Introduction

Metronomic chemotherapy (MCT) has been studied as a viable (neo)adjuvant therapy to illicit innate immune response and normalize tumor vasculature for additional treatment in different cancer types^1,2. The optimal biological dose is being studied at the biological level predominately; however, utilizing a noninvasive imaging technique such as dynamic contrast enhanced (DCE)-MRI may aid in assessing treatment response. Pharmacokinetic model analysis of DCE-MRI data can provide spatial heterogeneity of treatment and its longitudinal changes. Here we propose to utilize a previously reported MCT dose scheme³ of 70mg/kg of cyclophosphamide (CPA) intra-peritoneally (IP)-injected 3 times a week and assess for any heterogenous changes observed throughout the dose scheme by using DCE-MRI in an orthotopic murine breast cancer model.

Methods

Six to eight -week-old BALB/c mice (n=10) with 4T1luc2 carcinoma model were included in this study. The 4T1luc2 cells (10,000) were injected into the left 4^th set of the mammary fat pad and were scanned 8 days after for baseline measurements. The treated mice received IP injections of 70mg/kg of CPA 3 times a week, and the control received equivalent volume of saline. MRI experiments were performed on a Bruker 7T micro-MRI system, with a four-channel phased array receive-only MRI surface coil. DCE-MRI scan was performed using a custom-made 3D UTE pulse sequence with golden-angle projections⁴ (TR=5ms and TE=0.028ms) to achieve an isotropic spatial resolution and to minimize the T₂^* effect. It was continuously run to acquire 48,500 spokes (9,700 spokes each flip angle segment, total 14 segments, flip angels of segments in series were: 10^o-10^o-10^o-10^o-2^o-10^o-20^o-10^o-5^o-10^o-15^o-10^o-25^o-10^o) for 11 min 19 s. The multiple flip angles during the dynamic scan were used to estimate the pre-contrast T₁ along with the pharmacokinetic parameters, i.e., active contrast encoding MRI^5-7. Image reconstruction was conducted to have temporal frame resolution T= 5 s/frame, Image matrix = 128x128x128, field of view = 27x27x27 mm³ and the spatial resolution was 0.210x0.210x0.210 mm³. A bolus of gadobutrol (Gadavist, Bayer) in saline at the dose of 0.1 mmol/kg was injected through a tail vein catheter, starting 60 seconds after the start of data acquisition. Arterial Input Function (AIF) was obtained using the median of the 10 most enhancing vessels (selected by initial area under curve, IAUC) from the whole field of view (FOV). For conversion of AIF signal intensity to concentration, the pre-contrast T₁ values of those vascular voxels were obtained by minimizing the jumps in the concentration curve as demonstrated before.⁶ Pharmacokinetic model analysis was carried out with the two-compartment exchange model to simultaneously estimate pre-contrast T₁₀, and four pharmacokinetic parameters: PS (permeability surface area product), F_p (blood flow), v_e (extracellular space volume fraction), and v_p (vascular space volume fraction). K^trans (volume transfer constant) was calculated from the estimated PS and F_p.

Results and Discussion

Representative tumors, in three orthogonal planes (axial/coronal/sagittal), of a control mouse and a cyclophosphamide treated mouse one week after baseline (Week 2) are shown in Figure 1. The tumor volume measured using post-contrast MR images shows that the two groups did not have significant difference at Week1, but the CPA treated group had a significantly smaller tumor volume than the control group (Figure 1C). Figure 2A shows the signal enhancement ratio curve generated from an ROI of the mouse common iliac artery and Figure 2B shows the resulting AIF in concentration. The log-linear function fit to the AIF data was used for pharmacokinetic analysis. Figure 3 shows representative pharmacokinetic parameter maps which indicate that tumors have relatively high-enhancing (HE) portion near the rim (i.e., IAUC > 0.4) and low-enhancing (LE) area near the core of the tumors (i.e., IAUC < 0.4). The estimated parameters in the whole tumor as well as in HE and LE voxels are shown in Figure 4. It is observed that the treated group had a similar sample dataset of a control mouse and a metronomic CPA treated mouse from baseline (week 1) to one week after baseline. These maps show an increasing trend of v_p and F_p with the HE portion of the treated group. In addition, the control group had a bigger decrease of the HE portion of the tumor, compared to the treated group (Figure 5), suggesting the vascular normalization effect of the metronomic therapy.

Conclusion

This study demonstrates the utility of DCE-MRI and contrast kinetic analysis for monitoring the changes in tumor vasculature and permeability with MCT. While this study shows the changes between baseline and post 1 week of MCT (3 total doses), future studies will include a larger cohort of animals and further timepoints to better understand the full scope of MCT, together with further optimization of the AIF and histological assessment.

Acknowledgements

NIH R01CA160620, R01CA219964, UH3CA228699.