Purpose

Chronically hypoxic regions which are normally distant from the blood vessels are also known to show limited effectiveness of chemotherapies due to its acquired resistance to apoptosis induced by hypoxia.[1] Thus, several combination modes of Evofosfamide with chemotherapeutics such as doxorubicin, gemcitabine, bortezomib, etc have been evaluated pre-clinically and clinically.[2-4] For designing and evaluating such Evofosfamide combination therapies, it is crucial to understand the pO2 change in response to Evofosfamide in the tumor microenvironment because hypoxic fraction which is the target of Evofosfamide can be modified by the treatment itself. EPRI is one of the most reliable means of measuring pO2 available for live animal tissue among other modalities including 19F MRI, blood oxygen level dependent MRI, and photoacoustic imaging. EPRI requires the injection of non-toxic spin probe and absolute pO2 can be calculated from the linewidth of the distributed probe.[6-8] It allows the repeated measurement with minimum toxicity. By examining pO2 of each unique tumor and comparing the difference between before and after the treatment, it is feasible to obtain the detailed information of the effect of Evofosfamide treatment. In this study, to further understand the mechanism of the improved oxygenation by Evofosfamide treatment, tumor blood perfusion was examined using DCE MRI.[9, 10]

Methods

MIA Paca-2 cell line and Su.86.86 cell line (human pancreatic adenocarcinoma) were used in this study. Athymic nude mice were inoculated with the cells s.c. into the right leg. For treatment, approximately 600 mm3 tumor bearing mice were injected i.v. with 50 mg/kg of Evofosfamide five days a week. Tumor bearing mice in the control group were injected with same amount of buffer. EPRI: Parallel coil resonators tuned to 300MHz were used for EPRI. OX063 (1.125mmol/kg bolus) was injected i.v. to a mouse. The free induction decay (FID) signals were collected following the radiofrequency excitation pulses (65 ns) with a nested looping of the x, y, and z gradients, and each time point in the FID underwent phase modulation, enabling 3D spatial encoding. The repetition time was 8.0 μs. The number of averages was 4000. After EPRI measurement, anatomic T2-weighted MR images were collected with a 1T scanner. DCE-MRI: DCE-MRI studies were performed on a 1 T scanner. T1-weighted fast low-angle shot (FLASH) images were obtained with TR = 156 ms; TE = 4 ms; flip angle = 45˚; four slices; 0.44 x 0.44 mm resolution; 20-second acquisition time per image; and 98 repetitions. Gd-DTPA solution (4 mL/g of body weight of 50 mmol/L Gd-DTPA) was injected 1 minute after the start of the dynamic FLASH sequence. To determine the local concentrations of Gd-DTPA, T1 maps were calculated from four sets of Rapid Imaging with Refocused Echoes (RARE) images obtained with TR = 300, 600, 1000, and 2000 ms, with the acquisitions being made before running the FLASH sequence.

Results

The hypothesis of differential treatment response to Evofosfamide between MIA Paca-2 and Su.86.86 tumors was examined by evaluating the growth inhibition. Compared with the groups treated with vehicle (5% DMSO in PBS), MIA Paca-2 tumors showed a distinct growth inhibition by the treatment while Su.86.86 didn’t (Fig. 1A and Fig. 1B). The difference in tumor growth between Evofosfamide treated group and vehicle treated group was displayed as Kaplan-Meier plots (Fig. 1C and Fig. 1D) and statistically evaluated by log rank test. Tumor growth inhibition was significant only in MIA Paca-2 tumors. Intra-tumor pO2 of pre- and post- treatment with Evofosfamide. Figure 2A shows the representative images of pre-treated MIA Paca-2 tumor (top panels) and post-treated tumor (bottom panels). Although MIA Paca-2 tumor tends to exhibit the improved oxygenation, the change in pO2 between pre-treatment and post-treatment tumor showed a mixed profile (Fig. 2B, 2C). Considering the oxygen dependent activation of Evofosfamide, the treatment response in pO2 can also be dependent on pO2 prior to the treatment. In the scattered plot, only MIA Paca-2 tumors, which are Evofosfamide sensitive, showed statistically significant negative linear correlation between pre-treatment pO2 and ΔpO2 (Fig. 2D, 2F) and between pretreatment HF10 and ΔHF10 (Fig. 2E, 2G). The treatment effect of Evofosfamide on tumor perfusion was also investigated by dynamic contrast enhanced (DCE) MRI using Gd- DTPA as a contrast agent. Similar Ktrans value between pre- and post- treated tumors indicates the same level of accumulation of Gd-DTPA in the extracellular extravascular space, suggesting no significant difference in perfusion that reflects total effect of permeability of capillary and blood delivery. Figure 3A and 3B is the representative Ktrans map of pre- and post- Evofosfamide treated MIA Paca-2 tumor overlaid on anatomical images. No significant change in Ktrans was observed after Evofosfamide treatment in both MIA Paca-2 and Su.86.86 tumors (Fig. 3C, 3D).

Conclusion

pO2 improvement as well as decrease in hypoxic fraction were observed exclusively in hypoxic MIA Paca-2 tumors. Such effect was not observed when the intra-tumor pO2 was as high as 15 mmHg even in MIA Paca-2 tumors. The treatment didn’t affect the blood perfusion in the tumor regardless of the treatment effect. This study shows that pO2 changes but not perfusion changes reflect treatment effects with hypoxia activated prodrugs such as evofosfamide.

Acknowledgements

The study was funded by intramural research program of NCI/NIH (1ZIABC010476-15)

References

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