Detection of Treatment Response in Triple-Negative Breast Tumors to Paclitaxel Using MRI Cell Size Imaging.

BACKGROUND: Breast cancer treatment response evaluation using the response evaluation criteria in solid tumors (RECIST) guidelines, based on tumor volume changes, has limitations, prompting interest in novel imaging markers for accurate therapeutic effect determination. PURPOSE: To use MRI-measured cell size as a new imaging biomarker for assessing chemotherapy response in breast cancer. STUDY TYPE: Longitudinal; animal model. STUDY POPULATION: Triple-negative human breast cancer cell (MDA-MB-231) pellets (4 groups, n = 7) treated with dimethyl sulfoxide (DMSO) or 10 nM of paclitaxel for 24, 48, and 96 hours, and 29 mice with MDA-MB-231 tumors in right hind limbs treated with paclitaxel (n = 16) or DMSO (n = 13) twice weekly for 3 weeks. FIELD STRENGTH/SEQUENCE: Oscillating gradient spin echo and pulsed gradient spin echo sequences at 4.7 T. ASSESSMENT: MDA-MB-231 cells were analyzed using flowcytometry and light microscopy to assess cell cycle phases and cell size distribution. MDA-MB-231 cell pellets were MR imaged. Mice were imaged weekly, with 9, 6, and 14 being sacrificed for histology after MRI at weeks 1, 2, and 3, respectively. Microstructural parameters of tumors/cell pellets were derived by fitting diffusion MRI data to a biophysical model. STATISTICAL TESTS: One-way ANOVA compared cell sizes and MR-derived parameters between treated and control samples. Repeated measures 2-way ANOVA with Bonferroni post-tests compared temporal changes in MR-derived parameters. A P-value <0.05 was considered statistically significant. RESULTS: In vitro experiments showed that the mean MR-derived cell sizes of paclitaxel-treated cells increased significantly with a 24-hours treatment and decreased (P = 0.06) with a 96-hour treatment. For in vivo xenograft experiments, the paclitaxel-treated tumors showed significant decreases in cell size at later weeks. MRI observations were supported by flowcytometry, light microscopy, and histology. DATA CONCLUSIONS: MR-derived cell size may characterize the cell shrinkage during treatment-induced apoptosis, and may potentially provide new insights into the assessment of therapeutic response. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 4.

Experimental demonstration of diffusion limitations on resolution and SNR in MR microscopy.

PURPOSE: MR microscopy is in principle capable of producing images at cellular resolution (<10 µm), but various factors limit the quality achieved in practice. A recognized limit on the signal to noise ratio and spatial resolution is the dephasing of transverse magnetization caused by diffusion of spins in strong gradients. Such effects may be reduced by using phase encoding instead of frequency encoding read-out gradients. However, experimental demonstration of the quantitative benefits of phase encoding are lacking, and the exact conditions in which it is preferred are not clearly established. We quantify the conditions where phase encoding outperforms a readout gradient with emphasis on the detrimental effects of diffusion on SNR and resolution. METHODS: A 15.2 T Bruker MRI scanner, with 1 T/m gradients, and micro solenoid RF coils < 1 mm in diameter, were used to quantify diffusion effects on resolution and the signal to noise ratio of frequency and phase encoded acquisitions. Frequency and phase encoding's spatial resolution and SNR per square root time were calculated and measured for images at the diffusion limited resolution. The point spread function was calculated and measured for phase and frequency encoding using additional constant time phase gradients with voxels 3-15 µm in dimension. RESULTS: The effect of diffusion during the readout gradient on SNR was experimentally demonstrated. The achieved resolutions of frequency and phase encoded acquisitions were measured via the point-spread-function and shown to be lower than the nominal resolution. SNR per square root time and actual resolution were calculated for a wide range of maximum gradient amplitudes, diffusion coefficients, and relaxation properties. The results provide a practical guide on how to choose between phase encoding and a conventional readout. Images of excised rat spinal cord at 10 µm × 10 µm in-plane resolution demonstrate phase encoding's benefits in the form of higher measured resolution and higher SNR than the same image acquired with a conventional readout. CONCLUSION: We provide guidelines to determine the extent to which phase encoding outperforms frequency encoding in SNR and resolution given a wide range of voxel sizes, sample, and hardware properties.

Diffusion time dependency of extracellular diffusion.

PURPOSE: To quantify the variations of the power-law dependences on diffusion time t or gradient frequency f $$ f $$ of extracellular water diffusion measured by diffusion MRI (dMRI). METHODS: Model cellular systems containing only extracellular water were used to investigate the t / f $$ t/f $$ dependence of D ex $$ {D}_{ex} $$ , the extracellular diffusion coefficient. Computer simulations used a randomly packed tissue model with realistic intracellular volume fractions and cell sizes. DMRI measurements were performed on samples consisting of liposomes containing heavy water(D2 O, deuterium oxide) dispersed in regular water (H2 O). D ex $$ {D}_{ex} $$ was obtained over a broad t $$ t $$ range (∼1-1000 ms) and then fit power-law equations D ex ( t ) = D const + const · t - ϑ t $$ {D}_{ex}(t)={D}_{\mathrm{const}}+\mathrm{const}\cdotp {t}^{-{\vartheta}_t} $$ and D ex ( f ) = D const + const · f ϑ f $$ {D}_{ex}(f)={D}_{\mathrm{const}}+\mathrm{const}\cdotp {f}^{\vartheta_f} $$ . RESULTS: Both simulated and experimental results suggest that no single power-law adequately describes the behavior of D ex $$ {D}_{ex} $$ over the range of diffusion times of most interest in practical dMRI. Previous theoretical predictions are accurate over only limited t $$ t $$ ranges; for example, θ t = θ f = - 1 2 $$ {\theta}_t={\theta}_f=-\frac{1}{2} $$ is valid only for short times, whereas θ t = 1 $$ {\theta}_t=1 $$ or θ f = 3 2 $$ {\theta}_f=\frac{3}{2} $$ is valid only for long times but cannot describe other ranges simultaneously. For the specific t $$ t $$ range of 5-70 ms used in typical human dMRI measurements, θ t = θ f = 1 $$ {\theta}_t={\theta}_f=1 $$ matches the data well empirically. CONCLUSION: The optimal power-law fit of extracellular diffusion varies with diffusion time. The dependency obtained at short or long t $$ t $$ limits cannot be applied to typical dMRI measurements in human cancer or liver. It is essential to determine the appropriate diffusion time range when modeling extracellular diffusion in dMRI-based quantitative microstructural imaging.

Towards differentiation of brain tumor from radiation necrosis using multi-parametric MRI: Preliminary results at 4.7 T using rodent models.

BACKGROUND: It remains a clinical challenge to differentiate brain tumors from radiation-induced necrosis in the brain. Despite significant improvements, no single MRI method has been validated adequately in the clinical setting. METHODS: Multi-parametric MRI (mpMRI) was performed to differentiate 9L gliosarcoma from radiation necrosis in animal models. Five types of MRI methods probed complementary information on different scales i.e., T2 (relaxation), CEST based APT (probing mobile proteins/peptides) and rNOE (mobile macromolecules), qMT (macromolecules), diffusion based ADC (cell density) and SSIFT iAUC (cell size), and perfusion based DSC (blood volume and flow). RESULTS: For single MRI parameters, iAUC and ADC provide the best discrimination of radiation necrosis and brain tumor. For mpMRI, a combination of iAUC, ADC, and APT shows the best classification performance based on a two-step analysis with the Lasso and Ridge regressions. CONCLUSION: A general mpMRI approach is introduced to choosing candidate multiple MRI methods, identifying the most effective parameters from all the mpMRI parameters, and finding the appropriate combination of chosen parameters to maximize the classification performance to differentiate tumors from radiation necrosis.

Rodent Model of Brain Radionecrosis Using Clinical LINAC-Based Stereotactic Radiosurgery.

Purpose: Our purpose was to develop a rodent model of brain radionecrosis using clinical linear accelerator based stereotactic radiosurgery. Methods and Materials: Single fraction maximum prescription points in the mouse's left hemisphere were irradiated using linear accelerator-based stereotactic radiosurgery with multiple arcs at 60 (n = 5), 100 (n = 5), and 140 (n = 5) Gy. Rats (n = 6) were similarly treated with 140 Gy. Gadolinium (Gd)-enhanced magnetic resonance imaging (MRI) was used to track radiation injury in mice over weeks (100 and 140 Gy) or months (60 Gy). Target accuracy was measured by the distance from the prescription point to the center of the earliest Gd-MRI enhancement. Confirmation of necrosis via histology was performed at the subject endpoints. Results: Radiation injury as indicated by Gd-MRI was first identified at 2 weeks (140 Gy), 4 to 6 weeks (100 Gy), and 8 months (60 Gy). A volumetric time course showed rapid growth in the volume of Gd-MRI signal enhancement after the appearance of apparent necrosis. Histopathologic features were consistent with radionecrosis. Conclusions: The presented method uses a commonly available clinical linear accelerator to induce radiation necrosis in both mice and rats. The treatment is modeled after patient therapy for a more direct model of human tissue under a range of doses used in clinical neuro-ablation techniques. The short time to onset of apparent necrosis, accurate targeting of the prescription point, high incidence of necrosis, and similar pathologic features make this a suitable animal model for further research in radionecrosis.

Selective Cell Size MRI Differentiates Brain Tumors from Radiation Necrosis.

Brain metastasis is a common characteristic of late-stage lung cancers. High doses of targeted radiotherapy can control tumor growth in the brain but can also result in radiotherapy-induced necrosis. Current methods are limited for distinguishing whether new parenchymal lesions following radiotherapy are recurrent tumors or radiotherapy-induced necrosis, but the clinical management of these two classes of lesions differs significantly. Here, we developed, validated, and evaluated a new MRI technique termed selective size imaging using filters via diffusion times (SSIFT) to differentiate brain tumors from radiotherapy necrosis in the brain. This approach generates a signal filter that leverages diffusion time dependence to establish a cell size-weighted map. Computer simulations in silico, cultured cancer cells in vitro, and animals with brain tumors in vivo were used to comprehensively validate the specificity of SSIFT for detecting typical large cancer cells and the ability to differentiate brain tumors from radiotherapy necrosis. SSIFT was also implemented in patients with metastatic brain cancer and radiotherapy necrosis. SSIFT showed high correlation with mean cell sizes in the relevant range of less than 20 µm. The specificity of SSIFT for brain tumors and reduced contrast in other brain etiologies allowed SSIFT to differentiate brain tumors from peritumoral edema and radiotherapy necrosis. In conclusion, this new, cell size-based MRI method provides a unique contrast to differentiate brain tumors from other pathologies in the brain. SIGNIFICANCE: This work introduces and provides preclinical validation of a new diffusion MRI method that exploits intrinsic differences in cell sizes to distinguish brain tumors and radiotherapy necrosis.

Improving MR cell size imaging by inclusion of transcytolemmal water exchange.

The goal of the current study is to include transcytolemmal water exchange in MR cell size imaging using the IMPULSED model for more accurate characterization of tissue cellular properties (e.g., apparent volume fraction of intracellular space v in ) and quantification of indicators of transcytolemmal water exchange. We propose a heuristic model that incorporates transcytolemmal water exchange into a multicompartment diffusion-based method (IMPULSED) that was developed previously to extract microstructural parameters (e.g., mean cell size d and apparent volume fraction of intracellular space v in ) assuming no water exchange. For t diff ≤ 5 ms, the water exchange can be ignored, and the signal model is the same as the IMPULSED model. For t diff ≥ 30 ms, we incorporated the modified Kärger model that includes both restricted diffusion and exchange between compartments. Using simulations and previously published in vitro cell data, we evaluated the accuracy and precision of model-derived parameters and determined how they are dependent on SNR and imaging parameters. The joint model provides more accurate d values for cell sizes ranging from 10 to 12 microns when water exchange is fast (e.g., intracellular water pre-exchange lifetime τ in ≤ 100 ms) than IMPULSED, and reduces the bias of IMPULSED-derived estimates of v in , especially when water exchange is relatively slow (e.g., τ in > 200 ms). Indicators of transcytolemmal water exchange derived from the proposed joint model are linearly correlated with ground truth τ in values and can detect changes in cell membrane permeability induced by saponin treatment in murine erythroleukemia cancer cells. Our results suggest this joint model not only improves the accuracy of IMPULSED-derived microstructural parameters, but also provides indicators of water exchange that are usually ignored in diffusion models of tissues.

Does coffee, tea and caffeine consumption reduce the risk of incident breast cancer? A systematic review and network meta-analysis.

OBJECTIVE: We aimed to evaluate the association between coffee and/or tea consumption and breast cancer (BC) risk among premenopausal and postmenopausal women and to conduct a network meta-analysis. DESIGN: Systematic review and network meta-analysis. SETTING: We conducted a systematic review of electronic publications in the last 30 years to identify case-control studies or prospective cohort studies that evaluated the effects of coffee and tea intake. RESULTS: Forty-five studies that included more than 3 323 288 participants were eligible for analysis. Network meta-analysis was performed to determine the effects of coffee and/or tea consumption on reducing BC risk in a dose-dependent manner and differences in coffee/tea type, menopause status, hormone receptor and the BMI in subgroup and meta-regression analyses. According to the first pairwise meta-analysis, low-dose coffee intake and high-dose tea intake may exhibit efficacy in preventing ER(estrogen receptor)- BC, particularly in postmenopausal women. Then, we performed another pairwise and network meta-analysis and determined that the recommended daily doses were 2-3 cups/d of coffee or ≥5 cups/d of tea, which contained a high concentration of caffeine, particularly in postmenopausal women. CONCLUSIONS: Coffee and tea consumption is not associated with a reduction in the overall BC risk in postmenopausal women and is associated with a potentially lower risk of ER- BC. And the highest recommended dose is 2-3 cups of coffee/d or ≥5 cups of tea/d. They are potentially useful dietary protectants for preventing BC.

MRI-cytometry: Mapping nonparametric cell size distributions using diffusion MRI.

PURPOSE: This report introduces and validates a new diffusion MRI-based method, termed MRI-cytometry, which can noninvasively map intravoxel, nonparametric cell size distributions in tissues. METHODS: MRI was used to acquire diffusion MRI signals with a range of diffusion times and gradient factors, and a model was fit to these data to derive estimates of cell size distributions. We implemented a 2-step fitting method to avoid noise-induced artificial peaks and provide reliable estimates of tumor cell size distributions. Computer simulations in silico, experimental measurements on cultured cells in vitro, and animal xenografts in vivo were used to validate the accuracy and precision of the method. Tumors in 7 patients with breast cancer were also imaged and analyzed using this MRI-cytometry approach on a clinical 3 Tesla MRI scanner. RESULTS: Simulations and experimental results confirm that MRI-cytometry can reliably map intravoxel, nonparametric cell size distributions and has the potential to discriminate smaller and larger cells. The application in breast cancer patients demonstrates the feasibility of direct translation of MRI-cytometry to clinical applications. CONCLUSION: The proposed MRI-cytometry method can characterize nonparametric cell size distributions in human tumors, which potentially provides a practical imaging approach to derive specific histopathological information on biological tissues.

Relayed nuclear Overhauser enhancement sensitivity to membrane Cho phospholipids.

PURPOSE: Phospholipids are key constituents of cell membranes and serve vital functions in the regulation of cellular processes; thus, a method for in vivo detection and characterization could be valuable for detecting changes in cell membranes that are consequences of either normal or pathological processes. Here, we describe a new method to map the distribution of partially restricted phospholipids in tissues. METHODS: The phospholipids were measured by signal changes caused by relayed nuclear Overhauser enhancement-mediated CEST between the phospholipid Cho headgroup methyl protons and water at around -1.6 ppm from the water resonance. The biophysical basis of this effect was examined by controlled manipulation of head group, chain length, temperature, degree of saturation, and presence of cholesterol. Additional experiments were performed on animal tumor models to evaluate potential applications of this novel signal while correcting for confounding contributions. RESULTS: Negative relayed nuclear Overhauser dips in Z-spectra were measured from reconstituted Cho phospholipids with cholesterol but not for other Cho-containing metabolites or proteins. Significant contrast was found between tumor and contralateral normal tissue signals in animals when comparing both the measured saturation transfer signal and a more specific imaging metric. CONCLUSION: We demonstrated specific relayed nuclear Overhauser effects in partially restricted phospholipid phantoms and similar effects in solid brain tumors after correcting for confounding signal contributions, suggesting possible translational applications of this novel molecular imaging method, which we name restricted phospholipid transfer.

MicroRNA-375 exacerbates knee osteoarthritis through repressing chondrocyte autophagy by targeting ATG2B.

OBJECTIVE: This study aimed to explore the underlying mechanism of miR-375 in exacerbating osteoarthritis (OA). RESULTS: MiR-375 expression were upregulated in OA cartilage tissues, whereas ATG2B expression was decreased. MiR-375 targeted ATG2B 3' UTR and inhibited its expression in the chondrocytes, and then suppressed autophagy and promoted endoplasmic reticulum stress (ERs). The apoptosis rate of chondrocytes was increased after being transfected with miR-375 mimics. In vivo results further verified that inhibition of miR-375 could relieve OA-related symptoms. CONCLUSION: miR-375 can inhibit the expression of ATG2B in chondrocytes, suppress autophagy and promote the ERs. It suggests that miR-375 could be considered to be a key therapy target for OA. METHODS: Differential expression analyses for mRNA and miRNA microarray datasets from ArrayExpress were performed. MiR-375 and ATG2B expressions in cartilage tissues were detected by qRT-PCR. Dual luciferase assay was applied to verify the targeting relationship between ATG2B and miR-375. In vitro, the role of miR-375 on chondrocyte autophagy and ERs was investigated by western blot and immunofluorescence. The apoptotic rate was quantified by flow cytometry. In vivo, OA mice model was established, HE and Safranin O and Fast Green staining, as well as the OARSI and modified Mankin scores, were applied to measure the OA cartilage damage severity.

The Potential Targets and Mechanisms of a Carbazole and Pyrazole Containing Anticancer Compound.

AIMS: Characterization of a small anticancer compound. BACKGROUND: The development of small molecules as new anti-cancer therapeutics is necessary to improve anti-tumor efficacy and reduce toxicities, especially for the treatment of brain tumors, where only small molecules can effectively cross the brain-blood barrier. Several novel hits were previously selected by concurrently screening colon and glioma cancer cell lines with a sensorconjugated reporter system. Here, we focused on one of them. OBJECTIVE: Elucidating the potential target(s) of a novel anticancer compound. METHODS: Computer-assisted structural and motif analysis (least absolute shrinkage and selection operator or LASSO score) was used to assess compound's targets, then direct kinase activity assays were used for the confirmation; Western blot of phosphorylated kinases, as well as FACS and caspase 3/7 activity assays, were used to decipher the action mechanisms. Finally, the expression profiling of proteins involved in various G-protein pathways by real-time PCR was performed. RESULTS: The small chemical, (4E)-4-[2-(9-ethyl-9H-carbazol-3-yl)hydrazin-1-ylidene]-3-methyl- 4,5-dihydro-1H-pyrazol-5-one, with a formula C18H17N5O and MW of 319.36, designated as VUGX01, was predicted to be a ligand/inhibitor to receptor tyrosine kinases (RTKs) by computer analysis (least absolute shrinkage and selection operator or LASSO score). However, direct analysis with recombinant kinases showed that it is not an effective inhibitor to the popular receptor kinases at 1µM concentration. This compound can activate caspases in some tumor cell lines but has minimal effects on the cell cycle. Drug treatments lead to the changes in phosphorylation of AKT and c- RAF, as well as the expression level of MAP2K, suggesting this compound may interact with Gprotein coupled receptors (GPCRs). The expression profiling of 82 proteins involved in various Gprotein pathways by real-time PCR showed that the treatment up-regulates the expression of several proteins, including angiotensinogen, angiotensin II receptor, and IP3-kinase catalytic subunit gamma. CONCLUSION: VUGX01 can effectively block proliferation and induce apoptosis of certain types of cancer cells, even it is predicted by high LASSO score, but it is not an effective RTKs inhibitor, it may inhibit cell growth through acting as a novel ligand to one or several GPCRs.

Magnetic resonance imaging of mean cell size in human breast tumors.

PURPOSE: Cell size is a fundamental characteristic of all tissues, and changes in cell size in cancer reflect tumor status and response to treatments, such as apoptosis and cell-cycle arrest. Unfortunately, cell size can currently be obtained only by pathological evaluation of tumor tissue samples obtained invasively. Previous imaging approaches are limited to preclinical MRI scanners or require relatively long acquisition times that are impractical for clinical imaging. There is a need to develop cell-size imaging for clinical applications. METHODS: We propose a clinically feasible IMPULSED (imaging microstructural parameters using limited spectrally edited diffusion) approach that can characterize mean cell sizes in solid tumors. We report the use of a combination of pulse sequences, using different gradient waveforms implemented on clinical MRI scanners and analytical equations based on these waveforms to analyze diffusion-weighted MRI signals and derive specific microstructural parameters such as cell size. We also describe comprehensive validations of this approach using computer simulations, cell experiments in vitro, and animal experiments in vivo and demonstrate applications in preoperative breast cancer patients. RESULTS: With fast acquisitions (~7 minutes), IMPULSED can provide high-resolution (1.3 mm in-plane) mapping of mean cell size of human tumors in vivo on clinical 3T MRI scanners. All validations suggest that IMPULSED provides accurate and reliable measurements of mean cell size. CONCLUSION: The proposed IMPULSED method can assess cell-size variations in tumors of breast cancer patients, which may have the potential to assess early response to neoadjuvant therapy.

Substantive molecular and histological changes within the meniscus with tears.

BACKGROUND: The meniscus plays a vital role in the normal biomechanics of the knee. However, it is not well studied at the molecular level. The purpose of this study was to determine whether molecular and pathological changes in the meniscal tissue vary depending on the presence or absence of meniscal and/or anterior cruciate ligament tear (ACL). METHODS: Six normal menisci (group A), seven simple torn menisci (group B) and seven torn menisci with concomitant anterior cruciate ligament tears (group C) were collected. We observed the pathological changes in the menisci and used real-time polymerase chain reaction along with immunohistochemistry and in situ hybridisation to examine the levels of ACAN, ADAMTS5, COL10A1, CEBPß, MMP13 and miR-381-3p, miR-455-3p, miR-193b-3p, miR-92a-3p, respectively. Patients were scored preoperatively and postoperatively using the Lysholm Knee Scoring Scale and International Knee Documentation Committee Subjective Knee Evaluation Form. RESULTS: Compared with group A, the expression levels of ADAMTS5, COL10A1, CEBPß, and MMP13 and all the miRNAs were increased while ACAN was down-regulated in groups B and C. Additionally, the gene expression and miRNA levels were higher in group C than that in group B, except for ACAN, which was lower. Several fibrochondrocytes strongly expressed ADAMTS5, CEBPß, and MMP13 in groups B and C and had high levels of miR-381-3p and miR-455-3p than that in group A. Postoperative Lysholm and IKDC scores were higher in group B than in group C. CONCLUSIONS: Our findings suggest that the meniscus tended to degenerate after it was injured, especially when combined with a torn ACL. The miRNAs investigated in this study might also contribute to meniscus degeneration. Patients with a combined injury patterns might have relatively worse joint function.

The combination of Astragalus membranaceus extract and ligustrazine to improve the inflammation in rats with thrombolytic cerebral ischemia.

The purpose of the study was to evaluate the effect of Astragalus membranaceus extract and ligustrazine combination on ameliorating inflammation in cerebral ischemic rats that have undergone thrombolysis. Astragalus membranaceus and ligustrazine per se, or a combination of A. membranaceus and ligustrazine was administered by intraperitoneal injection immediately after surgery and sham surgery. After the induction of thrombolysis, the neurological function was measured and cerebral lesion volume was determined. The regulatory T cells in the spleen were measured by flow cytometry. To explore the protective effects of the combination drug on the neurological function and inflammation, the expression of transcription factor Foxp3 and cytokines, including transforming growth factor beta 1, interleukin 10, interleukin 4, interleukin 1 beta, interferon gamma, interleukin 17, in damaged brain was examined using reverse transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. The cerebral lesion volume was markedly reduced in the combination drug-treated rats compared to the rats treated with either A. membranaceus or ligustrazine alone (P < 0.05). The neurological function, regulatory T cells, transcription factor Foxp3, transforming growth factor beta 1, interleukin 10, and interleukin 4 were markedly elevated in the rats treated with combination drugs (P < 0.05). The expression of interleukin 1 beta, interferon gamma, and interleukin 17 was reduced in the rats treated with combination drug therapy (P < 0.05). Treatment with a combination of A. membranaceus and ligustrazine can ameliorate inflammation after thrombolysis and regulate the related cytokines by elevating the expression of endogenous regulatory T cells.

In vivo magnetic resonance imaging of treatment-induced apoptosis.

Imaging apoptosis could provide an early and specific means to monitor tumor responses to treatment. To date, despite numerous attempts to develop molecular imaging approaches, there is still no widely-accepted and reliable method for in vivo imaging of apoptosis. We hypothesized that the distinct cellular morphologic changes associated with treatment-induced apoptosis, such as cell shrinkage, cytoplasm condensation, and DNA fragmentation, can be detected by temporal diffusion spectroscopy imaging (TDSI). Cetuximab-induced apoptosis was assessed in vitro and in vivo with cetuximab-sensitive (DiFi) and insensitive (HCT-116) human colorectal cancer cell lines by TDSI. TDSI findings were complemented by flow cytometry and immunohistochemistry. Cell cycle analysis and flow cytometry detected apoptotic cell shrinkage in cetuximab-treated DiFi cells, and significant apoptosis was confirmed by histology. TDSI-derived parameters quantified key morphological changes including cell size decreases during apoptosis in responsive tumors that occurred earlier than gross tumor volume regression. TDSI provides a unique measurement of apoptosis by identifying cellular characteristics, particularly cell shrinkage. The method will assist in understanding the underlying biology of solid tumors and predict tumor response to therapies. TDSI is free of any exogenous agent or radiation, and hence is very suitable to be incorporated into clinical applications.

Spin-lock imaging of early tissue pH changes in ischemic rat brain.

We have previously reported that the dispersion of spin-lattice relaxation rates in the rotating frame (R1ρ ) of tissue water protons at high field can be dominated by chemical exchange contributions. Ischemia in brain causes changes in tissue pH, which in turn may affect proton exchange rates. Amide proton transfer (APT, a form of chemical exchange saturation transfer) has been shown to be sensitive to chemical exchange rates and able to detect pH changes non-invasively following ischemic stroke. However, the specificity of APT to pH changes is decreased because of the influence of several other factors that affect magnetization transfer. R1ρ is less influenced by such confounding factors and thus may be more specific for detecting variations in pH. Here, we applied a spin-locking sequence to detect ischemic stroke in animal models. Although R1ρ images acquired with a single spin-locking amplitude (ω1 ) have previously been used to assess stroke, here we use ΔR1ρ , which is the difference in R1ρ values acquired with two different locking fields to emphasize selectively the contribution of chemical exchange effects. Numerical simulations with different exchange rates and measurements of tissue homogenates with different pH were performed to evaluate the specificity of ΔR1ρ to detect tissue acidosis. Spin-lock and APT data were acquired on five rat brains after ischemic strokes induced via middle cerebral artery occlusions. Correlations between these data were analyzed at different time points after the onset of stroke. The results show that ΔR1ρ (but not R1ρ acquired with a single ω1 ) was significantly correlated with APT metrics consistent with ΔR1ρ varying with pH.

Assignment of the molecular origins of CEST signals at 2 ppm in rat brain.

PURPOSE: Chemical exchange saturation transfer effects at 2 ppm (CEST@2ppm) in brain have previously been interpreted as originating from creatine. However, protein guanidino amine protons may also contribute to CEST@2ppm. This study aims to investigate the molecular origins and specificity of CEST@2ppm in brain. METHODS: Two experiments were performed: (i) samples containing egg white albumin and creatine were dialyzed using a semipermeable membrane to demonstrate that proteins and creatine can be separated by this method; and (ii) tissue homogenates of rat brain with and without dialysis to remove creatine were studied to measure the relative contributions of proteins and creatine to CEST@2ppm. RESULTS: The experiments indicate that dialysis can successfully remove creatine from proteins. Measurements on tissue homogenates show that, with the removal of creatine via dialysis, CEST@2ppm decreases to approximately 34% of its value before dialysis, which indicates that proteins and creatine have comparable contribution to the CEST@2ppm in brain. However, considering the contribution from peptides and amino acids to CEST@2ppm, creatine may have much less contribution to CEST@2ppm. CONCLUSIONS: The contribution of proteins, peptides, and amino acids to CEST@2ppm cannot be neglected. The CEST@2ppm measurements of creatine in rat brain should be interpreted with caution. Magn Reson Med 78:881-887, 2017. © 2017 International Society for Magnetic Resonance in Medicine.

Impact of transcytolemmal water exchange on estimates of tissue microstructural properties derived from diffusion MRI.

PURPOSE: To investigate the influence of transcytolemmal water exchange on estimates of tissue microstructural parameters derived from diffusion MRI using conventional PGSE and IMPULSED methods. METHODS: Computer simulations were performed to incorporate a broad range of intracellular water life times τin (50-∞ ms), cell diameters d (5-15 µm), and intrinsic diffusion coefficient Din (0.6-2 µm2 /ms) for different values of signal-to-noise ratio (SNR) (10 to 50). For experiments, murine erythroleukemia (MEL) cancer cells were cultured and treated with saponin to selectively change cell membrane permeability. All fitted microstructural parameters from simulations and experiments in vitro were compared with ground-truth values. RESULTS: Simulations showed that, for both PGSE and IMPULSED methods, cell diameter d can be reliably fit with sufficient SNR (≥ 50), whereas intracellular volume fraction fin is intrinsically underestimated due to transcytolemmal water exchange. Din can be reliably fit only with sufficient SNR and using the IMPULSED method with short diffusion times. These results were confirmed with those obtained in the cell culture experiments in vitro. CONCLUSION: For the sequences and models considered in this study, transcytolemmal water exchange has minor effects on the fittings of d and Din with physiologically relevant membrane permeabilities if the SNR is sufficient (> 50), but fin is intrinsically underestimated. Magn Reson Med 77:2239-2249, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

MR imaging of a novel NOE-mediated magnetization transfer with water in rat brain at 9.4 T.

PURPOSE: To detect, map, and quantify a novel nuclear Overhauser enhancement (NOE)-mediated magnetization transfer (MT) with water at approximately -1.6 ppm [NOE(-1.6)] in rat brain using MRI. METHODS: Continuous wave MT sequences with a variety of radiofrequency irradiation powers were optimized to achieve the maximum contrast of this NOE(-1.6) effect at 9.4 T. The distribution of effect magnitudes, resonance frequency offsets, and line widths in healthy rat brains and the differences of the effect between tumors and contralateral normal brains were imaged and quantified using a multi-Lorentzian fitting method. MR measurements on reconstituted model phospholipids as well as two cell lines (HEK293 and 9L) were also performed to investigate the possible molecular origin of this NOE. RESULTS: Our results suggest that the NOE(-1.6) effect can be detected reliably in rat brain. Pixel-wise fittings demonstrated the regional variations of the effect. Measurements in a rodent tumor model showed that the amplitude of NOE(-1.6) in brain tumor was significantly diminished compared with that in normal brain tissue. Measurements of reconstituted phospholipids suggest that this effect may originate from choline phospholipids. CONCLUSION: NOE(-1.6) could be used as a new biomarker for the detection of brain tumor. Magn Reson Med 78:588-597, 2017. © 2016 International Society for Magnetic Resonance in Medicine.