Photoacoustic imaging for the evaluation of early tumor response to antivascular treatment

Abstract

Background: Photoacoustic imaging (PAI) provides real-time noninvasive and contrast agent-free monitoring of the concentrations of some endogenous compounds related to tumor vascularization and oxygenation. In this study, we used PAI to noninvasively evaluate tumor responses to antiangiogenic therapy.

Methods: In vivo studies were performed with the approval of our institutional animal ethics committee. We used a xenograft mouse model of 4T1 breast cancer treated with different doses of bevacizumab or vehicle. Seven days after implantation, tumor-bearing mice (with tumors ~5-8 mm diameter) were randomly divided into low-dose (10 mg/kg), high-dose (20 mg/kg) and vehicle groups (same dose of saline). Each experimental group was administered bevacizumab intraperitoneally only once. Before and after treatment, acoustic resolution-photoacoustic microscopy (AR-PAM), a type of PAI, was conducted in vivo consecutively from day 1 to day 5. PAI-derived quantitative parameters were calculated at each time point. Additional cohorts of mice were used to quantify CD31 and hypoxia by immunohistochemical assays.

Results: The values of the PAI parameters were not significantly different among the experimental and control groups at the same time point before treatment (all P>0.05). The total hemoglobin (HbT) levels in the treatment group gradually decreased from day 1 to day 2 (relative to those in the control group, P>0.05) and decreased significantly relative to those in the control group from day 3 to day 5 (P<0.05). The deoxyhemoglobin (HbR) levels in the treatment group decreased from day 1 to 5 after treatment. The high-dose group had significantly decreased HbR levels relative to the control group from day 1 to 5 (P<0.05). The low-dose group also showed a gradual and significant decrease in HbR levels on day 3 (P<0.05). CD31 was decreased in the low-dose group relative to the control group on day 1 (decreased by 34.05%, P=0.067) and day 3 (decreased by 45.27%, P=0.180), and the decrease in CD31 persisted on day 5 (decreased by 71.41%, P=0.000). CD31 decreased to a greater extent in the high-dose group than in the low-dose group. Tumor hypoxia was significantly increased on day 1 from day 0 in the treatment groups (P<0.05), especially in the high-dose group. Hypoxia was decreased on days 3 and 5 in the low-dose group (10.92±0.92 and 8.17±1.9, P=0.317) but continuously increased over time in the high-dose group. Significantly greater hypoxia was observed in the high-dose group than in the low-dose group (17.60±1.20 and 20.33±0.47, P<0.05).

Conclusions: PAI can be used to evaluate both vessel regression and hypoxia in response to anti-vascular treatment.

Keywords: Photoacoustic imaging; antiangiogenic; hypoxia; vascular normalization; vessel regression.

Figure 1

PAI MAP images from day 0 to day 5. (A,B,C) show maps of HbT. d-f show maps of HbR. The red areas represent ROIs. The white broken circles represent the tumor center. (A) and (D) represent the high-dose groups; (B) and (E) represent the low-dose groups; and (C) and (F) represent the control groups. The units of HbT and HbR are intensity arbitrary.

Figure 2

Longitudinal development of four types of parameters from PAI. The signal intensity of MAP 760 (A), MAP 840 (B), HbR (C) and HbT (D) in the treatment groups decreased after bevacizumab treatment from day 1 to 5, especially in the high-dose group.

Figure 3

Immunohistochemistry with pimonidazole staining for hypoxia. Tumor hypoxia was increased after bevacizumab treatment, especially in the high-dose group. Hypoxia in the high-dose group continued to increase, whereas in the low-dose group, hypoxia first increased and then gradually decreased (A). (B,C,D) show representative hypoxia on day 5; (B) shows hypoxia of the control group, (C) shows hypoxia of the low-dose group, and (D) shows hypoxia of the high-dose group. (magnification: ×20, scale: 100 µm). * and ** mean compared with the control group, P<0.05.

Figure 4

CD31 (A) and VMI (B) of tumor responses to the antiangiogenic therapy. CD31 staining was decreased after anti-vascular treatment and was decreased to a greater extent in the high-dose group than in the low-dose group. VMI increased from day 3 to 5 in the low-dose group, indicating that the tumor vessels gradually became mature and normalized. (C) shows double-marker immunofluorescence of CD31 (red) and α-SMA (green), and the blue area represents the nucleus. Compared with control the control group, *P<0.05; compared with the control group, **P>0.05.