Introduction

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and represents a leading cause of cancer mortality worldwide. Despite a range of treatment options, the prognosis for patients with HCC remains poor, with a 5-year survival rate of less than 5%.¹ The kinase inhibitor sorafenib is the main systemic therapy for patients with advanced HCC, but provides only modest improvements in overall survival by inhibiting proliferative and angiogenic signaling.² Effectiveness of sorafenib treatment is currently assessed by monitoring tumor size and contrast enhancement in anatomical images, however this approach can be slow to demonstrate treatment failure.² Functional imaging biomarkers of treatment response are therefore sorely needed to better asses the effects of sorafenib in individual patients. Blood oxygen level dependent (BOLD) MRI measures the effect of deoxygenated hemoglobin, which decreases the T2* relaxation time in tissue. This method has traditionally been utilized in functional imaging of the brain, but recent work has demonstrated it to provide sensitive and reproducible measures of blood oxygenation state in liver parenchyma and HCC tumors when combined with hyperoxic and hypercapnic challenges.³ In this work we evaluated BOLD MRI with hyperoxic challenge as a method for assessing treatment effects of sorafenib in an orthotopic rodent model of HCC.

Methods

N = 19 male Buffalo rats were implanted with 106 hepatoma McA-RH7777 cells in the inferior left liver. N = 9 rats were treated with a daily dose of 7.5 mg/kg sorafenib by oral gavage starting two weeks after tumor inoculation, while N = 10 rats served as untreated controls. All animals were imaged on a Bruker Biospec 4.7T preclinical MRI system four weeks after HCC cell implantation. 2D Multi-gradient echo (MGE) images of the liver were acquired (12 echoes, TE₁ = 2.8 ms, ΔTE = 3.4 ms, 30° excitation angle, 100 kHz BW, 6x6 cm FOV, 256x144 matrix, 12 slices, 1.5 mm slice thickness with 0.5 mm gap) while medical air was delivered via nose cone, then repeated approximately 10 minutes after switching animals to oxygen gas inhalation. Image acquisitions were respiratory gated and isoflurane at a concentration of 2-4% was delivered in both inhalation conditions to maintain a respiratory rate of ~30 bpm (~2 s TR). T2* maps were generated in ParaVision 5 by voxel-wise fitting of a 3-parameter transverse relaxation model to MGE images. BOLD effect was quantified as the difference in T2* relaxation time constant between oxygen and medical air inhalation for ROIs including tumor, normal liver tissue and paraspinal muscle. The Wilcoxon rank-sum test was used to compare the differences in T2* relaxation times between sorafenib-treated and untreated animals.

Results

Representative images are shown in figure 1. Increases in T2* were typically observed in the tumor, normal liver and other well-perfused tissues following hyperoxic challenge. In rats treated with sorafenib, this BOLD effect (ΔT2*) was reduced in HCC tumor (p = 0.0095) and background liver (p = 0.0016) compared to untreated rats. No significant change in T2* was observed in paraspinal muscle with treatment (p = 0.068), figure 2.

Discussion

The use of the BOLD MRI technique presented here to assess oxygenation state of HCC tumors is appealing because it is non-invasive and utilizes widely available medical gases. The changes in T2* quantified in this work can be a consequence of a variety of changes in tissue state following sorafenib treatment. We expect that the antiangionenic effects of sorafenib resulted in deficient tumor vasculature in treated rats, reducing blood volume and blood flow and causing a smaller change in T2* with hyperoxic challenge. The heterogeneity of BOLD effect seen in tumors could identify regions of hypoxia non-invasively and without administration of exogenous contrast agents. Our study demonstrated no significant change in T2* of paraspinal muscle, which served as an internal control and replicated results shown in preliminary studies using BOLD MRI in HCC patients.³ It should be noted that the orthotopic model of HCC used here does not perfectly recapitulate the vascular characteristics of HCC observed in patients, namely the predominance of blood supply via abnormal hepatic arteries.² Nevertheless, the significant reduction in BOLD effect we have observed in tumors is demonstrative of the potential for this technique to provide measurements sensitive to sorafenib treatment and inform clinical decision making for patients with HCC.

Acknowledgements

The authors acknowledge funding from CPRIT RP160229 and NIH P30-CA016672.