Monitoring the Process of Endostar-Induced Tumor Vascular Normalization by Non-contrast Intravoxel Incoherent Motion Diffusion-Weighted MRI

Abstract

Tumor vascular normalization has been proposed as a new concept in anti-tumor angiogenesis, and the normalization window is considered as an opportunity to increase the effect of chemoradiotherapy. However, there is still a lack of a non-invasive method for monitoring the process of tumor vascular normalization. Intravoxel incoherent motion diffusion-weighted magnetic resonance imaging (IVIM DW-MRI) is an emerging approach which can effectively assess microperfusion in tumors, without the need for exogenous contrast agents. However, its role in monitoring tumor vascular normalization still needs further study. In this study, we established a tumor vascular normalization model of CT26 colon-carcinoma-bearing mice by means of Endostar treatment. We then employed IVIM DW-MRI and immunofluorescence to detect the process of tumor vascular normalization at different times after treatment. We found that the D^* values of the Endostar group were significantly higher than those of the control group on days 4, 6, 8, and 10 after treatment, and the f values of the Endostar group were significantly higher than those of the control group on days 6 and 8. Furthermore, we confirmed through analysis of histologic parameters that Endostar treatment induced the CT26 tumor vascular normalization window starting from day 4 after treatment, and this window lasted for 6 days. Moreover, we found that D^* and f values were well correlated with pericyte coverage (r = 0.469 and 0.504, respectively; P < 0.001, both) and relative perfusion (r = 0.424 and 0.457, respectively; P < 0.001, both). Taken together, our findings suggest that IVIM DW-MRI has the potential to serve as a non-invasive approach for monitoring Endostar-induced tumor vascular normalization.

Keywords: Endostar; IVIM-DWI; MRI; perfusion; tumor vascular normalization.

Figure 1

Schematic diagram of the design and treatment process of the study. A total of 70 Balb/c were randomly divided into the Endostar and control groups (n = 35, each). Five mice in each group were randomly selected for IVIM-MRI and sacrifice for histologic analysis at each time point from days 0 to 12. IVIM, intravoxel incoherent motion; MRI, magnetic resonance imaging.

Figure 2

Mouse CT26 tumor growth is hindered by Endostar treatment. Tumor volumes in the Endostar group were significantly lower than those in the control group on days 2, 4, 6, 8, 10, and 12 after treatment. *P < 0.05.

Figure 3

IVIM-DWI MRI features of the CT26 colon cancer mouse model in the Endostar (A) and control (B) groups at different time points after treatment. The panel shows diffusion-weighted images (DWI) with b = 0 s/mm2 and parametric maps (D*, D, and f) of representative Endostar- and saline-treated tumors on days 0, 2, 4, 6, 8, 10, and 12 after treatment. IVIM, intravoxel incoherent motion; MRI, magnetic resonance imaging.

Figure 4

Comparison of D, D*, and f values between the Endostar and control groups at different time points after treatment. (A) The differences in D value between the two groups were significant on days 2, 4, and 12 after treatment (*P < 0.05). (B) The D*values of the Endostar group were significantly higher than those of the control group on days 4, 6, 8, and 10 after treatment (*P < 0.05). (C) On days 6 and 8 after treatment, the f values of the Endostar group were significantly higher than those of the control group (*P < 0.05).

Figure 5

Evaluation of the vascular normalization window by histologic analysis of the vascular structure on days 0, 2, 4, 6, 8, 10, and 12 after treatment. (A) CD31 (red), α-SMA (green), and DAPI (blue) staining of representative tumor sections of the Endostar and control groups at different time points. Scale bars: 200 μm. (B) Comparison of changes in vessel density (CD31+ area) between the Endostar and control groups at each time point (*P < 0.05; **P < 0.01). (C) Comparison of changes in pericyte coverage (CD31+ area/α-SMA+ area) between the Endostar and control groups at each time point (*P < 0.05; **P < 0.01). CD31, cluster of differentiation 31; SMA, smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 6

Evaluation of the vascular normalization window by histologic analysis of vascular perfusion. (A) Lectin perfusion (green) and CD31 (red) staining of tumor sections of the Endostar and control groups at different time points after treatment. (B) Changes and differences in relative perfusion (lectin+ area/CD31+ area) in the two groups at each time point (days 0, 2, 4, 6, 8, 10, and 12 after treatment; *P < 0.05; **P < 0.01). CD31, cluster of differentiation 31.

Figure 7

Correlation between IVIM-DWI MRI and histologic parameters for analysis of tumor vascular normalization. (A) There was no significant correlation between D value and pericyte coverage (r = 0.235; P = 0.051). (B,C) D*value (r = 0.469; P < 0.001) and f value (r = 0.504; P < 0.001) were significantly correlated with pericyte coverage during the process of tumor vascular normalization. (D) There was no significant correlation between D value and relative perfusion (r = 0.173; P = 0.151). (E) D*value and (F) f value were both positively correlated with relative perfusion (correlation coefficients, 0.424 and 0.457, respectively; P < 0.001). CD31, cluster of differentiation 31; SMA, smooth muscle actin; IVIM, intravoxel incoherent motion; DWI, diffusion-weighted imaging; MRI, magnetic resonance imaging.