Edward J. Delikatny

1H NMR visible lipids are induced by phosphonium salts and 5-fluorouracil in human breast cancer cells

Cationic lipophilic phosphonium salts (CLPS) selectively accumulate in the mitochondria of neoplastic cells and inhibit mitochondrial function. The effects of the CLPS p‐(triphenylphosphoniummethyl) benzaldehyde chloride (drug A), and [4‐(hydrazinocarboxy)‐1‐butyl] tris‐(4‐dimethylaminophenyl) phosphonium chloride (drug B), on human breast cells of differing biological properties were assessed using growth inhibition assays and 1H NMR. Drug A and, to a lesser extent, drug B demonstrated selective growth inhibition of the highly tumorigenic DU4475 breast carcinoma cell line compared to the transformed HBL‐100 human breast cell line. However, in contrast to previous studies using other cell lines, no synergistic activity was found when the drugs were used in combination. 1H NMR demonstrated significant increases in mobile lipid acyl chain resonances in both cell lines treated with cytotoxic doses (IC50, 48 h) of the drugs used either alone or in combination. Two‐dimensional NMR revealed accompanying decreases in phosphocholine/Lys levels in HBL‐100 cells treated with A, B, or a 1:1 combination A+B at the IC50, and in DU4475 cells treated with drug A (IC50). This was accompanied by significant increases in cho/Lys ratios with IC50 A or combination A+B treatment. Similar spectra were observed in cells treated with 5‐fluorouracil but not methotrexate, indicating that mobile lipid accumulation is a general but not universal response to cytotoxic insult. Magn Reson Med 45:1001–1010, 2001.

In vivo assessment of hepatic triglycerides in murine non-alcoholic fatty liver disease using magnetic resonance spectroscopy

In vivo(1)H magnetic resonance spectroscopy (MRS) was used to examine the progression of fatty liver in two murine models of progressive hepatic steatosis: leptin-deficient obese (ob/ob) mice and mice maintained on a diet deficient in methionine and choline (MCDD). Ob/ob mice displayed high levels of intracellular hepatic triglycerides as early as 9 weeks after birth, as observed with MRS and histopathology. Single voxel spectra of ob/ob liver displayed strong resonances arising from saturated (1.3 ppm) and unsaturated (2.8 and 5.3 ppm) fatty acyl chains that could be resolved in the absence of water suppression. Hepatic inflammation, induced by lipopolysaccharide administration, led to a significant increase in unsaturated and polyunsaturated fatty acyl chain resonances (P<0.05), indicating a change in the composition of hepatic triglycerides in lipid droplets. Mice maintained on the MCDD displayed histological evidence of hepatic steatosis as early as two weeks, progressing to macrovesicular steatohepatitis at 10 weeks. The histological changes were accompanied by significant increases in saturated and unsaturated fatty acyl chain resonances and a significant decrease in the lipid/(water+lipid) ratio (P<0.05). These results indicate that in vivo(1)H MRS may be a suitable method to monitor the progression of steatohepatitis.

In vivo MRS markers of response to CHOP chemotherapy in the WSU‐DLCL2 human diffuse large B‐cell lymphoma xenograft

To identify 1H‐MRS molecular biomarkers of early clinical therapeutic response in non‐Hodgkins lymphoma, an in vivo longitudinal study was performed on human non‐Hodgkins 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‐Hodgkins lymphoma and serves as a basis for future clinical implementation of these methods. Copyright

Decreased lactate concentration and glycolytic enzyme expression reflect inhibition of mTOR signal transduction pathway in B-cell lymphoma

The application of kinase inhibitors in cancer treatment is growing rapidly. However, methods for monitoring the effectiveness of the inhibitors are still poorly developed and currently rely mainly on the tracking of changes in the tumor volume, a rather late and relatively insensitive marker of the therapeutic response. In contrast, MRS can detect changes in cell metabolism and has the potential to provide early and patient‐specific markers of drug activity. Using human B‐cell lymphoma models and MRS, we have demonstrated that the inhibition of the mTOR signaling pathway can be detected in malignant cells in vitro and noninvasively in vivo by the measurement of lactate levels. An mTOR inhibitor, rapamycin, suppressed lactic acid production in lymphoma cell line cultures and also diminished steady‐state lactate levels in xenotransplants. The inhibition was time dependent and was first detectable 8 h after drug administration in cell cultures. In xenotransplants, 2 days of rapamycin treatment produced significant changes in lactic acid concentration in the tumor measured in vivo, which were followed by tumor growth arrest and tumor volume regression. The rapamycin‐induced changes in lactate production were strongly correlated with the inhibition of expression of hexokinase II, the key enzyme in the glycolytic pathway. These studies suggest that MRS or 18F‐fluorodeoxyglucose positron emission tomography (FDG PET) detection of changes in glucose metabolism may represent effective noninvasive methods for the monitoring of mTOR targeting therapy in lymphomas and other malignancies. Furthermore, the measurement of glucose metabolic inhibition by MRS or FDG PET imaging may also prove to be effective in monitoring the efficacy of other kinase inhibitors given that the rapamycin‐sensitive mTOR lies downstream of many oncogenic signaling pathways. Copyright

Magnetic resonance spectroscopy of the occipital cortex and the cerebellar vermis distinguishes individual cats affected with alpha-mannosidosis from normal cats

A genetic deficiency of lysosomal alpha‐mannosidase causes the lysosomal storage disease alpha‐mannosidosis (AMD), in which oligosaccharide accumulation occurs in neurons and glia. The purpose of this study was to evaluate the role of magnetic resonance spectroscopy (MRS) in detecting the oligosaccharide accumulation in AMD. Five cats with AMD and eight age‐matched normal cats underwent in vivo MRS studies with a single voxel short echo time (20 ms) STEAM spectroscopy sequence on a 4.7T magnet. Two voxels were studied in each cat, from the cerebellar vermis and the occipital cortex. Metabolites of brain samples from these regions were extracted with perchloric acid and analyzed by high resolution NMR spectroscopy. A significantly elevated unresolved resonance signal between 3.4 and 4. ppm was observed in the cerebellar vermis and occipital cortex of all AMD cats, which was absent in normal cats. This resonance was shown to be from carbohydrate moieties by high resolution NMR of tissue extracts. Resonances from the Glc‐NAc group (1.8–2.2 ppm) along with anomeric proton signals (4.6–5.4 ppm) from undigested oligosaccharides were also observed in the extract spectra from AMD cats. This MRS spectral pattern may be a useful biomarker for AMD diagnosis as well as for assessing responses to therapy. Copyright

In vivo1H MRS of WSU‐DLCL2 human non‐Hodgkin's lymphoma xenografts: response to rituximab and rituximab plus CHOP

In order to identify early 1H 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‐Hodgkins lymphoma (NHL) xenograft model. Human WSU‐DLCL2 NHL cells were subcutaneously implanted into flanks of female severe combined immunodeficient mice. When tumor volumes reached ∼600 mm3, 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 (∼5 days), whereas treatment with rituximab plus CHOP (RCHOP) led to ∼20% tumor regression after three cycles of therapy. After one cycle of rituximab, the tCho/H2O ratio had decreased significantly (5%, P = 0.003), whereas the Lac/H2O ratio had not changed (P = 0.58). Both Lac/H2O and tCho/H2O 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 ∼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/H2O are more sensitive indices of response to rituximab, whereas decreases in Lac/H2O are more sensitive to response to CHOP combination therapy. Copyright

Proton and Phosphorous MR Spectroscopy in Squamous Cell Carcinomas of the Head and Neck

RATIONALE AND OBJECTIVES Phosphorous magnetic resonance spectroscopy ((31)P MRS) has been used to evaluate and predict treatment response in squamous cell carcinoma of the head and neck (HNSCC). Several studies have also shown the potential of proton MRS ((1)H MRS) in assessing response in HNSCC. In view of the inherent limitations associated with performing (31)P MRS in clinical settings, the current study was performed to explore whether (1)H MRS could provide similar or complementary metabolic information in HNSCC. MATERIALS AND METHODS Fifteen patients with HNSCC underwent pretreatment magnetic resonance imaging. Both (1)H MRS and (31)P MRS were performed on viable solid parts of the metastatic lymph nodes of these patients. Peak areas of total choline (tCho) and unsuppressed water as observed on (1)H MRS and phosphomonoester (PME) and beta-nucleotide triphosphate (beta-NTP) on (31)P MRS were computed. Pearsons correlation coefficient was used to correlate the tCho/water and PME/beta-NTP ratios. RESULTS In all patients, the metastatic nodes appeared hyperintense on T2-weighted images and hypointense on T1-weighted images with variable signal intensity. A prominent resonance of tCho on (1)H MRS and a resonance of PME on (31)P MRS from the metastatic nodes of all patients were observed. A moderate correlation of 0.31 was observed between tCho/water and PME/beta-NTP (P > .05). CONCLUSIONS The biochemical pathways involved in (1)H MRS of tCho may be different from the phospholipid metabolites seen on (31)P MRS of head and neck cancers, and thus the two MRS techniques may be complementary to each other.

Direct Inhibition of Choline Kinase by a Near-Infrared Fluorescent Carbocyanine

Choline kinase alpha (ChoK) expression is increasingly being recognized as an important indicator of breast cancer prognosis; however, previous efforts to noninvasively measure ChoK status have been complicated by the spectral limitations of in vivo magnetic resonance spectroscopy (MRS) and the complex network of enzymes involved in choline metabolism. The most effective ChoK inhibitors are symmetric and contain quaternary ammonium groups within heterocyclic head groups connected by an aliphatic spacer. Characterization of these bis-pyridinium and bis-quinolinium compounds has led to phase I clinical trials to assess small-molecule inhibitors of ChoK for solid tumor treatment. We report the development of a novel carbocyanine dye, JAS239, whose bis-indolium structure conforms to the parameters established for ChoK specificity and whose spacer length confers fluorescence in the near-infrared (NIR) window. Fluorimetry and confocal microscopy were used to demonstrate that JAS239 rapidly enters breast cancer cells independent of the choline transporters, with accumulation in the cytosolic space where ChoK is active. Radio-tracing and 1H MRS techniques were used to determine that JAS239 binds and competitively inhibits ChoK intracellularly, preventing choline phosphorylation while inducing cell death in breast cancer cell lines with similar efficacy to known ChoK inhibitors. Fluorescent molecules that report on ChoK status have potential use as companion diagnostics for noninvasive breast tumor staging, because NIR fluorescence allows for detection of real-time probe accumulation in vivo. Furthermore, their ability as novel ChoK inhibitors may prove effective against aggressive, therapy-resistant tumors. Mol Cancer Ther; 13(9); 2149–58. ©2014 AACR.

Intraoperative Molecular Imaging Combined With Positron Emission Tomography Improves Surgical Management of Peripheral Malignant Pulmonary Nodules

Objective: To determine if intraoperative molecular imaging (IMI) can improve detection of malignant pulmonary nodules. Background: 18-Fluorodeoxyglucose positron emission tomography (PET) is commonly utilized in preoperative assessment of patients with solid malignancies; however, false negatives and false positives remain major limitations. Using patients with pulmonary nodules as a study model, we hypothesized that IMI with a folate receptor targeted near-infrared contrast agent (OTL38) can improve malignant pulmonary nodule identification when combined with PET. Methods: Fifty patients with pulmonary nodules with imaging features suspicious for malignancy underwent preoperative PET. Patients then received OTL38 before pulmonary resection. During resection, IMI was utilized to evaluate known pulmonary nodules and identify synchronous lesions. Tumor size, PET standardized uptake value, and IMI tumor-to-background ratios were compared for known and synchronous nodules via paired and unpaired t tests, when appropriate. Test characteristics of PET and IMI with OTL38 were compared. Results: IMI identified 56 of 59 (94.9%) malignant pulmonary nodules identified by preoperative imaging. IMI located an additional 9 malignant lesions not identified preoperatively. Nodules only detected by IMI were smaller than nodules detected preoperatively (0.5 vs 2.4 cm; P < 0.01), but displayed similar fluorescence (tumor-to-background ratio 3.3 and 3.1; P = 0.50). Sensitivity of IMI and PET were 95.6% and 73.5% (P = 0.001), respectively; and positive predictive values were 94.2% and 89.3%, respectively (P > 0.05). Additionally, utilization of IMI clinically upstaged 6 (12%) subjects and improved management of 15 (30%) subjects. Conclusions: These data suggest that combining IMI with PET may provide superior oncologic outcomes for patients with resectable lung cancer.

Cerenkov specific contrast agents for detection of pH in vivo

We report the design, testing, and in vivo application of pH-sensitive contrast agents designed specifically for Cerenkov imaging. Radioisotopes used for PET emit photons via Cerenkov radiation. The multispectral emission of Cerenkov radiation allows for selective bandwidth quenching, in which a band of photons is quenched by absorption by a functional dye. Under acidic conditions, 18F-labeled derivatives emit the full spectrum of Cerenkov light. Under basic conditions, the dyes change color and a wavelength-dependent quenching of Cerenkov emission is observed. Methods: Mono- and di-18F-labeled derivatives of phenolsulfonphthalein (phenol red) and meta-cresolsulfonphthalein (cresol purple) were synthesized by electrophilic fluorination. Cerenkov emission was measured at different wavelengths as a function of pH in vitro. Intramolecular response was measured in fluorinated probes and intermolecular quenching by mixing phenolphthalein with 18F-FDG. Monofluorocresol purple (MFCP) was tested in mice treated with acetazolamide to cause urinary alkalinization, and Cerenkov images were compared with PET images. Results: Fluorinated pH indicators were produced with radiochemical yields of 4%–11% at greater than 90% purity. Selective Cerenkov quenching was observed intramolecularly with difluorophenol red or monofluorocresol purple and intermolecularly in phenolphthalein 18F-FDG mixtures. The probes were selectively quenched in the bandwidth closest to the indicator’s absorption maximum (λmax) at pHs above the indicator pKa (the negative logarithm of the acid dissociation constant). Addition of acid or base to the probes resulted in reversible switching from unquenched to quenched emission. In vivo, the bladders of acetazolamide-treated mice exhibited a wavelength-dependent quenching in Cerenkov emission, with the greatest reduction occurring near the λmax. Ratiometric imaging at 2 wavelengths showed significant decreases in Cerenkov emission at basic pH and allowed the estimation of absolute pH in vivo. Conclusion: We have created contrast agents that selectively quench photons emitted during Cerenkov radiation within a given bandwidth. In the presence of a functional dye, such as a pH indicator, this selective quenching allows for a functional determination of pH in vitro and in vivo. This method can be used to obtain functional information from radiolabeled probes using multimodal imaging. This approach allows for the imaging of nonfluorescent chromophores and is generalizable to any functional dye that absorbs at suitable wavelengths.