (ck) and human LDL receptor gene (hLDLr) linked by an internal ribosomal entry site sequence. Intravenous injection of the adenovirus into LDLr(/)mice (1 x 10(11) viral particles/mouse) resulted in transduction of more than 90% of hepatocytes in the liver. Simultaneous expression of ck and LDLr was confirmed by Western analysis of the transduced livers. Through precise regulation of transgene expression in hepatocytes in vitro, an excellent correlation (R(2) = 0.96) between LDLr and ck expression was demonstrated over a wide range of viral dose. In vivo 31P MRS was employed to detect the metabolic product (i.e., phosphocreatine) of the creatine kinase protein (CK) reaction. CK activity, which is a true measure of ck gene expression, was quantified in vivo by magnetization transfer. Because ck is expressed abundantly in human muscle and brain but is absent from the liver, ck is useful to monitor any liver directed gene transfer. Use of the ck reporter would facilitate the clinical translation of gene therapy by providing a nondestructive readout of the level and duration of therapeutic gene expression.

PURPOSE To compare strain analysis and wall thickening (WT) analysis in differentiating the infarcted, adjacent, and remote zones in a rat model of myocardial infarction (MI). MATERIAL AND METHODS Three normal (NL) and ten rats subjected to myocardial infarction were imaged on a 4.7T scanner. Gradient-echo and SPAMM-tagged cine images were acquired at three short axis levels of the left ventricle (LV). A homogenous strain analysis (principal strains lambda1 and lambda2, displacement D, angle beta) and a WT-analysis (mm- and %-thickening) were performed in all slices demonstrating MI. Regional function was compared between infarcted rats (infarcted, adjacent and remote zone) and corresponding regions in the NL rats. Additional segmental analysis was performed in the NL rats for the anterior, lateral, inferior and septal wall. RESULTS In the NL rats, lambda (1) (greatest radial thickening) was greatest in the lateral and anterior wall. WT-analysis showed a pattern of function similar to lambda1, however, regional differences using WT-analysis were not significant. lambda (2) (greatest circumferential shortening) was most negative in the anterior wall. D was greatest in the lateral and inferior wall. The angle beta was radially directed in all segments. In the infarcted rats, both strain and WT-analyses revealed significant impairment in function in the infarcted and adjacent zones as compared to NL (p < 0.001). However, only the strain analysis (lambda1, lambda2, p < 0.001) detected significant remote myocardial dysfunction. Myocardial function differed significantly between the infarcted and adjacent and between the infarcted and remote regions. Strain analysis (lambda2, D, beta, p < 0.001) also identified significant functional differences between the adjacent and remote zones, however, no statistically significant differences were found using WT-analysis. CONCLUSION Strain analysis is superior to WT-analysis in detecting regional functional variations in NL rats and in discriminating function in the infarcted, adjacent and remote zones post MI.

Rationale and objectives Over-expression of low-density lipoprotein receptors (LDLRs) occurs in many types of malignancies and is related to the requirement for lipids for rapid proliferation of the tumors. On the other hand, LDLRs that are unable to bind LDL are found on hepatocytes of patients with familial hypercholesterolemia (FH), a genetic disease that leads to premature atherosclerosis and death. The highly selective binding of LDL to LDLR makes these particles ideal carriers of therapeutic and diagnostic contrast agents into the targeted cells. The objectives of this paper are to examine whether a prototype contrast agent (PTIR267) with dual detection properties is suitable for labeling of LDL particles for in vivo detection of LDLR by magnetic resonance imaging (MRI) and for in vitro monitoring of cellular localization by confocal fluorescence microscopy. Materials and methods PTIR267 is a lipophilic GdDTPA derivative conjugated to a fluorescent dye. The conjugated dye molecule makes the probe sufficiently water soluble to allow labeling of LDL by a brief incubation of LDL with PTIR267 dissolved in PBS at 37°C (mole ratio LDL: PTIR267 = 0.09:1). The molar relaxivity of PTIR267 in saline is 26 mM−1s−1. Specific LDLR-mediated uptake of PTIR267-labeled LDL was demonstrated in vitro by confocal fluorescence imaging of B16 melanoma cells using confocal fluorescence imaging. In vivo uptake of PTIR267-labeled LDL by a subcutaneously implanted B16 melanoma in mice leads to 30% decrease in longitudinal relaxation time (T1) in the tumor. In vivo uptake of PTIR267-labeled LDL leads to 70% decrease in T1 in a normal C57BL/6 mouse liver; however, in the liver of LDL receptor gene knockout (LDLr−/−) mice with C57BL/6 background, only 12% decrease in T1 is observed. Conclusions The dual fluorescence and MR imaging properties of PTIR267, combined with the ease of LDL labeling, suggest that it will be a useful tool for optimization of LDLR-targeted cancer diagnosis or therapy and for monitoring the efficacy of gene therapy of FH.

One of the biochemical "hallmarks" of malignancy is enhanced tumor glycolysis, which is primary due to the overexpression of glucose transporters (GLUTs) and the increased activity of mitochondria-bound hexokinase in tumors. Easy methods for assessing glucose utilization in vitro and in vivo should find widespread application in biological and biomedical studies, as illustrated by the adoption of FDG PET imaging in medicine. We have recently synthesized a new NIR fluorescent pyropheophorbide conjugate of 2-deoxyglucose (2DG), Pyro-2DG, as a GLUT-targeted photosensitizer. In this study, we have evaluated the in vivo uptake of Pyro-2DG and found that Pyro-2DG selectively accumulated in two tumor models, 9L glioma in the rat and c-MYC-induced mammary tumor in the mouse, compared to surrounding normal muscle tissues at a ratio of about 10:1. By simultaneously performing redox ratio and fluorescence imaging, a high degree of correlation between the PN/(Fp+PN) redox ratio, where PN denotes reduced pyridine nucleotides (NADH) and Fp denotes oxidized flavoproteins, and the Pyro-2DG uptake was found in both murine tumor models, indicating that Pyro-2DG could serve as an extrinsic NIR fluorescent metabolic index for the tumors. The fact that only a low level of correlation was observed between the redox ratio and the uptake of Pyro-acid (the free fluorophore without the 2-deoxyglucose moiety) supports the hypothesis that Pyro-2DG is an index of the mitochondrial status (extent of PN reduction) of a tumor.

Synergistic efforts in the developments of molecular specific imaging probes and advancements of optical imaging technologies (including the novel instrumentation and imaging algorithms) that lead to a new tool for early disease diagnosis and drug discovery are described.

Rationale and objectives The purpose of this study was to define and characterize carbocyanine labeled low-density lipoprotein (LDL) to be used in the optical imaging of LDL receptor (LDLr)-overexpressing tumor models. Materials and methods 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was used to label LDL (DiI-LDL). Scatchard plots were generated to determine the maximum binding capacity Bmax and dissociation constants KD of DiI-LDL in B16 melanoma (B16) and hepatoblastoma G2 (HepG2) cell lines. Selective uptake of DiI-LDL into both tumor cells and corresponding subcutaneous tumors in mice were demonstrated by confocal microscopy and three-dimensional Cryo-imaging, respectively. Results The labeling efficiency of DiI-LDL was 61 ng DiI/μg LDL protein (34 mol DiI/mol LDL protein). Bmax and KD for B16 cells were 6.311 ng LDL/mg cell protein and 60.38 μg protein/mL (117 nM), respectively. Bmax and KD were 7.573 ng LDL/mg cell protein and 26.79 μg protein/mL (52 nM) for HepG2 cells, respectively. Confocal microscopic images showed specific uptake of DiI-LDL throughout the cytoplasm in the B16/HepG2 cells. Cryo-imaging demonstrated preferential accumulations of DiI-LDL in the viable tumor regions of both B16 and HepG2 tumors compared with their adjacent normal tissues and corresponding necrotic tumor regions. In addition, uptake of DiI-LDL by the HepG2 tumor was much higher than that of the B16 tumor, consistent with the fact that the probe binding affinity for LDLrs of HepG2 cells is 2.3 times that of B16 cells. Conclusion This study suggested that carbocyanine labeled LDL could be used for optical imaging of tumors overexpressing LDLr.

A noninvasive technique for simultaneous measurement of the arterial input function (AIF) for gadodiamide (Omniscan) and its uptake in tumor was demonstrated in mice. Implantation of a tumor at a suitable location enabled its visualization in a cardiac short axis image. Sets of gated, low-resolution saturation recovery images were acquired from each of five tumor-bearing mice following intravenous administration of a bolus of contrast agent (CA). The AIF was extracted from the signal intensity changes in left ventricular blood using literature values of the CA relaxivity and a precontrast T1 map. The time-dependent 1H2O relaxation rate constant (R1 = 1/T1) in the tumor was modeled using the BOLus Enhanced Relaxation Overview (BOLERO) method in two modes regarding the equilibrium transcytolemmal water exchange system: 1) constraining it exclusively to the fast exchange limit (FXL) (the conventional assumption), and 2) allowing its transient departure from FXL and access to the fast exchange regime (FXR), thus designated FXL/FXR. The FXL/FXR analysis yielded better fittings than the FXL-constrained analysis for data from the tumor rims, whereas the results based on the two modes were indistinguishable for data from the tumor cores. For the tumor rims, the values of Ktrans (the rate constant for CA transfer from the vasculature to the interstitium) and ve (volume fraction of the tissue extracellular and extravascular space) returned from FXL/FXR analysis are consistently greater than those from the FXL-constrained analysis by a factor of 1.5 or more corresponding to a CA dose of 0.05 mmole/kg. Magn Reson Med 52:248–257, 2004. © 2004 Wiley-Liss, Inc.

Rational and Objectives. Dynamic contrast-enhanced (DCE) MRI offers the potential to provide quantitative maps of tumor perfusion parameters and is therefore expected to play an important role in the study of cancer in small animal models. Extraction of such information from DCE-MRI data requires a methodology for determination of the arterial input function (AIF) for the target tissues. An MRI based method for observation of the AIF in a mouse model is demonstrated in the present report. Materials and Methods. A series of short-axis cardiac images was acquired during the first pass of a bolus of Gadodiamide using a low-resolution, EEG-gated, saturation-recovery gradient echo imaging sequence. The AIF was then extracted from the observed signal intensity changes in the left ventricle (LV) blood pool. Results. The proposed technique provides sufficient temporal and spatial resolution to accurately characterize the AIF of Gadodiamide in mouse models. The AIF was observed in 4 mice and was found to be qualitatively similar to that previously observed in larger animals. However, significant inter-animal variability in the precise form of the AIF was evident. Conclusions. The proposed method for determination of AIF in mice has been shown to be effective and reliable. The inter-animal variability observed in the present study suggests that the AIF should be measured in each animal undergoing analysis by DCE-MRI.

Mouse models are expected to play an important role in future investigations of human cardiac diseases. In the present report, MRI methods for determining global and regional cardiac function in the mouse are demonstrated. ECG-gated cine images were acquired in five C57BL/6 mice at physiological temperatures (37°C) and heart rates of 500 ± 50 beats per minute. Left ventricular mass, ejection fraction, and cardiac output were estimated from the resulting images. Regional myocardial function was also determined in three animals by application of 2D SPAtial Modulation of Magnetization (SPAMM) in combination with the cine protocol. The quality of the tagged images was sufficient to allow mapping of myocardial strains and displacements. The results of the regional strain analysis were consistent with similar studies in larger animals. This work demonstrates the first characterization of regional myocardial function in the mouse via SPAMM techniques. Magn Reson Med 49:760–764, 2003. © 2003 Wiley-Liss, Inc.

The purpose of this study was to develop a reliable, noninvasive method for early detection of tumor response to therapy that would facilitate optimization of treatment regimens to the needs of the individual patient. In the present study, the effects of cyclophosphamide (Cp, a widely used alkylating agent) were monitored in a murine radiation induced fibrosarcoma (RIF-1) using in vivo (1)H NMR spectroscopy and imaging to evaluate the potential of these techniques towards early detection of treatment response. Steady-state lactate levels and Gd-DTPA uptake kinetics were measured using selective multiple quantum coherence (Sel-MQC) transfer spectroscopy and dynamic contrast enhanced imaging, respectively in RIF-1 tumors before, 24 and 72 h after 300 mg/kg of Cp administration. High-resolution (1)H NMR spectra of perchloric acid extracts of the tumor were correlated with lactate and glucose concentrations determined enzymatically. In vivo NMR experiments showed a decrease in steady-state lactate to water ratios (5.4 +/- 1.6 to 0.6 +/- 0.5, p < 0.05) and an increase in Gd-DTPA uptake kinetics following treatment response. The data indicate that decreases in lactate result from decreased glycolytic metabolism and an increase in tumor perfusion/permeability. Perchloric acid extracts confirmed the lower lactate levels seen in vivo in treated tumors and also indicated a higher glycerophosphocholine/phosphocholine (GPC/PC) integrated intensity ratio (1.39 +/- 0.09 vs 0.97 +/- 0.04, p < 0.01), indicative of increased membrane degradation following Cp treatment. Steady-state lactate levels provide metabolic information that correlates with changes in tumor physiology measured by Gd-DTPA uptake kinetics with high spatial and temporal resolution. Both of these parameters may be useful for monitoring early tumor response to therapy.

Perfusion parameters, such as blood flow, are critical properties of tumors related to angiogenesis, drug delivery, radiosensitivity, bioenergetic status, and steady state levels of metabolites, such as lactate, that have been proposed as indices of tumor response to therapy. The existing MR methods for measuring tumor blood flow (TBF) have limitations related to sensitivity, spatial resolution, or dependence on other physiological properties such as vascular permeability. To address many of these difficulties, this study introduces the use of an 17O-enriched tracer in conjunction with high-resolution, indirect MRI to measure TBF. To demonstrate the advantages of this technique, relative TBF was measured in subcutaneous RIF-1 tumors in C3H mice by monitoring the uptake of H217O with a resolution of 0.16 × 0.31 × 3 mm in 13 sec. At this resolution, tumor heterogeneity with respect to blood flow is clearly visible. Measurement of the tracer arterial input function, which is necessary for determination of absolute blood flow, may be facilitated with improved temporal resolution. Magn Reson Med 49:1–6, 2003. © 2003 Wiley-Liss, Inc.

Contains fulltext : 142496.pdf (Publisher’s version ) (Closed access) RATIONALE AND OBJECTIVES: Combinations of chemotherapy and fractionated radiation therapy are the currently preferred nonsurgical treatment methods for squamous cell carcinoma of the head and neck, but to the authors' knowledge there is no reliable marker for predicting therapeutic response. Early identification of nonresponders would allow prompt replacement of ineffective, toxic therapy by alternative, potentially more effective procedures. Frequent regional node involvement facilitates surface coil investigation with phosphorus-31 magnetic resonance spectroscopy. MATERIALS AND METHODS: P-31 magnetic resonance spectra were acquired from 12 patients before radiation therapy or chemotherapy. In vivo three-dimensional localized P-31 nuclear magnetic resonance chemical shift imaging was performed with a 1.5-T clinical imager and a dual-tuned H-1/P-31 surface coil. Proton decoupling and nuclear Overhauser enhancement were used to improve sensitivity and resolve overlapping signals in the phosphomonoester region of the spectrum. RESULTS: The average pretreatment ratio of phosphomonoester to beta-nucleoside triphosphate was significantly smaller in complete responders (n = 4) than in incomplete responders (partial responders plus nonresponders, n = 8) (0.0 +/- 0.0 vs 1.22 +/- 0.17 [P = .004]). CONCLUSION: Results of this preliminary study suggest that H-1-decoupled P-31 magnetic resonance spectroscopy may prove to be a useful predictor of therapeutic response in head and neck cancers.

Rationale and Objectives Cancer cells generate more lactate than normal cells under both aerobic and hypoxic conditions—exhibiting the so-called Warburg effect. However, the relationship between the Warburg effect and tumor metastatic potential remains controversial. We intend to investigate whether the higher lactate reflects higher tumor metastatic potential. Materials and Methods We used hyperpolarized 13 C-pyruvate magnetic resonance spectroscopy (MRS) to compare lactate 13 C-labeling in vivo in mouse xenografts of the highly metastatic (MDA-MB-231) and the relatively indolent (MCF-7) human breast cancer cell lines. We obtained the kinetic parameters of the lactate dehydrogenase (LDH)–catalyzed reaction by three methods of data analysis including the differential equation fit, q-ratio fit, and ratio fit methods. Results Consistent results from the three methods showed that the highly metastatic tumors exhibited a smaller apparent forward rate constant ( k + = 0.060 ± 0.004 s −1 ) than the relatively indolent tumors ( k + = 0.097 ± 0.013 s −1 ). The ratio fit generated the greatest statistical significance for the difference ( P = .02). No significant difference in the reverse rate constant was found between the two tumor lines. Conclusions The result indicates that the less metastatic breast tumors may produce more lactate than the highly metastatic ones from the injected 13 C-pyruvate and supports the notion that breast tumor metastatic risk is not necessarily associated with the high levels of glycolysis and lactate production. More studies are needed to confirm whether and how much the measured apparent rate constants are affected by the membrane transporter activity and whether they are primarily determined by the LDH activity.

Rationale and Objectives The authors performed this study to evaluate the selective acidification of a human melanoma xenograft in mice with severe combined immunodeficiency with the induction of hyperglycemia (mean blood glucose level ± standard error of the mean, 26 mmol/L ± 1) and the intraperitoneal administration of metaiodobenzylguanidine (MIBG, 30 mg/kg), α-cyano-4-hydroxycinnamate (CNCn, 300 mg/kg), lonidamine (100 mg/kg), cariporide (HOE642, 160 mg/kg), or 4,4′-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS, 50 mg/kg). Materials and Methods The intra- and extracellular pH levels of tumor were estimated from the chemical shifts of inorganic phosphate and 3-aminopropylphosphonate, respectively, with phosphorus-31 nuclear magnetic resonance (MR) spectroscopy. The relative level of steady-state lactate was monitored with hydrogen-1 MR spectroscopy. Results In small tumors (≤8.0 mm), hyperglycemia decreased the intra- and extracellular pH levels by less than 0.2. The combination of hyperglycemia and MIBG decreased the intra- and extracellular pH levels by approximately 0.4 and 0.6, respectively, and lowered the β-nucleoside triphosphate (NTP)/inorganic phosphate (P i ) ratio of tumor and liver by about 60% and 25%, respectively. The combination of hyperglycemia, MIBG, and CNCn produced a transient decrease in the intracellular pH of about 0.6. The combination of hyperglycemia and lonidamine produced a sustained (>3 hours) 0.8-unit decrease in intracellular pH and an 83% and 100% decrease in PCr/P i and β-NTP/P i ratios, respectively. The combination of hyperglycemia, MIBG, cariporide, and DIDS produced a gradual decrease in intra- and extracellular pH by 1.1 and 1.0, respectively. The relative level of steady-state lactate concentration in tumors increased 10% with hyperglycemia alone, about 20% with MIBG plus hyperglycemia, and increased more than twofold when hyperglycemia was combined with MIBG and CNCn administration. Conclusion These preliminary data suggest that hyperglycemia and combinations of respiratory and ion transport inhibitors can be used to selectively acidify tumors and, thereby, sensitize them to hyperthermia or other pH-sensitive therapeutic modalities.

In vivo assessment of gene expression is desirable to obtain information on the extent and duration of transduction of tissue after gene delivery. We have developed an in vivo , potentially noninvasive, method for detecting virally mediated gene transfer to the liver. The method employs an adenoviral vector carrying the gene for the brain isozyme of murine creatine kinase (CK-B), an ATP-buffering enzyme expressed mainly in muscle and brain but absent from liver, kidney, and pancreas. Gene expression was monitored by 31 P magnetic resonance spectroscopy (MRS) using the product of the CK enzymatic reaction, phosphocreatine, as an indicator of transfection. The vector was administered into nude mice by tail vein injection, and exogenous creatine was administered in the drinking water and by i.p. injection of 2% creatine solution before 31 P MRS examination, which was performed on surgically exposed livers. A phosphocreatine resonance was detected in livers of mice injected with the vector and was absent from livers of control animals. CK expression was confirmed in the injected animals by Western blot analysis, enzymatic assays, and immunofluorescence measurements. We conclude that the syngeneic enzyme CK can be used as a marker gene for in vivo monitoring of gene expression after virally mediated gene transfer to the liver.