Optimization of Early Response Monitoring and Prediction of Cancer Antiangiogenesis Therapy via Noninvasive PET Molecular Imaging Strategies of Multifactorial Bioparameters

Abstract

Objective: Antiangiogenesis therapy (AAT) has provided substantial benefits regarding improved outcomes and survival for suitable patients in clinical settings. Therefore, the early definition of therapeutic effects is urgently needed to guide cancer AAT. We aimed to optimize the early response monitoring and prediction of AAT efficacy, as indicated by the multi-targeted anti-angiogenic drug sunitinib in U87MG tumors, using noninvasive positron emission computed tomography (PET) molecular imaging strategies of multifactorial bioparameters. Methods: U87MG tumor mice were treated via intragastric injections of sunitinib (80 mg/kg) or vehicle for 7 consecutive days. Longitudinal MicroPET/CT scans with ¹⁸F-FDG, ¹⁸F-FMISO, ¹⁸F-ML-10 and ¹⁸F-Alfatide II were acquired to quantitatively measure metabolism, hypoxia, apoptosis and angiogenesis on days 0, 1, 3, 7 and 13 following therapy initiation. Tumor tissues from a dedicated group of mice were collected for immunohistochemical (IHC) analysis of key biomarkers (Glut-1, CA-IX, TUNEL, α_νβ₃ and CD31) at the time points of PET imaging. The tumor sizes and mouse weights were measured throughout the study. The tumor uptake (ID%/g_max), the ratios of the tumor/muscle (T/M) for each probe, and the tumor growth ratios (TGR) were calculated and used for statistical analyses of the differences and correlations. Results: Sunitinib successfully inhibited U87MG tumor growth with significant differences in the tumor size from day 9 after sunitinib treatment compared with the control group (P < 0.01). The uptakes of ¹⁸F-FMISO (reduced hypoxia), ¹⁸F-ML-10 (increased apoptosis) and ¹⁸F-Alfatide II (decreased angiogenesis) in the tumor lesions significantly changed during the early stage (days 1 to 3) of sunitinib treatment; however, the uptake of ¹⁸F-FDG (increased glucose metabolism) was significantly different during the late stage. The PET imaging data of each probe were all confirmed via ex vivo IHC of the relevant biomarkers. Notably, the PET imaging of ¹⁸F-Alfatide II and ¹⁸F-FMISO was significantly correlated (all P < 0.05) with TGR, whereas the imaging of ¹⁸F-FDG and ¹⁸F-ML-10 was not significantly correlated with TGR. Conclusion: Based on the tumor uptake of the PET probes and their correlations with MVD and TGR, ¹⁸F-Alfatide II PET may not only monitor the early response but also precisely predict the therapeutic efficacy of the multi-targeted, anti-angiogenic drug sunitinib in U87MG tumors. In conclusion, it is feasible to optimize the early response monitoring and efficacy prediction of cancer AAT using noninvasive PET molecular imaging strategies of multifactorial bioparameters, such as angiogenesis imaging with ¹⁸F-Alfatide II, which represents an RGD-based probe.

Keywords: 18F-ML-10; 18F-Alfatide II; 18F-FDG; 18F-FMISO; Antiangiogenesis therapy; Molecular imaging probe; PET; Sunitinib.; Therapy response.

Figure 1

Experimental design for longitudinal MicroPET/CT imaging, tumor sampling and treatment protocols of sunitinib. Notes: ^{# 18}F-FDG, ¹⁸F-FMISO, ¹⁸F-ML-10 and ¹⁸F-Alfatide II were used for MicroPET/CT imaging of U87MG tumor mice.

Figure 2

Effects of sunitinib on antitumor activity (A) and body weight (B) in U87MG tumor mice over time. U87MG tumor-bearing mice were treated with sunitinib or vehicle. There was significant difference for the tumor size between the sunitinib group and control group post day 9 (P < 0.01). On the other hand, there was no difference for the mice weight between the sunitinib group and control group. ** P < 0.01.

Figure 3

¹⁸F-FDG MicroPET/CT imaging of U87MG tumor-bearing mice, IHC staining for Glut-1 of tumor tissues and the relevant quantitative and correlation analysis. (A) Representative MicroPET/CT images at 1.0 h after intravenous injection of ¹⁸F-FDG (5.55 MBq per mouse) on days 0, 1, 3, 7 and 13 post treatment. (B, C) Quantitative %ID/g_max of tumor uptake and the ratios of tumor to contralateral muscle (T/M) in the sunitinib and control groups based on ROIs analysis from ¹⁸F-FDG MicroPET/CT. (D, E) IHC and quantitative analysis of tumor sections about Glut-1. (F) Correlations between ¹⁸F- FDG T/M and Glut-1 expression of tumor. Notes: The tumors were indicated by arrows. * P < 0.05, ** P < 0.01, within the sunitinib group, compared to day 0. # # P < 0.01, between the sunitinib group and the control group.

Figure 4

¹⁸F-FMISO MicroPET/CT imaging of U87MG tumor-bearing mice, IHC staining for CA-IX of tumor tissues and the relevant quantitative and correlation analysis. (A) Representative MicroPET/CT images at 1.0 h after intravenous injection of ¹⁸F-FMISO (5.55 MBq per mouse) on days 0, 1, 3, 7 and 13 post treatment. (B, C) Quantitative %ID/g_max of tumor uptake and the ratios of tumor to contralateral muscle (T/M) in the sunitinib and control groups based on ROIs analysis from ¹⁸F-FMISO MicroPET/CT. (D, E) IHC and quantitative analysis of tumor sections about CA-IX. (F) Correlations between ¹⁸F-FMISO T/M and CA-IX expression of tumor. Notes: The tumors were indicated by arrows. * P < 0.05, ** P < 0.01, within the sunitinib group, compared to day 0. + P < 0.05, + + P < 0.01, within the control group, compared to day 0. # # P < 0.01, between the sunitinib group and the control group.

Figure 5

¹⁸F-ML-10 MicroPET/CT imaging of U87MG tumor-bearing mice, IHC staining for TUNEL of tumor tissues and the relevant quantitative and correlation analysis. (A) Representative MicroPET/CT images at 1.0 h after intravenous injection of ¹⁸F-ML-10 (5.55 MBq per mouse) on days 0, 1, 3, 7 and 13 post treatment. (B, C) Quantitative %ID/g_max of tumor uptake and the ratios of tumor to contralateral muscle (T/M) in the sunitinib and control groups based on ROIs analysis from ¹⁸F-ML-10 MicroPET/CT. (D, E) IHC and quantitative analysis of tumor sections about TUNEL. (F) Correlations between ¹⁸F-ML-10 T/M and AI from TUNEL. Notes: The tumors were indicated by arrows. * P < 0.05, ** P < 0.01, within the sunitinib group, compared to day 0. + + P < 0.01, within the control group, compared to day 0. # # P < 0.01, between the sunitinib group and the control group.

Figure 6

¹⁸F-Alfatide II MicroPET/CT imaging of U87MG tumor-bearing mice, IHC staining for integrin α_vβ₃ of tumor tissues and the relevant quantitative and correlation analysis. (A) Representative MicroPET/CT images at 1.0 h after intravenous injection of ¹⁸F-Alfatide II (5.55 MBq per mouse) on days 0, 1, 3, 7 and 13 post treatment. (B, C) Quantitative %ID/g_max of tumor uptake and the ratios of tumor to contralateral muscle (T/M) in the sunitinib and control groups based on ROIs analysis from ¹⁸F-Alfatide II MicroPET/CT. (D, E) IHC and quantitative analysis of tumor sections about integrin α_vβ₃. (F) Correlations between ¹⁸F-Alfatide II T/M and integrin α_vβ₃ expression of tumor. Notes: The tumors were indicated by arrows. ** P < 0.01, within the sunitinib group, compared to day 0. # P < 0.05, # # P < 0.01, between the sunitinib group and the control group.

Figure 7

IHC analysis of tumor sections about CD31 on days 0, 1, 3, 7 and 13 post therapy and the correlations with T/M of each probe. (A) Representative captures of CD31 staining of tumor tissues in sunitinib and control groups (40×10). CD31 in the sunitinib group revealed effective antiangiogenic activity from days 1 to 7. MVD in sunitinib group lowered significantly than that in control group. (B-E) Correlations between MVD and T/M of each probe (¹⁸F-FDG, ¹⁸F-FMISO, ¹⁸F-ML-10, ¹⁸F-Alfatide II, from Left to Right).

Figure 8

Correlations between tumor growth ratios (TGR) and T/M of ¹⁸F-Alfatied II (A) and ¹⁸F-FMISO (B) on days 1, 3, 7 and 13 (from Left to Right). There were significantly positive correlations between the TGR and the T/M of ¹⁸F-Alfatide II and ¹⁸F-FMISO.