Introduction

Evofosfamide is a prodrug that selectively cytotoxic under hypoxic conditions where other anticancer treatment is less effective. This drug is not effective to all cancer subtypes in pancreatic ductal adenocarcinoma. MIA Paca-2 tumors respond to evofosfamide, while SU.86.86 tumors do not.1 On the other hand, MIA Paca-2 tumors do not respond to gemcitabine (GEM), while SU.86.86 tumors do.2, 3 Despite the difference of pO2 level in two tumors, combination treatment of evofosfamide and GEM reduces tumor growth than either GEM/evofosfamide alone in both tumors.4 The underlying mechanism of this combination treatment is not fully understood. Therefore, the aim of this study is to evaluate the mechanism of the synergistic effect of evofosfamide and GEM using multimodal imaging methods.

Methods

Athymic nude mice were inoculated with 2 x 106 MIA Paca-2 and SU.86.86 tumor cells adjacent to the right tibial periosteum. For treatment, approximately 400 mm3 tumor bearing mice were injected i.p. with 50 mg/kg of evofosfamide on day 1 through day 5 and 150 mg/kg of GEM on day 1. Tumor bearing mice in the control group were injected with same amount of vehicle. EPRI: Parallel coil resonators tuned to 300MHz were used for EPRI. OX063 (1.125 mmol/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 4,000. 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 (Bruker BioSpin MRI GmbH). 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 through a tail vein cannula 2 minutes after the start of the dynamic FLASH sequence. To determine the local concentrations of Gd-DTPA, T1 maps were calculated from three sets of Rapid Imaging with Refocused Echoes (RARE) images obtained with TR = 300, 600, 1,000, and 2,000 ms, with the acquisitions being made before running the FLASH sequence. Blood volume (BV) imaging: MRI scanning was conducted a 1 T scanner (Bruker BioSpin MRI GmbH). For BV calculation, spoiled gradient echo sequence images were collected before and 5 minutes after injection of ultra-small superparamagnetic iron oxide (USPIO) contrast (1.2 μL/g of body weight). The imaging parameters included the following: FOV = 28 x 28 mm; matrix = 128 x 128; echo time (TE) = 5.6 ms; TR = 200 ms; and number of average = 12

Results

Combination treatment of evofosfamide and GEM showed a synergistic effect on both MIA Paca-2 and SU.86.86 tumors (Fig. 1). Cell cytotoxicity assay showed that MIA Paca-2 tumor cells have a higher sensitivity to GEM than SU.86.86 in vitro. Also, sensitivity of evofosfamide was similar between both tumor cells (Fig. 2). MIA Paca-2 tumors showed hypoxic profile compared with SU.86.86 tumors (Fig. 3). In MIA Paca-2 cells, the combination treatment with evofosfamide and GEM had only an additive effect (Fig. 4A). Thus, the in vivo synergistical effect can be attributed to the changes in TME. EPRI and DCE-MRI analysis showed that GEM caused hypoxia and decreased perfusion in MIA Paca-2 tumors (Fig 4.B-I). GEM did not affect the blood volume (Fig. 4J and 4K). In SU.86.86 cells, the combination treatment with evofosfamide and GEM had a synergistic effect in vitro, which may explain the synergistical effect in vivo. Further analysis on TME by multimodal imagings revealed that evofosfamide improved the pO2 and HF10 when treated with evofosfamide (Fig. 5B-E). Evofosfamide improved the blood volume (Fig. 5J and 5K) though it did not affect perfusion (Fig. 5G-I).

Conclusion

Multi-model imaging showed that GEM induced hypoxia and led to synergistical effect with evofosfamide in MIA Paca-2 tumors. On the other hand, combination of evofosfamide and GEM showed synergistic effect and enhanced angiogenesis by evofosamide which led to improvement in HF10. These data can provide imaging biomarkers to detect early response to cancer therapy.

Acknowledgements

No acknowledgement found.

References

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