Novel FAP-Targeted Heptamethine Cyanines for NIRF Imaging Applications

Abstract

Fibroblast activation protein (FAP) is a pan-cancer target that is useful for imaging, ideally all epithelial cancers. This work aimed to develop, characterize, and validate two novel FAP-targeted probes for optical imaging, both in vitro and in vivo. IRDye800CW and FNIRTag heptamethine cyanines were conjugated to the NH precursor of the well-known FAP inhibitor FAPI-46, which is widely employed in nuclear medicine. In addition to synthesis, the dyes were characterized in terms of physicochemical properties, biodistribution, and imaging performances in a breast cancer tumor model. FAPI-FNIRTag showed a stronger fluorescence and higher photostability compared to FAPI-IRDye800CW. Notably, both compounds exhibited strong tumor accumulation in TUBO breast cancer-bearing mice 24 h postadministration, suggesting potential for further investigation as fluorescence-guided surgery (FGS) agents.

Keywords: FAP; breast cancer; heptamethine cyanines; near-infrared fluorescence imaging; optical imaging.

Figure 1

Synthetic protocol for FAPI-IRDye800CW and FAPI-FNIRTag.

Figure 2

Fluorescence excitation and emission spectra. Fluorescence excitation and emission spectra in PBS (pH = 7.4) of (A) FAPI-FNIRTag (λ_ex = 740 nm, λ_em = 788 nm) and (B) FAPI-IRDye800CW (λ_ex = 730 nm, λ_em = 790 nm).

Figure 3

Fluorescence linearity and phantom imaging. (A) Average radiance efficiency comparison between FAPI-FNIRTag and FAPI-IRDye800CW at different concentrations (IVIS measurements and phantom imaging). (B) Fluorescence emission comparison (cps) between FAPI-FNIRTag and FAPI-IRDye800CW based on fluorimeter measurements (λ_ex = 740 nm for FAPI-FNIRTag, λ_ex = 730 nm for FAPI-IRDye800CW). (C) Phantom imaging comparison between FAPI-FNIRTag (on the left) and FAPI-IRDye800CW (on the right) (λ_ex = 740 nm, λ_em = 790 nm).

Figure 4

Photostability. (A) FAPI-IRDye800CW excitation spectra before and after irradiation. (B) FAPI-IRDye800CW emission spectra before and after irradiation. (C) FAPI-FNIRTag excitation spectra before and after irradiation. (D) FAPI-FNIRTag emission spectra before and after irradiation. (E) The time course of normalized fluorescence intensity decreases after irradiation (2 h, every 60 s) for FAPI-FNIRTag and FAPI-IRDye800CW.

Figure 5

Serum albumin binding. Albumin binding data after 1 h incubation ([dye] = 0.15 μM, PBS pH 7.4) at RT. (A) FAPI-FNIRTag. (B) FAPI-IRDye800CW.

Figure 6

Fluorescent dye uptake on FAP-expressing cells. Fluorescence emission on FAP-expressing cells of dyes incubated at 0.5 μM (monochromatic histograms), dyes together with 100× excess of competitor (unconjugated FAPI-46) (angled patterned histograms), without any dyes (gray), and dyes on no-FAP-expressing cells (horizontal patterned histograms). Dot plots of acquired representative samples are fully reported in the Supporting Information. Data were shown as means ± SD **** p < 0.0001 (n = 3) t-test (α = 0.05).

Figure 7

Flow cytometry results of FAP-expressing cells in TUBO tumors. (A) Percentage of FAP-expressing cells in live cells extracted from TUBO tumors in Balb/c mice. Control results refer to unstained FAP-expressing cells. Data were shown as means ± SD *** p = 0.0005 (n = 4) t-test (α = 0,05). (B) Dot plot of the TUBO tumor sample (BalB/c mice), gating on live cells.

Figure 8

Immunohistochemical evaluation of excised tumor tissues from TUBO tumor-bearing Balb/C mice. Brown signals correspond to anti-FAP staining; nuclei were counterstained with hematoxylin. Immunohistochemical staining of excised tumor tissue in the absence of primary antibody anti-FAP above and immunohistochemical staining of excised tumor tissue (anti-FAP primary antibody) below.

Figure 9

Fluorescence imaging. (A) Kinetics of in vivo average radiance efficiency found for tumor tissue and (B) muscle (background). (C) Tumor-to-background ratio. * p = 0.0297 t-test (α = 0.05) (n = 3). (D) In vivo fluorescence imaging of TUBO tumor-bearing mice intravenously injected with 5 nmol of FAPI-IRDye800CW and FAPI-FNIRTag 24 h-postinjection (white arrows indicate the tumors; green arrow indicates the kidneys). (E) In vivo fluorescence imaging of TUBO tumor-bearing mice intravenously injected with 5 nmol of FAPI-FNIRTag at different time points. (F) In vivo fluorescence imaging of TUBO tumor-bearing mice intravenously injected with 5 nmol of FAPI-IRDye800CW at different time points.

Figure 10

Ex vivo biodistribution study. (A) Ex vivo fluorescence imaging of the main organs excised from TUBO tumor-bearing mice i.v. injected with 5 nmol of FAPI-FNIRTag or FAPI-IRDye800CW (1: muscle, 2: tumor, 3: heart, 4: lung, 5: liver, 6: spleen, 7: pancreas, 8: intestine, 9: kidneys, 10: blood). (B) Biodistribution for FAPI-FNIRTag and FAPI-IRDye800CW (5 nmol) in TUBO tumor-bearing athymic mice 24 h postinjection. p = 0.0161 for FAPI-FNIRTag tumor/muscle; p = 0.0127 for FAPI-IRDye800CW tumor/muscle; p = 0.0030 for kidneys comparison; and p = 0.0014 for liver comparison. (t-test α = 0.05). (C) Percentage of dye-bound cells in live cells extracted from TUBO tumors in nude mice excised 24 h after i.v. injection of 5 nmol of FAPI-IRDye800CW or FAPI-FNIRTag. p = 0.024 (t-test α = 0.05).