Application of IVIM-DWI in Detecting the Tumor Vasculogenic Mimicry Under Antiangiogenesis Combined With Oxaliplatin Treatment

Abstract

Objectives: This study aimed to detect the time window of vascular normalization during anti-vascular treatment using intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI). Simultaneously, we evaluated the tumor invasiveness and vasculogenic mimicry and performed synthetic assessment of treatment efficacy of angiogenesis inhibitor combined with conventional chemotherapy using IVIM-DWI. Materials and Methods: HCT116 cells were subcutaneously administered into the right flank of BALB/C nude mice to build a colon cancer xenograft model. Thirty-two tumor-bearing mice were randomly divided into four groups and intraperitoneally administered with normal saline (Group A or control group), bevacizumab (Group B), oxaliplatin monotherapy (Group C), and oxaliplatin combined with bevacizumab (Group D). The IVIM-DWI was performed on days 0, 3, 6, 9, 12, and 15 after the treatments. Another 51 tumor-bearing mice were included in the pathological examinations. α-Smooth muscle actin (SMA) and CD31 double-staining, periodic acid-Schiff (PAS) and CD31 double-staining, hematoxylin and eosin (HE), Ki-67, and E-cadherin staining were performed. The tumor growth and dynamic change of each parameter were noted. Results: The mice in Group D manifested the smallest tumor volume and highest tumor inhibition rate. Microvessel density was significantly decreased but accompanied by increased vasculogenic mimicry after antiangiogenic treatment. The trend was reversed by oxaliplatin treatment. Treated with bevacizumab, the vessel maturity index shared a similar trend with D ^* and f-values during days 3-12, which slowly increased from days 0 to 9 and then decreased briefly. D-value significantly correlated with vasculogenic mimicry and Ki-67, while D ^* and f-values showed positive correlations with microvessel density and E-cadherin, an indicator of epithelial-mesenchymal transition. Conclusion: Oxaliplatin performed an inhibited effect on vasculogenic mimicry. Bevacizumab can enhance the tumor chemotherapy through vascular normalization within a transient time period, which can be detected by IVIM-DWI. D ^* and f-values are able to predict the tumor invasiveness while D is superior in reflecting vasculogenic mimicry and Ki-67 expression during antitumor treatment.

Keywords: MRI; colon cancer; epithelial–mesenchymal transition; intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI); vascular normalization; vasculogenic mimicry.

Figure 1

Axial T1-weighted imaging (T1WI), T2WI, and pseudocolor maps of D, D*, and f-values at different time points treated with bevacizumab and oxaliplatin (Group D). D-value is the pure diffusion of water molecule. D* value refers to perfusion-related diffusion. F-value is the perfusion fraction. The white circles plot the tumor area. The tumor showed a much slow growth during the 3rd−15th days. A small patch of high signal was observed in the tumor core on T2WI as early as 6 days after treatment. Color ranging from blue to red represents values ranging from low to high. D-value increased significantly after 6 days and reached a peak on day 15. D* and f-values increased to a peak on day 9, and then fell back until day 15.

Figure 2

Longitudinal monitoring of intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) and pathological parameters as well as tumor growth in the four groups. The data point indicated mean and standard deviation. *P < 0.05, **P < 0.01, and ***P < 0.001 were generated from comparisons between the four groups at each time point using one-way analysis of variance. Graphs show trends for tumor growth (A), and MRI parameters–D value (B), D^* value (C), and f value (D), and vessel maturity index (E), vasculogenic mimicry (F), microvessel density (G), Ki-67 index (H) and E-cadherin (I).

Figure 3

Hematoxylin and eosin staining (×100) of a representative tumor section from the four groups at different time points. More necrosis areas were observed in Group D and obvious on days 12 and 15. The tumor cells in Group A were dense. A few of necrosis areas were observed as the tumor grew. In Group B, a small amount of necrosis and cell apoptosis occurred in the tumor core. More karyopyknosis and nuclear fragmentation with homogeneous red-stained areas were observed in the later time points. In Group C, small patches of hemorrhage and necrosis appeared on day 3, which mainly distributed in the tumor core. More necrosis areas were observed in Group D, especially on days 12 and 15.

Figure 4

α-Smooth muscle actin (SMA) and CD31 immunofluorescent double-staining (×100) of a representative tumor section from the four groups at different time points. α-SMA was stained in green and CD31 in red. Elevated expressions of green-stained α-SMA were observed 3 days after treatment in both Groups B and D. The vessel maturity index rose to a peak on day 6 and 9, respectively, and returned to a lower level afterward in Groups B and D. No meaningful changes were observed in Groups A and C over time.

Figure 5

Periodic acid-Schiff (PAS) and CD31 immunohistochemical double-staining (×100) of a representative tumor section from the four groups at different time points. Both Groups B and D showed a decreased trend in microvessel density after treatment, with increased vasculogenic mimicry number in Group B. However, the numbers declined in Groups C and D 6 days after administration. Both Groups B and D showed a decreased trend in microvessel density after treatment. The number of vasculogenic mimicry in Group B increased in different speeds with time. However, downward trends were observed in Groups C and D 6 days after administration. No significant change was observed in Group A regarding microvessel density and vasculogenic mimicry.

Figure 6

Ki-67 immunofluorescent staining (×100) of a representative tumor section from the four groups at different time points. Groups C and D showed lower Ki-67 expressions than those of the other groups, especially on days 12 and 15. Group D showed the lowest Ki-67 expression on day 15 compared to that of the other groups, indicating that combination therapy could significantly suppress the proliferation activity of tumor cells after treatment.

Figure 7

E-cadherin immunofluorescent staining (×100) of a representative tumor section from the four groups at different time points. Bright red-stained E-cadherin was the most obvious in Group D in the late time points. Groups B and C also showed an increase of E-cadherin expression after treatment. The E-cadherin expression rapidly increased in both Groups B and D, especially in Group D, indicating more stable intercellular connectivity and lower likelihood of epithelial–mesenchymal transition after treatment. A mild increase was also observed in Group C, but a slow decline was observed in Group A.