Preclinical imaging of epidermal growth factor receptor with ABY-029 in soft-tissue sarcoma for fluorescence-guided surgery and tumor detection

Abstract

Background and objectives: Fluorescence-guided surgery using epidermal growth factor receptor (EGFR) targeting has been performed successfully in clinical trials using a variety of fluorescent agents. We investigate ABY-029 (anti-EGFR Affibody^® molecule labeled with IRDye 800CW) compared with a small-molecule perfusion agent, IRDye 700DX carboxylate, in a panel of soft-tissue sarcomas with varying levels of EGFR expression and vascularization.

Methods: Five xenograft soft-tissue sarcoma cell lines were implanted into immunosuppressed mice. ABY-029 and IRDye 700DX were each administered at 4.98 μM. Fluorescence from in vivo and ex vivo (fresh and formalin-fixed) fixed tissues were compared. The performance of three fluorescence imaging systems was assessed for ex vivo tissues.

Results: ABY-029 is retained longer within tumor tissue and achieves higher tumor-to-background ratios both in vivo and ex vivo than IRDye 700DX. ABY-029 fluorescence is less susceptible to formalin fixation than IRDye 700DX, but both agents have disproportional signal loss in a variety of tissues. The Pearl Impulse provides the highest contrast-to-noise ratio, but all systems have individual advantages.

Conclusions: ABY-029 demonstrates promise to assist in wide local excision of soft-tissue sarcomas. Further clinical evaluation of in situ or freshly excised ex vivo tissues using fluorescence imaging systems is warranted.

Keywords: Affibody® molecule; IRDye 700DX; IRDye 800CW; Odyssey CLx; Pearl Impulse; Solaris.

Figure 1.

Fluorescence intensity of ABY-029 (solid lines) and IRDye 700DX (dashed lines) over 24 hours in various soft tissue sarcoma xenograft models and normal tissues. The displayed images are for the Pearl imaging system. Similar trends were observed for the Solaris and Odyssey imaging systems (not shown).

Figure 2.

In vivo soft tissue sarcoma tumors display varying levels of ABY-029 and IRDye 700DX uptake at 4 hours post-administration. Images were collected on the Pearl and are scaled to the same relative intensities to show differences in uptake. The animal was covered with a black drape with a small opening cut into the fabric to exclude high fluorescent signal from abdominal organs. The white dashed line indicates the tumor region, all other visible fluorescence within the field is normal muscle.

Figure 3.

Ex vivo xenograft soft tissue sarcoma tumors demonstrate varying levels of EGFR expression and corresponding uptake of ABY-029 and IRDye 700DX at 4 hours post-administration imaged on the Odyssey scanner. Odyssey scanner images are displayed on the same scale (right) for each fluorophore to demonstrate the varying uptake observed between tumor types.

Figure 4.

Effects of formalin fixation on ABY-029 (solid) and IRDye 700DX (dashed) over time. Top: Changes in fluorescence intensity from fresh (t = 0) to 24 hours post-fixation. Bottom: The change in tumor-to-background ratio (TBR) from fresh to 24 hours post-formalin fixation.

Figure 5.

Tumor-to-background ratio (TBR) varies over time and tissue type imaged on the Pearl Impulse. In vivo measurements (left column) demonstrate that all tumors have significantly greater TBR than the autofluorescence values at t = 0 h for ABY-029 (white box) for at least one time point, but mainly at 4 hours or greater. IRDye 700DX TBR (grey box) only showed statistical significance at 8-hours in the SK-NEP-1 tumor line. No in vivo TBR showed statistical significance at t ≤ 2 h. Ex vivo TBR (right column) demonstrated significant differences from the autofluorescence control at many time points for both ABY-029 and IRDye 700DX.

Figure 6.

Comparing three fluorescence imaging systems using contrast-to-variance ratio (CVR). A) A representative ABY-029 fluorescence image from all three systems is provided, using the 4-hour time point and the 800-nm channel for SK-LMS-1 tumor and normal tissues. B) The Pearl performed the best for both ABY-029 and IRDye 700DX imaging agents, but the Solaris was comparable. Note that the Odyssey displays the most fluorescence detail, such that the large variation in fluorescence intensity observed causes low CVR. The highest CVRs were observed between 4 and 24 hours for all imaging systems.