Mn-alginate gels as a novel system for controlled release of Mn2+ in manganese-enhanced MRI

The aim of the present study was to test alginate gels of different compositions as a system for controlled release of manganese ions (Mn2+) for application in manganese‐enhanced MRI (MEMRI), in order to circumvent the challenge of achieving optimal MRI resolution without resorting to high, potentially cytotoxic doses of Mn2+. Elemental analysis and stability studies of Mn‐alginate revealed marked differences in ion binding capacity, rendering Mn/Ba‐alginate gels with high guluronic acid content most stable. The findings were corroborated by corresponding differences in the release rate of Mn2+ from alginate beads in vitro using T1‐weighted MRI. Furthermore, intravitreal (ivit) injection of Mn‐alginate beads yielded significant enhancement of the rat retina and retinal ganglion cell (RGC) axons 24 h post‐injection. Subsequent compartmental modelling and simulation of ivit Mn2+ transport and concentration revealed that application of slow release contrast agents can achieve a significant reduction of ivit Mn2+ concentration compared with bolus injection. This is followed by a concomitant increase in the availability of ivit Mn2+ for uptake by RGC, corresponding to significantly increased time constants. Our results provide proof‐of‐concept for the applicability of Mn‐alginate gels as a system for controlled release of Mn2+ for optimized MEMRI application

Dynamic contrast enhanced MRI detects early response to adoptive NK cellular immunotherapy targeting the NG2 proteoglycan in a rat model of glioblastoma

There are currently no established radiological parameters that predict response to immunotherapy. We hypothesised that multiparametric, longitudinal magnetic resonance imaging (MRI) of physiological parameters and pharmacokinetic models might detect early biological responses to immunotherapy for glioblastoma targeting NG2/CSPG4 with mAb9.2.27 combined with natural killer (NK) cells. Contrast enhanced conventional T1-weighted MRI at 7±1 and 17±2 days post-treatment failed to detect differences in tumour size between the treatment groups, whereas, follow-up scans at 3 months demonstrated diminished signal intensity and tumour volume in the surviving NK+mAb9.2.27 treated animals. Notably, interstitial volume fraction (ve), was significantly increased in the NK+mAb9.2.27 combination therapy group compared mAb9.2.27 and NK cell monotherapy groups (p = 0.002 and p = 0.017 respectively) in cohort 1 animals treated with 1 million NK cells. ve was reproducibly increased in the combination NK+mAb9.2.27 compared to NK cell monotherapy in cohort 2 treated with increased dose of 2 million NK cells (p<0.0001), indicating greater cell death induced by NK+mAb9.2.27 treatment. The interstitial volume fraction in the NK monotherapy group was significantly reduced compared to mAb9.2.27 monotherapy (p<0.0001) and untreated controls (p = 0.014) in the cohort 2 animals. NK cells in monotherapy were unable to kill the U87MG cells that highly expressed class I human leucocyte antigens, and diminished stress ligands for activating receptors. A significant association between apparent diffusion coefficient (ADC) of water and ve in combination NK+mAb9.2.27 and NK monotherapy treated tumours was evident, where increased ADC corresponded to reduced ve in both cases. Collectively, these data support histological measures at end-stage demonstrating diminished tumour cell proliferation and pronounced apoptosis in the NK+mAb9.2.27 treated tumours compared to the other groups. In conclusion, ve was the most reliable radiological parameter for detecting response to intralesional NK cellular therapy

Characterization of tumor microvascular structure and permeability: comparison between magnetic resonance imaging and intravital confocal imaging

Solid tumors are characterized by abnormal blood vessel organization, structure, and function. These abnormalities give rise to enhanced vascular permeability and may predict therapeutic responses. The permeability and architecture of the microvasculature in human osteosarcoma tumors growing in dorsal window chambers in athymic mice were measured by confocal laser scanning microscopy (CLSM) and dynamic contrast enhanced magnetic resonance imaging (DCE-MRI). Dextran (40 kDa) and Gadomer were used as molecular tracers for CLSM and DCE-MRI, respectively. A significant correlation was found between permeability indicators. The extravasation rate Ki as measured by CLSM correlated positively with DCE-MRI parameters, such as the volume transfer constant Ktrans and the initial slope of the contrast agent concentration-time curve. This demonstrates that these two techniques give complementary information. Extravasation was further related to microvascular structure and was found to correlate with the fractal dimension and vascular density. The structural parameter values that were obtained from CLSM images were higher for abnormal tumor vasculature than for normal vessels

Expression of glial cell markers and NK cell ligands on U87MG tumour cells.

<p>(<b>A</b>) Mean Fluorescence Intensity (MFI) histograms and % cells expressing glial markers (GFAP,Nestin, Vimentin, and A2B5), <b>(B)</b> MFI histograms and % cells expressing class I HLA ligands (HLA-A,-B,-C), non-classical HLA-G and HLA-E, as well as HLA-DR,DP,DQ. <b>(C)</b> MFI histograms and % cells expressing ULBP 1, 3, 2/5/6, MICA and MICB activating ligands. Dark histograms represent negative control, light histograms represent stained cells.</p

Longitudinal T1-weighted images and tumour growth.

<p>(<b>A</b>) Longitudinal axial post-contrast T1-weighted images of nude rats bearing U87MG tumours treated with combination NK+mAb9.2.27, NK cell monotherapy, mAb9.2.27 monotherapy, and vehicle controls, showing the same animal after 7 days, 17 days and 3 months post NK+mAb9.2.27 treatment. (<b>B</b>) Tumour volumes (#voxels) quantified on post-contrast T1-weighted images, before and after 7 and 17 days treatment. Data represents mean ±SEM of all animals treated.</p

Immunohistochemical staining and cell proliferation.

<p>(<b>A</b>, top panel) haematoxylin and eosin staining showing leucocyte packed necrosis in U87MG tumours treated with NK+mAb9.2.27 and mAb9.2.27 monotherapy, (arrows), Magnification 200X; Scale bar 100 µm. Cellular dense tumours treated with NK cell monotherapy and haemorrhaging control, untreated tumours. (<b>A</b>, middle panel) Ki67 staining of proliferating tumour cells (<b>A</b>, bottom panel). Magnification 200X; scale bar 100 µm. Tunel stained apoptotic cells, Magnification 200X; Scale bar 100 µm. (<b>B</b>) Quantified Ki67 labelling index, data represents mean ±SEM of all animals treated. (<b>C</b>) Quantified Tunel labelling index, data represents mean ±SEM of all animals treated,*p<0.05; **p<0.001, ***p<0.0001. D Ratio of proliferation: apoptosis index.</p

Perfusion parameters and maps.

<p>(<b>A</b>) Elevated extravascular extracellular volume fraction v_e, in NK+mAb9.2.27 compared to monotherapy animals from cohort 1 (received 1 million NK cells), *p<0.05, **p<0.001 ***p<0.0001. (<b>A left panels</b>) Parametric maps visualising intratumoral heterogeneity in v_e in representative control, monotherapy and NK+mAb9.2.27 treated animals at 7 days. Intensity scale shows minimum (blue voxels) and maximum (red voxels) intensity levels. (<b>B</b>) Increased interstitial extracellular volume fraction, v_e, elevated in NK+mAb9.2.27 compared to NK cells and mAb9.2.27 monotherapy animals, and decreased v_e in NK cell monotherapy compared to untreated control from cohort 2 (treated with 2 million NK cells), *p<0.05, **p<0.001, ***p<0.0001. (<b>B, left panels</b>) Parametric maps visualising intratumoural heterogeneity in v_e in control, monotherapy and NK+mAb9.2.27 treated animals at 17 days. (<b>C</b>) Significant association between ADC and v_e in the NK+mAb9.2.27 (R² = 0.798, p = 0.041) and NK cell monotherapy animals (R² = 0.993, p = 0.004). Graphs in A and B represent estimated marginal mean±95% confidence intervals.</p