A molecular imaging paradigm to rapidly profile response to angiogenesis-directed therapy in small animals

Abstract

Purpose: The development of novel angiogenesis-directed therapeutics is hampered by the lack of non-invasive imaging metrics capable of assessing treatment response. We report the development and validation of a novel molecular imaging paradigm to rapidly assess response to angiogenesis-directed therapeutics in preclinical animal models.

Procedures: A monoclonal antibody-based optical imaging probe targeting vascular endothelial growth factor receptor-2 (VEGFR2) expression was synthesized and evaluated in vitro and in vivo via multispectral fluorescence imaging.

Results: The optical imaging agent demonstrated specificity for the target receptor in cultured endothelial cells and in vivo. The agent exhibited significant accumulation within 4T1 xenograft tumors. Mice bearing 4T1 xenografts and treated with sunitinib exhibited both tumor growth arrest and decreased accumulation of NIR800-alphaVEGFR2ab compared to untreated cohorts (p = 0.0021).

Conclusions: Molecular imaging of VEGFR2 expression is a promising non-invasive biomarker for assessing angiogenesis and evaluating the efficacy of angiogenesis-directed therapies.

Fig. 1

Spectroscopic characterization NIR800-αVEGFR2ab imaging probe in aqueous medium revealed peak absorbance at 777 nm and peak fluorescence emission at 798 nm.

Fig. 2

a Immunoblot for VEGFR2 expression and actin control in cultured endothelial (bEnd3) and breast tumor (4T1) cells. b Endothelial (bEnd3) cells were incubated with serial dilutions of NIR800-αVEGFR2ab probe and non-specific NIR800-IgG probe, washed, and imaged for fluorescence. c Fluorescence intensity was assessed from bEnd3 cells incubated with NIR800-αVEGFR2ab, bEnd3 cells blocked with excess unlabeled VEGR2 antibody followed by incubation with NIR800-αVEGFR2ab, and bEnd3 cells incubated with non-specific NIR800-IgG.

Fig. 3

a Representative fluorescence image of a mouse bearing a 4T1 xenograft tumor on the right hind limb. Image was collected 24 h after administration of NIR800-αVEGFR2ab demonstrating significant accumulation of the imaging probe within the tumor region. Single channel unmixed image of NIR800-αVEGFR2ab uptake (left) as well as multi-channel fluorescence composite image of autofluoresence (white) and NIR800-αVEGFR2ab uptake (red) are shown (right). b Representative fluorescence image of a mouse bearing a similar 4T1 xenograft tumor 24 h after administration of non-specific NIR800-IgG probe. Retention of the non-specific probe was not observed within the tumor region. Single channel unmixed image of NIR800-IgG uptake (left) as well as composite image of autofluoresence (white) and NIR800-IgG (red) are shown (right). c Quantified fluorescence intensity of the tumor region normalized to the contralateral flank for mice injected with the NIR800-αVEGFR2ab probe (n=20) and the non-specific NIR800-IgG probe (n=6).

Fig. 4

a Tumor size, as assessed by caliper measurement of the longest dimension, for animals treated with sunitinib (n=9) and treated with vehicle (n=10) before and after the treatment period (5 days). b Fluorescence intensity from NIR800-αVEGFR2ab accumulation in the tumor normalized to the contralateral hind limb for sunitinib-treated animals and animals receiving vehicle before and after the treatment period (5 days).

Fig. 5

a Immunohistochemical staining for VEGFR2 expression (brown) within the tumors of vehicle-treated animals (a) and sunitinib-treated animals (b). Immunohistochemical staining for CD34 (brown) within the tumors of vehicle-treated animals (c) and sunitinib-treated animals (d). Immunohistochemical staining with hematoxylin and eosin within the tumors of vehicle-treated animals (e) and sunitinib-treated animals (f). g Morphometric analysis of CD34 and VEGFR2 expression in vehicle- and sunitinib-treated mice. h Morphometric analysis of VEGFR2 expression versus fluorescence intensity from NIR800-αVEGFR2ab accumulation in the tumor normalized to the contralateral hind limb.