Near-infrared fluorescence imaging of tumor integrin alpha v beta 3 expression with Cy7-labeled RGD multimers

Abstract

Purpose: Cell adhesion molecule integrin alpha v beta 3 is an excellent target for tumor interventions because of its unique expression on the surface of several types of solid tumor cells and on almost all sprouting tumor vasculatures. Here, we describe the development of near-infrared (NIR) fluorochrome Cy7-labeled RGD peptides for tumor integrin targeting.

Procedures: Mono-, di-, and tetrameric RGD peptides were synthesized and conjugated with Cy7. The integrin specificity of these fluorescent probes was tested in vitro for receptor binding assay and fluorescence microscopy and in vivo for subcutaneous U87MG tumor targeting.

Results: The tetrameric RGD peptide probe with the highest integrin affinity showed the highest tumor activity accumulation and strongest tumor-to-normal tissue contrast. This uptake is integrin-specific as the signal accumulated in the tumor can be effectively blocked by unconjugated RGD peptide antagonist of integrin alpha v beta 3.

Conclusions: Noninvasive NIR fluorescence imaging is able to detect and semiquantify tumor integrin expression based upon the highly potent tetrameric RGD peptide probe.

Fig. 1

Chemical structures of Cy7-c(RGDyK), Cy7-E[c(RGDyK)]₂, and Cy7-E{E[c(RGDyK)]₂}₂.

Fig. 2

(A) In vitro inhibition of ¹²⁵I-echistatin binding to integrin α_vβ₃ expressed on U87MG cells by c(RGDyK) (▪), E[c(EGDyK)]₂ (▴), and E{E[c(EGDyK)]₂}₂ (▾). (B) In vitro inhibition of ¹²⁵I-echistatin binding to integrin α_vβ₃ expressed on U87MG cells by Cy7-c(RGDyK) (□), Cy7-E[c(RGDyK)]₂ (▵), and Cy7-E{E[c(RGDyK)]₂}₂ (▽). All data points were acquired from triplicates.

Fig. 3

Fluorescence microscopy images of U87MG cells stained with 1 μM of Cy7, Cy7-c(RGDyK), Cy7-E[c(RGDyK)]₂, and Cy7-E{E[c(RGDyK)]₂}₂ without (Experiment A–D: brightfield images; Experiment E–H: fluorescence images) and with (Block I–L: brightfield images; Block M–P: fluorescence images) 20 μM c(RGDyK).

Fig. 4

Direct comparison of whole-body NIR fluorescence imaging of subcutaneous U87MG tumor-bearing mice injected with 500 pmol Cy7, Cy7-c(RGDyK), Cy7-E[c(RGDyK)]₂, or Cy7-E{E[c(RGDyK)]₂}₂ at 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours, p.i. Tumors are indicated by arrows and all images are normalized to the same scale.

Fig. 5

(A) Fluorescence intensity (p/s/cm²/sr) of the tumor as a function of time after injection of Cy7 (○), Cy7-c(RGDyK) (▴), Cy7-E[c(RGDyK)]₂ (▾), or Cy7-E{E[c(RGDyK)]₂}₂ (•). (B) Tumor contrast (tumor/normal tissue ratio) as a function of time postadministration of the Cy7-RGD conjugates.

Fig. 6

(A) Representative in vivo NIR fluorescence imaging (90° mounting angle) of U87MG tumor-bearing mice injected with Cy7-RGD conjugates (500 pmol) with (Block) and without (Control) coinjection of 200 μg/mouse of c(RGDyK) at 2 hours, p.i. Tumors are indicated by arrows. (B) Representative fluorescence images of dissected U87MG tumor and major organs and tissues after noninvasive imaging at 2 hours p.i.. From left to right: U87MG tumor, muscle, liver, kidney, and intestine.

Fig. 7

(A) ROI analysis of fluorescence intensity in vivo of U87MG tumor with (Block) and without (Experiment) coinjection of blocking dose of c(RGDyK) at 2 hours, p.i. (B) ROI analysis of dissected tumors of the same mice described in A. (C) Serum stability of Cy7-c(RGDyK). Fluorescence intensity at 775 nm over time was plotted (excitation: 720 nm).