Multi-b-value DWI to evaluate the synergistic antiproliferation and anti-heterogeneity effects of bufalin plus sorafenib in an orthotopic HCC model

Abstract

Background: Multi-b-value diffusion-weighted imaging (DWI) with different postprocessing models allows for evaluating hepatocellular carcinoma (HCC) proliferation, spatial heterogeneity, and feasibility of treatment strategies. We assessed synergistic effects of bufalin+sorafenib in orthotopic HCC-LM3 xenograft nude mice by using intravoxel incoherent motion (IVIM), diffusion kurtosis imaging (DKI), a stretched exponential model (SEM), and a fractional-order calculus (FROC) model.

Methods: Twenty-four orthotopic HCC-LM3 xenograft mice were divided into bufalin+sorafenib, bufalin, sorafenib treatment groups, and a control group. Multi-b-value DWI was performed using a 3-T scanner after 3 weeks' treatment to obtain true diffusion coefficient D_t, pseudo-diffusion coefficient D_p, perfusion fraction f, mean diffusivity (MD), mean kurtosis (MK), distributed diffusion coefficient (DDC), heterogeneity index α, diffusion coefficient D, fractional order parameter β, and microstructural quantity μ. Necrotic fraction (NF), standard deviation (SD) of hematoxylin-eosin staining, and microvessel density (MVD) of anti-CD31 staining were evaluated. Correlations of DWI parameters with histopathological results were analyzed, and measurements were compared among four groups.

Results: In the final 22 mice, f positively correlated with MVD (r = 0.679, p = 0.001). Significantly good correlations of MK (r = 0.677), α (r = -0.696), and β (r= -0.639) with SD were observed (all p < 0.010). f, MK, MVD, and SD were much lower, while MD, α, β, and NF were higher in bufalin plus sorafenib group than control group (all p < 0.050).

Conclusion: Evaluated by IVIM, DKI, SEM, and FROC, bufalin+sorafenib was found to inhibit tumor proliferation and angiogenesis and reduce spatial heterogeneity in HCC-LM3 models.

Relevance statement: Multi-b-value DWI provides potential metrics for evaluating the efficacy of treatment in HCC.

Key points: • Bufalin plus sorafenib combination may increase the effectiveness of HCC therapy. • Multi-b-value DWI depicted HCC proliferation, angiogenesis, and spatial heterogeneity. • Multi-b-value DWI may be a noninvasive method to assess HCC therapeutic efficacy.

Keywords: Bufalin; Carcinoma (hepatocellular); Diffusion magnetic resonance imaging; Liver neoplasms; Sorafenib.

Fig. 1

Multi-b-value DWI images and corresponding histopathological images of hepatocellular carcinoma in the bufalin plus sorafenib treatment group. a A transverse T2-weighted image shows the slice with the maximum tumor diameter. b Apparent diffusion coefficient map for outlining the tumor. c–i D_t, D_p, f, mean diffusivity, mean kurtosis, distributed diffusion coefficient, and α tumor maps and (j–l) D, β, and µ tumor maps. m Hematoxylin-eosin staining showing patchy necrosis (black arrows, ×10). n Anti-CD31 staining image showing sparse microvessels (black arrows, ×20)

Fig. 2

Grayscale images of two whole hematoxylin-eosin -stained slides and corresponding histograms of pixel distribution after treatment. a Grayscale image of a tumor in the bufalin plus sorafenib treatment group (histopathological standard deviation 10.475). b Grayscale image of a tumor in the control group (histopathological standard deviation 13.795). c Histograms of pixel distributions in the grayscale images of the tumors in the bufalin plus sorafenib treatment (blue) and control groups (orange)

Fig. 3

Comparisons of diffusion-weighted imaging and histopathological parameters of hepatocellular carcinoma LM3 models among the four groups. The results are expressed as the mean ± standard deviation (^*p < 0.05). B Bufalin treatment, BS Bufalin plus sorafenib treatment, C Control, DDC Distributed diffusion coefficient, MD Mean diffusivity, MK Mean kurtosis, MVD Microvessel density, NF Necrotic fraction, S Sorafenib treatment, SD Histopathological standard deviation