(1)H NMR signal amplification by reversible exchange (SABRE) was observed for pyridine and pyridine-d5 at 9.4 T, a field that is orders of magnitude higher than what is typically utilized to achieve the conventional low-field SABRE effect. In addition to emissive peaks for the hydrogen spins at the ortho positions of the pyridine substrate (both free and bound to the metal center), absorptive signals are observed from hyperpolarized orthohydrogen and Ir-complex dihydride. Real-time kinetics studies show that the polarization build-up rates for these three species are in close agreement with their respective (1)H T1 relaxation rates at 9.4 T. The results suggest that the mechanism of the substrate polarization involves cross-relaxation with hyperpolarized species in a manner similar to the spin-polarization induced nuclear Overhauser effect. Experiments utilizing pyridine-d5 as the substrate exhibited larger enhancements as well as partial H/D exchange for the hydrogen atom in the ortho position of pyridine and concomitant formation of HD molecules. While the mechanism of polarization enhancement does not explicitly require chemical exchange of hydrogen atoms of parahydrogen and the substrate, the partial chemical modification of the substrate via hydrogen exchange means that SABRE under these conditions cannot rigorously be referred to as a non-hydrogenative parahydrogen induced polarization process.
Spin order obtained in the strong coupling regime of protons from parahydrogen-induced hyperpolarization (PHIP) is initially captured as an ensemble of singlet states. For biomedical applications of PHIP, locking this spin order on long-lived heteronuclear storage nuclei increases spectral dispersion, reduces background interference from water protons, and eliminates the need to synchronize subsequent detection pulse sequences to accrued singlet-state evolution. A variety of traditional sequences such as INEPT or HMQC are available to interconvert heteronuclear single quantum coherences at high field, but new approaches are required for converting singlet states into heteronuclear single quantum coherences at low field in the strong coupling regime of protons. Described here is a consolidated pulse sequence that achieves this transformation of singlet-state spin order into heteronuclear magnetization across a wide range of scalar couplings in AA'X spin systems. Analytic solutions to the spin evolution are presented, and performance was validated experimentally in the parahydrogen addition product, 2-hydroxyethyl 1-(13)C-propionate-d(3). Hyperpolarized carbon-13 signals were enhanced by a factor of several million relative to Boltzmann polarization in a static magnetic field of 47.5 mT (~13% polarization). We anticipate that this pulse sequence will provide efficient conversion of parahydrogen spin order over a broad range of emerging PHIP agents that feature AA'X spin systems.
Selective inhibition of oncogenic targets and associated signaling pathways forms the basis of personalized cancer medicine. The clinical success of (V600E)BRAF inhibition in melanoma, coupled with the emergence of acquired resistance, underscores the importance of rigorously validating quantitative biomarkers of treatment response in this and similar settings. Because constitutive activation of BRAF leads to proliferation in tumors, we explored 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET to noninvasively quantify changes in tumor proliferation that are associated with pharmacologic inhibition of (V600E)BRAF downstream effectors and that precede changes in tumor volume.
Human colorectal cancer (CRC) cell lines expressing (V600E)BRAF were used to explore relationships between upregulation of p27 and phosphorylation of BRAF downstream effectors on small-molecule (V600E)BRAF inhibitor exposure. Athymic nude mice bearing (V600E)BRAF-expressing human CRC cell line xenografts were treated with a small-molecule (V600E)BRAF inhibitor (or vehicle) daily for 10 d. Predictive (18)F-FLT PET was conducted before changes in tumor volume occurred. Correlations were evaluated among PET, inhibition of phosphorylated MEK (p-MEK) and phosphorylated-ERK (p-ERK) by Western blot, tumor proliferation by histology, and small-molecule exposure by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS).
Treatment of CRC cell lines with PLX4720 reduced proliferation associated with target inhibition and upregulation of p27. In vivo, PLX4720 treatment reduced (18)F-FLT uptake, but not (18)F-FDG uptake, in Lim2405 xenografts before quantifiable differences in xenograft volume. Reduced (18)F-FLT PET reflected a modest, yet significant, reduction of Ki67 immunoreactivity, inhibition of p-MEK and p-ERK, and elevated tumor cell p27 protein levels. Both (18)F-FLT PET and (18)F-FDG PET accurately reflected a lack of response in HT-29 xenografts, which MALDI imaging mass spectrometry suggested may have stemmed from limited PLX4720 exposure.
We used preclinical models of CRC to demonstrate (18)F-FLT PET as a sensitive predictor of response to (V600E)BRAF inhibitors. Because (18)F-FLT PET predicted reduced proliferation associated with attenuation of BRAF downstream effectors, yet (18)F-FDG PET did not, these data suggest that (18)F-FLT PET may represent an alternative to (18)F-FDG PET for quantifying clinical responses to BRAF inhibitors.
Magnetization transfer (MT) provides an indirect means to detect noninvasively variations in macromolecular contents in biological tissues, but, so far, there have been only a few quantitative MT (qMT) studies reported in cancer, all of which used off-resonance pulsed saturation methods. This article describes the first implementation of a different qMT approach, selective inversion recovery (SIR), for the characterization of tumor in vivo using a rodent glioma model. The SIR method is an on-resonance method capable of fitting qMT parameters and T1 relaxation time simultaneously without mapping B0 and B1 , which is very suitable for high-field qMT measurements because of the lower saturation absorption rate. The results show that the average pool size ratio (PSR, the macromolecular pool versus the free water pool) in rat 9 L glioma (5.7%) is significantly lower than that in normal rat gray matter (9.2%) and white matter (17.4%), which suggests that PSR is potentially a sensitive imaging biomarker for the assessment of brain tumor. Despite being less robust, the estimated MT exchange rates also show clear differences from normal tissues (19.7 Hz for tumors versus 14.8 and 10.2 Hz for gray and white mater, respectively). In addition, the influence of confounding effects, e.g. B1 inhomogeneity, on qMT parameter estimates is investigated with numerical simulations. These findings not only help to better understand the changes in the macromolecular contents of tumors, but are also important for the interpretation of other imaging contrasts, such as chemical exchange saturation transfer of tumors.
Ionizing radiation treatment is used in over half of all cancer patients, thus determining the mechanisms of response or resistance is critical for the development of novel treatment approaches.
In this report, we utilize a high-content peptide array platform that performs multiplex kinase assays with real-time kinetic readout to investigate the mechanism of radiation response in vascular endothelial cells. We applied this technology to irradiated human umbilical vein endothelial cells (HUVEC).
We identified 49 specific tyrosine phosphopeptides that were differentially affected by irradiation over a time course of 1h. In one example, the Tropomyosin receptor kinase (Trk) family members, TrkA and TrkB, showed transient activation between 2 and 15 min following irradiation. When we targeted TrkA and TrkB using small molecule inhibitors, HUVEC were protected from radiation damage. Conversely, stimulation of TrkA using gambogic amide promoted radiation enhancement.
Thus, we show that our approach not only can identify rapid changes in kinase activity but also identify novel targets such as TrkA. TrkA inhibition resulted in radioprotection that correlated with enhanced repair of radiation-induced damage while TrkA stimulation by gambogic amide produced radiation sensitization.
A new approach has been developed to quantify cell sizes and intracellular volume fractions using temporal diffusion spectroscopy with diffusion-weighted acquisitions.
Temporal diffusion spectra may be used to characterize tissue microstructure by measuring the effects of restrictions over a range of diffusion times. Oscillating gradients have been used previously to probe variations on cellular and subcellular scales, but their ability to accurately measure cell sizes larger than 10 μm is limited. By combining measurements made using oscillating gradient spin echo (OGSE) and a conventional pulsed gradient spin echo (PGSE) acquisition with a single, relatively long diffusion time, we can accurately quantify cell sizes and intracellular volume fractions.
Based on a two compartment model (incorporating intra- and extracellular spaces), accurate estimates of cell sizes and intracellular volume fractions were obtained in vitro for (i) different cell types with sizes ranging from 10 to 20 μm, (ii) different cell densities, and (iii) before and after anticancer treatment.
Hybrid OGSE-PGSE acquisitions sample a larger region of temporal diffusion spectra and can accurately quantify cell sizes over a wide range. Moreover, the maximum gradient strength used was lower than 15 G/cm, suggesting that this approach is translatable to practical MR imaging.
A temporal diffusion MRI spectroscopy based approach has been developed to quantify cancer cell size and density in vivo.
A novel imaging microstructural parameters using limited spectrally edited diffusion (IMPULSED) method selects a specific limited diffusion spectral window for an accurate quantification of cell sizes ranging from 10 to 20 μm in common solid tumors. In practice, it is achieved by a combination of a single long diffusion time pulsed gradient spin echo (PGSE) and three low-frequency oscillating gradient spin echo (OGSE) acquisitions. To validate our approach, hematoxylin and eosin staining and immunostaining of cell membranes, in concert with whole slide imaging, were used to visualize nuclei and cell boundaries, and hence, enabled accurate estimates of cell size and cellularity.
Based on a two compartment model (incorporating intra- and extracellular spaces), accurate estimates of cell sizes were obtained in vivo for three types of human colon cancers. The IMPULSED-derived apparent cellularities showed a stronger correlation (r = 0.81; P < 0.0001) with histology-derived cellularities than conventional ADCs (r = -0.69; P < 0.03).
The IMPULSED approach samples a specific region of temporal diffusion spectra with enhanced sensitivity to length scales of 10-20 μm, and enables measurements of cell sizes and cellularities in solid tumors in vivo. Magn Reson Med 78:156-164, 2017. © 2016 International Society for Magnetic Resonance in Medicine.