Radiation impairs perineural invasion by modulating the nerve microenvironment.

PURPOSE: Perineural invasion (PNI) by cancer cells is an ominous clinical event that is associated with increased local recurrence and poor prognosis. Although radiation therapy (RT) may be delivered along the course of an invaded nerve, the mechanisms through which radiation may potentially control PNI remain undefined. EXPERIMENTAL DESIGN: An in vitro co-culture system of dorsal root ganglia (DRG) and pancreatic cancer cells was used as a model of PNI. An in vivo murine sciatic nerve model was used to study how RT to nerve or cancer affects nerve invasion by cancer. RESULTS: Cancer cell invasion of the DRG was partially dependent on DRG secretion of glial-derived neurotrophic factor (GDNF). A single 4 Gy dose of radiation to the DRG alone, cultured with non-radiated cancer cells, significantly inhibited PNI and was associated with decreased GDNF secretion but intact DRG viability. Radiation of cancer cells alone, co-cultured with non-radiated nerves, inhibited PNI through predominantly compromised cancer cell viability. In a murine model of PNI, a single 8 Gy dose of radiation to the sciatic nerve prior to implantation of non-radiated cancer cells resulted in decreased GDNF expression, decreased PNI by imaging and histology, and preservation of sciatic nerve motor function. CONCLUSIONS: Radiation may impair PNI through not only direct effects on cancer cell viability, but also an independent interruption of paracrine mechanisms underlying PNI. RT modulation of the nerve microenvironment may decrease PNI, and hold significant therapeutic implications for RT dosing and field design for patients with cancers exhibiting PNI.

Preclinical study of treatment response in HCT-116 cells and xenografts with (1) H-decoupled (31) P MRS.

The topoisomerase I inhibitor, irinotecan, and its active metabolite SN-38 have been shown to induce G(2) /M cell cycle arrest without significant cell death in human colon carcinoma cells (HCT-116). Subsequent treatment of these G(2) /M-arrested cells with the cyclin-dependent kinase inhibitor, flavopiridol, induced these cells to undergo apoptosis. The goal of this study was to develop a noninvasive metabolic biomarker for early tumor response and target inhibition of irinotecan followed by flavopiridol treatment in a longitudinal study. A total of eleven mice bearing HCT-116 xenografts were separated into two cohorts where one cohort was administered saline and the other treated with a sequential course of irinotecan followed by flavopiridol. Each mouse xenograft was longitudinally monitored with proton ((1) H)-decoupled phosphorus ((31) P) magnetic resonance spectroscopy (MRS) before and after treatment. A statistically significant decrease in phosphocholine (p = 0.0004) and inorganic phosphate (p = 0.0103) levels were observed in HCT-116 xenografts following treatment, which were evidenced within twenty-four hours of treatment completion. Also, a significant growth delay was found in treated xenografts. To discern the underlying mechanism for the treatment response of the xenografts, in vitro HCT-116 cell cultures were investigated with enzymatic assays, cell cycle analysis, and apoptotic assays. Flavopiridol had a direct effect on choline kinase as measured by a 67% reduction in the phosphorylation of choline to phosphocholine. Cells treated with SN-38 alone underwent 83 ± 5% G(2) /M cell cycle arrest compared to untreated cells. In cells, flavopiridol alone induced 5 ± 1% apoptosis while the sequential treatment (SN-38 then flavopiridol) resulted in 39 ± 10% apoptosis. In vivo (1) H-decoupled (31) P MRS indirectly measures choline kinase activity. The decrease in phosphocholine may be a potential indicator of early tumor response to the sequential treatment of irinotecan followed by flavopiridol in noninvasive and/or longitudinal studies.

Fat-free MRI based on magnetization exchange.

An MRI technique is proposed for complete fat signal elimination. This approach exploits the fact that water rapidly exchanges magnetization with protons in protein and membrane phospholipid of tissue and cells but does not exchange magnetization with triglyceride or fat protons in the tissue. Saturation of the proton signal from protein and membrane phospholipid thus results in partial saturation of the water proton signal, allowing acquisition of an image including a portion of the water signal and the full fat signal. Subtraction of this image from the standard image, containing both water and fat signals, results in an image in which all fat signal is cancelled. This fat-free image is sensitive to magnetization transfer and to water density and relaxation time, providing the possibility of additional contrast. Unlike most fat suppression techniques, this method is not compromised by the static or radiofrequency field heterogeneity and is equally efficient for all fat resonances independent of their chemical shift frequency.

Slc25a12 disruption alters myelination and neurofilaments: a model for a hypomyelination syndrome and childhood neurodevelopmental disorders.

BACKGROUND: SLC25A12, a susceptibility gene for autism spectrum disorders that is mutated in a neurodevelopmental syndrome, encodes a mitochondrial aspartate-glutamate carrier (aspartate-glutamate carrier isoform 1 [AGC1]). AGC1 is an important component of the malate/aspartate shuttle, a crucial system supporting oxidative phosphorylation and adenosine triphosphate production. METHODS: We characterized mice with a disruption of the Slc25a12 gene, followed by confirmatory in vitro studies. RESULTS: Slc25a12-knockout mice, which showed no AGC1 by immunoblotting, were born normally but displayed delayed development and died around 3 weeks after birth. In postnatal day 13 to 14 knockout brains, the brains were smaller with no obvious alteration in gross structure. However, we found a reduction in myelin basic protein (MBP)-positive fibers, consistent with a previous report. Furthermore, the neocortex of knockout mice contained abnormal neurofilamentous accumulations in neurons, suggesting defective axonal transport and/or neurodegeneration. Slice cultures prepared from knockout mice also showed a myelination defect, and reduction of Slc25a12 in rat primary oligodendrocytes led to a cell-autonomous reduction in MBP expression. Myelin deficits in slice cultures from knockout mice could be reversed by administration of pyruvate, indicating that reduction in AGC1 activity leads to reduced production of aspartate/N-acetylaspartate and/or alterations in the dihydronicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide(+) ratio, resulting in myelin defects. CONCLUSIONS: Our data implicate AGC1 activity in myelination and in neuronal structure and indicate that while loss of AGC1 leads to hypomyelination and neuronal changes, subtle alterations in AGC1 expression could affect brain development, contributing to increased autism susceptibility.

Proton MRS detects metabolic changes in hormone sensitive and resistant human prostate cancer models CWR22 and CWR22r.

17-Allylamino, 17-demethoxygeldanamycin (17-AAG), an effective inhibitor of the heat shock protein hsp90, preferentially inhibiting tumor hsp90 compared to hsp90 from normal cells, has shown promising results against several cancers, including hormone-resistant prostate cancer. Levels of several oncogenic proteins critical to tumor growth and progression, such as androgen receptor and HER2/neu, were reduced 4 h post 17-allylamino, 17-demethoxygeldanamycin treatment. Posttreatment metabolic changes have also been observed in several tumor cell lines. In this study, total choline distributions in hormone sensitive CWR22 and hormone resistant CWR22r prostate cancer xenograft tumors in mice were measured before and at 4 h and 48 h after a single-bolus 17-allylamino, 17-demethoxygeldanamycin treatment at 100 mg/kg, using proton MR spectroscopy. Our results show that tumor total choline levels declined 4 h after the treatment for CWR22 (P = 0.001) and 48 h post treatment for CWR22r (P = 0.003). Metabolic changes, in particular of total choline intensity detected by proton magnetic resonance spectroscopic imaging (MRSI), are consistent with the observed immunohistochemistry changes, tumor growth inhibition for CWR22r (P = 0.01 at 14 days post treatment), and a constant prostate specific antigen level versus increasing prostate specific antigen for control CWR22 (P = 0.01). Metabolic changes in total choline by proton MRSI can be used as an early biomarker of response for advanced-stage prostate cancer in targeted therapy such as 17-allylamino, 17-demethoxygeldanamycin.

Imaging transgene activity in vivo.

The successful translation of gene therapy for clinical application will require the assessment of transgene activity as a measure of the biological function of a therapeutic transgene. Although current imaging permits the noninvasive detection of transgene expression, the critical need for quantitative imaging of the action of the expressed transgene has not been met. In vivo magnetic resonance spectroscopic imaging (MRSI) was applied to quantitatively delineate both the concentration and activity of a cytosine deaminase-uracil phosphoribosyltransferase (CD-UPRT) fusion enzyme expressed from a transgene. MRSI enabled the generation of anatomically accurate maps of the intratumoral heterogeneity in fusion enzyme activity. We observed an excellent association between the CD-UPRT concentration and activity and the percentage of CD-UPRT(+) cells. Moreover, the regional levels of UPRT activity, as measured by imaging, correlated well with the biological affect of the enzyme. This study presents a translational imaging paradigm for precise, in vivo measurements of transgene activity with potential applications in both preclinical and clinical settings.

In vivo 5-fluorouracil and fluoronucleotide T1 relaxation time measurements using the variable nutation angle method.

19Fluorine NMRS has the potential to enable noninvasive predictions of tumor response to 5-fluorouracil (5FU) therapy based on tumor pharmacokinetics. Knowledge of the T1's of 5FU and its fluoronucleotide anabolites (FNuc) is required for quantitative spectral analysis and selection of optimal pulse parameters. We used the variable nutation angle (VNA) method to determine T1's of 5FU and FNuc in subcutaneous Walker 256 rat mammary carcinosarcoma tumors transfected with a cytosine deaminase/uracil phosphoribosyltransferase fusion gene. We calibrated in vivo NAs using methoxydifluoroacetate to ensure the accuracy of these measurements. The T1's were calculated based on signal intensities acquired with NAs of 20 degrees, 35 degrees, 45 degrees, 60 degrees, and 75 degrees. The acquisition order of these NAs was shuffled to reduce the effect of signal variations. The determined T1's for 5FU and FNuc (2.3 +/- 0.1 s and 1.3 +/- 0.1 s, respectively) represent the first reported in vivo measurements for these metabolites in tumor.