Nanodiamond as a New Hyperpolarizing Agent and Its ¹³C MRS

In this work, we have hyperpolarized carbonaceous nanoparticles (<i>D</i> ≈ 10 nm), that is, “nanodiamonds”, with 1.1% ¹³C (natural abundance) using dynamic nuclear polarization (DNP). The polarization buildup curve showed a signal enhancement with relative intensity up to 4700 at 1.4 K and 100 mW microwave power. ¹³C magnetic resonance spectra (MRS) were obtained from the sample at 7 T, and the signal decayed with a <i>T</i>₁ of 55 ± 3s. Notably, polarization was possible in the absence of added radical, consistent with previous results showing endogenous unpaired electrons in natural nanodiamonds. These likely contribute to the shorter <i>T</i>₁’s compared to those of highly pure diamond. Despite the relatively short <i>T</i>₁, these observations suggest that natural nanodiamonds may be useful for in vivo applications

Time-course imaging result of DCE-MRI and DW-MRI.

<p>A) Time-courses of normalized mean K^trans values. The mean values of normalized K^trans decreased 69% for TH-302 treated mice in Hs766t tumors, decreased 46% for Mia PaCa-2 tumors and increased 5% in SU.86.86 tumors. B) Changes in normalized mean tumor ADC values over time. A substantial increase in relative mean ADCs was observed for the TH-302 treated group at post- 24 and 48 hours (29% increasing for 24h, p<0.01; 17% increasing for 48h, p<0.01). MIA PaCa-2 is not statistically significant different between conducted groups (8% increasing for 24h, <i>P</i>>0.05; 4% increasing for 48h, p>0.05). For SU.86.86, no significant change was detected by DW-MRI for both TH-302 and control group (3% decreasing for 24h, p>0.05; 0.5% increasing for 48h, p>0.05). The normalization was calculated by the average of the difference of pre- and post-treatment in Th302 group relative to average of the difference of pre- and post-treatment in control group. Error bars stand for standard deviation.</p

ADC maps and histograms of DW-MRI from representative animals at different time points pre- and post-treatment initiation.

<p>A dramatically increased ADCs observed in HS766T, but not in the other two tumor types.</p

Representative K^trans maps for HS766t, Mia PaCa-2 and Su.86.86 tumor type from DCE-MRI (L-R).

<p>As demonstrated in the histogram, K^trans generated from HS766t and Mia-PaCa-2 tumors were dramatically decreased 48 hours after TH-302 treatment compared to pre-treatment values. There were no significant changes observed for a SU.86.86 mouse at the same time point.</p

Diffusion MRI and Novel Texture Analysis in Osteosarcoma Xenotransplants Predicts Response to Anti-Checkpoint Therapy

<div><p>Combinations of targeted drugs have been employed to treat sarcomas, however, response rates have not improved notably, therefore emphasizing the need for novel treatments. In addition, imaging approaches to assess therapeutic response is lacking, as currently measurable indices, such as volume and/or diameter, do not accurately correlate with changes in tumor biology. In this study, quantitative and profound analyses of magnetic resonance imaging (MRI) were developed to evaluate these as imaging biomarkers for MK1775 and Gem in an osteosarcoma xenotransplant model at early time-points following treatment. Notably, we showed that Gem and Gem+MK1775 groups had significantly inhibited tumor growth by day 4, which was presaged by elevations in mean ADC by 24 hours post treatment. Significant differences were also observed at later time points for the Gem+MK1775 combination and MK1775 therapy. ADC distribution and entropy (randomness of ADC values) were also elevated by 24 hours following therapy. Immunohistochemistry demonstrated that these treatment-related increases in ADC correlated with apoptosis and observed cell condensations (dense- and exploded bodies). These findings underline the role of ADC as a quantitative imaging biomarker for therapy-induced response and show promising clinical relevance in the sarcoma patient population.</p></div

The randomness of ADC values within tumors pre- and post therapy were visualized and quantified using Shannon Entropy in Matlab.

<p>(<b>A</b>) ADC-entropy plots as obtained by texture-based analysis of post-treatment ADC-maps herein demonstrating the evident differences in ADC values and distribution within the perimeters of the tumor (see color bar). The axes represent spatial dimensions (<i>i.e.</i> pixel coordinates). (<b>B</b>) Corresponding change in average entropy values from pre-treatment for all four groups displaying statistically larger variations in ADC values for the Gem group compared to both controls (p = 0.022) and MK1775 (p = 0.023).</p

P-values obtained from a one-way analysis of variance (ANOVA) followed by the Tukey test for comparison of mean values between the stated groups on the specified time points for (A) tumor volumes, (B) percent change in mean ADC, (C) ADC-AUC analysis and (D) CC3 and γH2AX staining.

<p>% was chosen and statistical significance determined at p<0.05. A confidence interval of 95</p><p>*indicates significant differences between the stated groups which were abbreviated as follows; C =  controls, M =  MK1775, G =  gemcitabine and X =  combination group. Each group was composed of 4 mice with two flank tumors each, thus N = 8 per group.</p

Quantitative analysis of unusually dense and “exploded” masses demonstrating a correlation with treatment response.

<p>(<b>A</b>) A standard H&E section of a Gem-treated tumor on day 4 imaged with 20X zoom and (<b>B</b>) quantitative analysis showing percent dense bodies in comparison with normal cells. (<b>C</b>) Insert demonstrates a higher burden of unusually small and dense cells (black arrows) and large bulgy masses herein denoted as “exploded cells” (blue arrows). (<b>D</b>) Amount exploded bodies per tumor section indicating an increase with treatment response. (<b>E</b>) A CC3 stained tumor section demonstrating the amount of apoptosis as indicated by positively stained cells (brown) and (<b>F</b>) quantitative analysis of percent “exploded dense bodies” that stain fully for CC3 and are thus apoptotic.</p

Analysis of ADC distribution within tumors prior to- and following therapy.

<p>(<b>A–D</b>) Pixel-by-pixel histograms demonstrating the ADC distribution pre- (blue) and post-treatment on day 2 (red) for select animals from all four treatment groups. (<b>E–H</b>) Corresponding incremental ADC pixel fraction plots for representative animals showing a shift of ADCs towards higher values in Gem and Combo treated animals compared to MK1775 and control. Quantitative analysis comparing pre-treatment to day 2 of (<b>K</b>) relative skewness and (<b>L</b>) kurtosis and comparison with (<b>I</b>) percent change in mean ADC, (<b>J</b>) AUC values.</p

Assessment of diffusion properties and potential alterations between treatment groups following therapy.

<p>(<b>A</b>) Parametric ADC maps demonstrating restricted tumor diffusivity for all four groups comparing pre-treatment to day 2 and 4 post-therapy. (<b>B</b>) Quantitative analysis of mean ADC plotted as percent change from baseline (pre-treatment) over the experimental time-line showing statistical significance between groups already by day 2 (24 hours following the first treatment on day 1). (<b>C</b>) To compare across mice, ADC-AUC for each mouse was quantified by subtracting pre-treatment ADC pixel fraction curves from post-therapy and plotted as the average value for each group at all time points.</p