Real-time near-infrared fluorescence imaging using cRGD-ZW800-1 for intraoperative visualization of multiple cancer types

Henricus J M Handgraaf, Martin C Boonstra, Hendrica A J M Prevoo, Joeri Kuil, Mark W Bordo, Leonora S F Boogerd, Babs G Sibinga Mulder, Cornelis F M Sier, Maaike L Vinkenburg-van Slooten, A Rob P M Valentijn, Jacobus Burggraaf, Cornelis J H van de Velde, John V Frangioni, Alexander L Vahrmeijer, Henricus J M Handgraaf, Martin C Boonstra, Hendrica A J M Prevoo, Joeri Kuil, Mark W Bordo, Leonora S F Boogerd, Babs G Sibinga Mulder, Cornelis F M Sier, Maaike L Vinkenburg-van Slooten, A Rob P M Valentijn, Jacobus Burggraaf, Cornelis J H van de Velde, John V Frangioni, Alexander L Vahrmeijer

Abstract

Incomplete resections and damage to critical structures increase morbidity and mortality of patients with cancer. Targeted intraoperative fluorescence imaging aids surgeons by providing real-time visualization of tumors and vital structures. This study evaluated the tumor-targeted zwitterionic near-infrared fluorescent peptide cRGD-ZW800-1 as tracer for intraoperative imaging of multiple cancer types. cRGD-ZW800-1 was validated in vitro on glioblastoma (U-87 MG) and colorectal (HT-29) cell lines. Subsequently, the tracer was tested in orthotopic mouse models with HT-29, breast (MCF-7), pancreatic (BxPC-3), and oral (OSC-19) tumors. Dose-ranging studies, including doses of 0.25, 1.0, 10, and 30 nmol, in xenograft tumor models suggest an optimal dose of 10 nmol, corresponding to a human equivalent dose of 63 μg/kg, and an optimal imaging window between 2 and 24 h post-injection. The mean half-life of cRGD-ZW800-1 in blood was 25 min. Biodistribution at 4 h showed the highest fluorescence signals in tumors and kidneys. In vitro and in vivo competition experiments showed significantly lower fluorescence signals when U-87 MG cells (minus 36%, p = 0.02) or HT-29 tumor bearing mice (TBR at 4 h 3.2 ± 0.5 vs 1.8 ± 0.4, p = 0.03) were simultaneously treated with unlabeled cRGD. cRGD-ZW800-1 visualized in vivo all colorectal, breast, pancreatic, and oral tumor xenografts in mice. Screening for off-target interactions, cRGD-ZW800-1 showed only inhibition of COX-2, likely due to binding of cRGD-ZW800-1 to integrin αVβ3. Due to its recognition of various integrins, which are expressed on malignant and neoangiogenic cells, it is expected that cRGD-ZW800-1 will provide a sensitive and generic tool to visualize cancer during surgery.

Keywords: RGD; fluorescence-guided surgery; in vivo diagnosis; integrins; preclinical validation.

Conflict of interest statement

CONFLICTS OF INTEREST

John V. Frangioni is currently CEO of Curadel, Curadel ResVet Imaging, and Curadel Surgical Innovations, which are for-profit companies that have licensed FLARE ® technology from Beth Israel Deaconess Medical Center. All other authors have no conflicts of interests or financial ties to disclose.

Figures

Figure 1. Binding assay and in vitro…
Figure 1. Binding assay and in vitro and in vivo competition experiments
(A) Binding assay of cRGD-ZW800-1 on intermediate integrin-expressing HT-29 cells shows an almost linear increase in fluorescence intensity with increasing applied concentrations. In addition, fluorescence signals are increased when cells are incubated at 37°C compared to 4°C with the various concentrations (p < 0.0001). This may be the result of enhanced internalization of the agent. (B) In vitro competition experiment (1:1 ratio) using unlabeled cRGD (competition group) showing decreased NIR fluorescent signals in both the αvβ3 positive U-87 MG (*p < 0.05) as well as the αvβ3 negative HT-29 cell line compared to the control without unlabeled cRGD. Healthy colon was used as background. (C) In vivo competition experiment using a 200 times higher dose of unlabeled cRGD compared to the dose of cRGD-ZW800-1 (1.24 mg, 2.0 μmol vs. 15.5 μg, 10 nmol) in the orthotopic HT-29 model. Compared to mice in the control group, a significant decrease in TBR was seen at 4 h (*p = 0.02) and 24 h (** p = 0.007). Healthy colon was used as background.
Figure 2. Near-infrared fluorescence imaging of colorectal…
Figure 2. Near-infrared fluorescence imaging of colorectal cancer (HT-29)
(A) Representative images captured using the original FLARE® prototype of the various concentrations at 4 h post injection. For the 30 nmol dose group the exposure time had to be set at 50 msec due to saturation of the near-infrared fluorescence images. For the other dose groups 200 msec was sufficient. NIR fluorescence signals are clearly visible in 10 and 30 nmol groups only. Arrow = tumor; arrowhead = bladder. (B) Near-infrared fluorescence imaging at 4 h post injection showed sufficient tumor-to-background ratios (i.e. ≥ 2) already in the 0.25 nmol (0.39 μg) dose group using the Pearl®. Surrounding tissue was used as background. (C) Near-infrared fluorescence imaging using the Pearl® at 24 h post injection still showed sufficient tumor-to-background ratios in the 1.0, 10 and 30 nmol dose groups. Healthy tongue tissue was used as background.
Figure 3. Near-infrared fluorescence imaging of pancreatic…
Figure 3. Near-infrared fluorescence imaging of pancreatic (BxPC-3) and oral cancer (OSC-19)
(A) Upper panel: example showing the in vivo images of 30 nmol cRGD-ZW800-1 in the orthotopic pancreas model at 4 h post injection using the original FLARE® prototype. Arrow = tumor. Lower panel: NIR fluorescence microscopy of the tumor shows fluorescence inside the cells. (B) Mean tumor-to-background ratios at 4 h post injection in the BXPC-3 model with the Pearl®. Skin was used as background. (C) Upper panel: example of images from the orthotopic OSC-19 model at 4 h using the original FLARE® prototype. Arrow = tumor. Lower panel: NIR fluorescence microscopy shows the border of a resected OSC-19 tumor. Clear fluorescent demarcation between normal and tumor tissue is shown (white dotted line). T = tumor; N = normal tissue surrounding the tumor. (D) Mean tumor-to-background ratios of the optimal dose 10 nmol at 4 h in the orthotopic head-and-neck cancer model using the Pearl®.
Figure 4. Near-infrared fluorescence imaging of breast…
Figure 4. Near-infrared fluorescence imaging of breast cancer (MCF-7)
(A) Representative images captured with the Pearl® of the various time points after administration of 10 nmol cRGD-ZW800-1 in a xenograft breast cancer model. Arrowhead: bladder, arrows: tumors. All images are identically scaled. (B) Tumor-to-background ratios over time measured using the Pearl®. (C) Fluorescence intensity of tumors compared with 0.5 h post injection using the Pearl®.
Figure 5. Biodistribution of cRGD-ZW800-1
Figure 5. Biodistribution of cRGD-ZW800-1
(A) In vivo fluorescence intensity of BxPC-3 and HT-29 tumors at 4 h post injection of several doses of cRGD-ZW800-1 using the Pearl®. Significant differences (*p < 0.05; **p < 0.01) were seen in the 10 nmol dose group (p < 0.05). (B) The ex vivo fluorescence intensity of BxPC-3 tumors were used to calculate the organ-to-tumor ratios at 4 h post injection. Compared to the 10 nmol dose group, significant differences were seen between kidneys (1.0 nmol: p < 0.05; 30 nmol: p < 0.01 ), liver (1.0 nmol: p < 0.05) and large intestines (1.0 nmol: p < 0.05). In all dose groups, tumors were more than twice as fluorescent compared to skin, intestines, lungs, muscle, brain, gallbladder, and blood. (C) In vivo fluorescence intensity of HT-29 tumors at 4 and 24 h post injection of several doses. Significant differences (*) were seen in the 10 nmol dose group (p < 0.05). (D) The ex vivo fluorescence intensity of HT-29 tumors were used to calculate the organ-to-tumor ratios at 24 h post injection. Compared to the 10 nmol dose group, significant differences (*) were seen between kidneys (1.0 nmol and 30 nmol: p < 0.05). In all dose groups, tumors were more than twice as fluorescent compared to lungs, pancreas, muscle, gallbladder, brain, and blood.
Figure 6. Pharmacokinetics of 10 nmol cRGD-ZW800-1
Figure 6. Pharmacokinetics of 10 nmol cRGD-ZW800-1
Graph shows the mean absolute concentrations (in μM) after intravenous administration of 10 nmol cRGD-ZW800-1 up to 240 min post injection. 1 AUC calculated via trapezoid rule. 2 Half-life calculated via ln(2)/k10.

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