Synopsis

Keywords: Quantitative Imaging, Preclinical

Pancreatic ductal adenocarcinoma (PDAC) is the leading cause of cancer deaths worldwide. Quantitative multiparametric magnetic resonance imaging (mpMRI) allows for measuring the tumor's physiological characteristics, such as cellularity and vascularity/permeability. This study aimed to evaluate tumor physiology using mpMRI after single-fraction irradiation in a mouse model. The results demonstrated the changes in the functional status of mpMRI metrics. They were validated with in vivo histology markers of tumor perfusion (Hoechst 33342) and tissue morphology (Hematoxylin and eosin staining).

Purpose

The stroma, a hallmark of pancreatic ductal adenocarcinoma (PDAC), forms a mechanistic barrier to effective drug delivery ¹. Multiparametric (mp) magnetic resonance imaging (MRI), including diffusion-weighted (DW)- and dynamic contrast-enhanced (DCE)-MRI, exhibits the potential to assess tumor physiology in a mouse model of PDAC ^2-4. Significant changes in relative apparent diffusion coefficient (ADC, surrogate of tumor cellularity) and volume transfer constant (K^trans, surrogate of tumor vascularity/permeability) values one day after a single fraction of irradiation have been reported in animal models of cerebral tumors^5,6. The present study aimed to evaluate the changes in tumor vascularity and cellularity within 20 hours after a single fraction of irradiation (10 Gy) using mpMRI in a mouse model of PDAC.

Methods

Animals and Tumor Models: All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Memorial Sloan Kettering Cancer Center. Tumors were established by injecting 2×105 KPC 4662 subcutaneously into the right shoulder region of athymic mice (n=5). The cells were originally derived from a murine pancreatic tumor, genotype Pdx1-Cre; LSL KRASG12D; Trp53R172H/wt7. The mpMRI was performed 10-12 days after tumor inoculation. MRI data were acquired before (pre-treatment [Tx]) and within 20 hours after irradiation (post-Tx).

MRI Data Acquisition: DW and DCE, followed by the T2-weighted MRI acquisition, were performed on a 7.0 T (Bruker BioSpin MRI GmbH). DW images were acquired with 7 b-values with TR/TE = 1500/ 20.12 ms, NS = 8-10; MS = 192x96, NA =1, NS=6, slice thickness=0.8 mm⁸. DCE-MRI images were acquired using a FLASH sequence (TR/TE=54.63/1.29 ms, NA=1, NS=6, flip angle (FA)=15°, MS=132×106). After acquiring 20 precontrast images, 0.1 mL of contrast agent was injected at a constant rate via a tail vein catheter8. Four TR values, i.e., 100, 200, 800, and 2000 ms, were used for T10 mapping, and other scanning parameters were the same as mentioned above.

Irradiation: Tumor-bearing mice were locally irradiated with a dose of 10 Gy administered by a dedicated small animal radiotherapy device (Precision X-Ray Inc, Madison, CT)⁹. Treatment was delivered using a photon beam (225 kV, 13 mA, 3 mm Cu), with a dose rate of approximately 3 Gy/min when using a 10 mm diameter collimator. Mice received continuous isoflurane gas anesthesia (2% isoflurane, 1l/minute in air). Histology: Tumor-bearing mice were injected intravenously with 0.1 ml Hoechst 33342 (10 mg/ml in saline) and euthanized by CO2 inhalation after 1 minute. Tumors were removed, frozen in OCT, and sectioned at 10 um thickness. Fluorescent images of unfixed sections were acquired using an Olympus microscope, where blue fluorescence indicates the presence of perfused vessels. Sections were subsequently stained with hematoxylin and eosin (H&E) and reimaged on brightfield.

Image Analysis: Using ITK-SNAP, regions of interest (ROIs) were manually contoured on DW- and DCE-MRI images⁸. The DW data were fitted to a monoexponential model (i.e., ADC (mm²/s) and intravoxel incoherent motion (IVIM) model (i.e., true and pseudo diffusion coefficients (D and D^* [mm²/s]) and perfusion fraction (f). The arterial input function (taken from the neck carotid artery) was incorporated into the time course of tissue concentration data to estimate K^trans [min^-1], v_e, and v_p¹⁰. The mpMRI metrics values at pre-Tx and post-Tx were compared from the same mice using the paired t-test. A P-value ≤0.05 was considered statistically significant.

Results

The post-Tx ADC and D values increased significantly from pre-TX (P=0.02 for ADC and P=0.04 for D, Figure 1A). Mean pre-Tx K^trans and v_p values were higher than the post-Tx (P=0.042 for K^trans and P= 0.048 for v_p, Figure 1B). D*, f, and ve exhibited a tendency towards substantial differences but were insignificant (P>0.05). The relative percentage change (%) of quantitative metric values is shown in Figure 1C. Quantitative metrics values from DW and DCE-MRI data are given in Table 1. Parametric maps of ADC, D, and K^trans exhibited a decrease in tumor cellularity and showed reduced vessel permeability after irradiation (Figures 2 and 3). The in vivo histology markers Hoechst 33342 and H&E stained images showed that the tumors have heterogeneous perfusion and tissue morphologies (Figure 4).

Discussion

Mean post-Tx ADC and D values differed from the pre-Tx by 21% and 22% after a single 10 Gy irradiation dose. In other studies on experimental PDAC tumors and a MEK inhibitor, changes in ADC correlate with tumor apoptosis². In the present study, K^trans, v_e, and v_p reduced by 37%, 20%, and 34%, respectively, consistent with Brown et al. results in a rat brain tumor after a single fraction 20 Gy stereotactic radiosurgery⁶, indicating radiation attributed to microvasculature destruction6. Cao et al. demonstrated that the DCE-MRI-derived metrics could reveal the sustained effect of combination drug treatment (PEGPH20+ gemcitabine)⁴. Tumor functional status and heterogeneity exhibited by the parametric maps are validated by in vivo histology markers of tumor vasculature (Hoechst 33342) and tissue morphology (H&E staining).

Conclusion

The mpMRI-derived metrics exhibited the potential to evaluate early changes in tumor physiology (i.e., cellularity and vascularity) in response to irridation in a mouse model of PDAC.

Acknowledgements

We acknowledge funding support from NCI R01 CA194321.

References

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