PMC

Direct comparison of the %ID tumor uptake (top) and %ID/cm³ tumor uptake (bottom) of ^99mTc-3P-RGD₂ in the vehicle (n = 5) and linifanib-treated (n = 5) groups. The tumor uptake values were calculated from quantification of SPECT/CT images. *: p < 0.05, significantly different from the vehicle-treated group. #: p < 0.01, significantly different from the vehicle-treated group.

Left: Linear relationship between the tumor size (cm³) and the tumor uptake (%/ID for total radioactivity accumulation) determined by SPECT/CT quantification (A) and biodistribution (C). Right: Relationship between the tumor size (g) and tumor uptake (%/ID/g or %/ID/cm³ for radioactivity concentration) obtained from SPECT/CT quantification (B) and biodistribution (D: n = 26).

Microscopic images of frozen tumor slices from the xenografted MDA-MB-435 tumors (0.05 g, 0.13 g, 0.28 g, 0.55 g and 0.88 g) after immunohistochemical staining for integrin β₃ and CD31. CD31 was used as a biomarker for tumor endothelial cells. Integrin β₃ was visualized with Cy3 (red), and CD31 with FITC (green). Yellow color indicates the integrin β₃ on new blood vessels.

Tumor growth curves (A) and body weight changes (B) for vehicle (n = 5) and RGD₂-treated (n = 5) groups in the xenografted MDA-MB-435 breast tumor model. Tumor size was calculated on the basis of caliper measurements according the formula (length × width²)/2. Body weight was measured by balance. RGD₂ treatment had to be stopped after five doses of RGD₂ because of toxicity, as indicated by significant weight loss. The body weight recovered after termination of RGD₂ treatment.

Top: The 3D and transverse views of SPECT/CT images of tumor-bearing mice before RGD₂ treatment, as well as on day 21 and 35 after inoculation of MDA-MB-435 cells (or on day 7 and 21 after final dose of RGD₂). SPECT image at day 7 was used only for comparison purposes. Bottom: SPECT/CT quantification data, including tumor volumes (A), %ID tumor uptake (B), %ID/cm³ tumor uptake (C), and T/M ratios (D) in vehicle (n = 5) and RGD₂-treated (n = 5) groups. *p < 0.05 and # p < 0.01: significantly different from the vehicle-treated group.

Comparison of changes in %ID/cm³ tumor uptake (A) and T/M ratios (B) of ^99mTc-3P-RGD₂ after linifanib treatment in three different tumor-bearing animal models (U87MG: high integrin α_vβ₃ expression on tumor cells and neovasculature; MDA-MB-435: moderate integrin α_vβ₃ expression on tumor cells and neovasculature; PC-3: low integrin α_vβ₃ expression on tumor cells and neovasculature). Tumor uptake values were calculated from quantification of SPECT/CT images. The %ID/cm³ tumor uptake and T/M ratios of ^99mTc-3P-RGD₂ in the U87MG and PC-3 models were obtained from our previous report . The changes in %ID/cm³ tumor uptake and T/M ratios were calculated by subtracting the value on a specific date (+1, +4 and +11) from the value on day -1. *: p < 0.05, significantly different from those in the U87MG and PC-3 models. ^#: p < 0.01, significantly different from those in the U87MG and MDA-MB-435 models.

A: Comparison of tumor volumes in vehicle and linifanib-treated in the MDA-MB-435 models. Tumor volume was determined by caliper measurements. *: p < 0.05, significantly different from the vehicle-treated group. #: p < 0.01, significantly different from the vehicle-treated group from that specific date until the end of study. B: Transverse views of selected SPECT/CT images from athymic nude mice bearing MDA-MB-435 breast tumors in the vehicle (upper panel) and linifanib-treated groups (lower panel). SPECT/CT images were obtained at -1, 1, 4 and 11 days after initiation of linifanib therapy to illustrate the early tumor response in terms of tumor uptake and integrin a_vβ₃ expression levels. Arrows indicate the presence of tumors.

A: Overlay images of MDA-MB-435 tumor tisues after immunohistochemical staining for integrin β₃ and CD31 in vehicle (upper panel) and linifanib-treated groups (lower panel) to illustrate the changes in integrin β₃ expression level and blood vessel density during linifanib therapy. Tumor tissues were harvested at -1, 1, 4 and 11 days after initiation of linifanib therapy. CD31 was the marker for tumor blood vessels (both mature and newly formed), which was visualized with FITC (green color). Integrin β₃ was visualized with Cy3 (red color). The yellow color in overlay images indicates the presence of integrin β₃ on new blood vessels. B: Images of selected histological slice (H&E stained) of tumor tissues from animals in the vehicle (upper panels) and linifanib-treated (lower panels) groups to illustrate vascular density changes after only one day of linifanib therapy. Red color indicates the presence of blood vessels.

Linear relationship between the %ID/cm³ tumor uptake change at days 1 (top), 4 (middle) and 11 (bottom) after linifanib therapy and the expression levels of integrin α_vβ₃ (left) and CD31 (right) in three animal models. The %ID/cm³ tumor uptake values of ^99mTc-3P-RGD₂ were calculated from SPECT/CT quantification, and reported as the mean plus/minus standard error of the mean based on results from five animals (n = 5). The %ID/cm³ tumor uptake values and integrin α_vβ₃/CD31 expression levels in the U87MG and PC-3 tumors were obtained from our previous report . The %ID/cm³ tumor uptake change was calculated by deducting the %ID/cm³ tumor uptake of ^99mTc-3P-RGD₂ on days 1, 4 and 11 from its original value on -1 day (before linifanib therapy) in the same animal. The average %ID/cm³ tumor uptake change is used as an indicator of tumor response to linifanib antiangiogenesis treatment.

A: The 3D (upper panel) and transverse (lower panel) views of SPECT/CT images of a mouse with bearing MDA-MB-435 breast cancer xenografts at 5, 7, 14, 21, 28 and 35 days after inoculation of MDA-MB-435 human breast cancer cells. The animal was administered with 37 - 55.5 MBq of ^99mTc-3P-RGD₂ via the lateral tail vein. B: Microscopic images (Original magnification: ×200) of tumor slice selected from the necrotic and viable regions after immunohistochemical staining for integrin β₃ and CD31. The tumor tissue was harvested on day 28 days after inoculation of MDA-MB-435 cells. CD31 was used as a biomarker for endothelial cells on blood vessels (both mature vasculature and neovasculture). Integrin β₃ was visualized with Cy3 (red) and CD31 was visualized with FITC (green). The yellow color in overlay images indicates the presence of integrin β₃ on neovasculature.