In vivo albumin labeling and lymphatic imaging

Abstract

The ability to accurately and easily locate sentinel lymph nodes (LNs) with noninvasive imaging methods would assist in tumor staging and patient management. For this purpose, we developed a lymphatic imaging agent by mixing fluorine-18 aluminum fluoride-labeled NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid)-conjugated truncated Evans blue ((18)F-AlF-NEB) and Evans blue (EB) dye. After local injection, both (18)F-AlF-NEB and EB form complexes with endogenous albumin in the interstitial fluid and allow for visualizing the lymphatic system. Positron emission tomography (PET) and/or optical imaging of LNs was performed in three different animal models including a hind limb inflammation model, an orthotropic breast cancer model, and a metastatic breast cancer model. In all three models, the LNs can be distinguished clearly by the apparent blue color and strong fluorescence signal from EB as well as a high-intensity PET signal from (18)F-AlF-NEB. The lymphatic vessels between the LNs can also be optically visualized. The easy preparation, excellent PET and optical imaging quality, and biosafety suggest that this combination of (18)F-AlF-NEB and EB has great potential for clinical application to map sentinel LNs and provide intraoperative guidance.

Keywords: Evans blue; PET; albumin; lymph node; optical imaging.

Fig. 1.

(A) Representative reconstructed coronal PET images of inflamed popliteal (Upper) and sciatic (Lower) LNs in the turpentine oil-induced hind limb inflammation model. LNs were pointed out by white arrows. (B) T₂-weighted MRI shows an enlarged inflamed popliteal LN, as indicated by a white arrow. (C) Overlay of PET with a 2D X-ray image. The LN is indicated by a white arrow and the injection sites by arrowheads. (D) Quantitative analysis based on the PET images. There is significantly higher total tracer uptake in inflamed popliteal LNs than that of contralateral normal LNs at 0.5, 1, 2, and 3 h after tracer injection (*P < 0.05). (E) Quantitative analysis of tracer uptake in sciatic LNs. No statistical significance was found between LNs in the left and right side.

Fig. 2.

(A–C) Representative ¹⁸F-AlF-NEB PET images of an axillary LN in the orthotropic breast cancer model (A, transaxial; B, sagittal; C, coronal images). PET scans were performed at 30 min after tracer injection. Arrows indicate tumor-draining axillary LNs, and arrowheads indicate primary tumors. A white dotted line was added to indicate animal contour. (D and E) Photograph (D) and ex vivo PET image (E) confirmed tracer uptake of an ipsilateral axillary LN after intratumoral injection of ¹⁸F-AlF-NEB. (F) Coronal image shows a cervical LN. Arrows indicate tumor-draining axillary LNs, and arrowheads indicate primary tumors.

Fig. 3.

(A) Representative BLI imaging of a metastatic popliteal LN (white arrow) located near the primary tumor (white arrowhead). (B) Axial T₂-weighted MRI shows an enlarged metastatic popliteal LN, as indicated by a white arrow. (C) Immunofluorescence staining confirmed the existence of metastasis in the popliteal LN. Yellow dashed line differentiates metastasis (Upper) from normal lymphatic tissue (Lower). (D) Representative coronal PET images of metastatic popliteal LNs (white arrows) at different time points after local injection of ¹⁸F-AlF-NEB. White arrowheads indicate the injection site. (E) Autoradiography confirmed the metastasis (cold area) in the popliteal LN. (F) Quantitative analysis of the total tracer uptake in tumor-draining LN (TLN) and right side normal LN (RLN). The value was corrected by the weights of LNs (*P < 0.05).

Fig. 4.

(A and B) Representative PET images show high tracer uptake in sciatic LN (A) or inguinal LN (B). Three sections of the same LN were presented (from left to right, transaxial, coronal, and sagittal). (C and D) H&E stain of a healthy popliteal LN (C) and a metastatic LN (D). Yellow dashed line delineates metastasis foci at the subscapular sinus area.

Fig. 5.

(A) LN mapping with EB dye in a turpentine oil-induced hind limb inflammation model. White arrows indicate popliteal LNs with blue color, and red arrows show the light blue left sciatic LN. (B) Photograph of excised LNs. The upper two are popliteal LNs, and the lower two are sciatic LNs. LNs on the left side are harvested from the inflamed hind limb, whereas those on the right side are from a normal limb. (C) Quantitative analysis of LN size based on its weight (*P < 0.05). (D) UV measurement showed the difference of EB amount in different LNs (*P < 0.05).

Fig. 6.

(A) Longitudinal fluorescence imaging of the lymphatic system after hock injection of ¹⁸F-AlF-NEB/EB. LNs and lymphatic vessels can be clearly seen with the migration of the tracer along with time. (B) Ex vivo optical imaging of LNs without skin. (C) Photograph of the same mice to show the blue color within the LNs. (D) Coregistration of optical image (Left) and PET image (Middle) to present the popliteal LNs, indicated by a white arrow. (E) Coregistration of optical image (Left) and PET image (Middle) to present the sciatic LNs, indicated by a white arrow. The mice were euthanized at 90 min after hock injection of ¹⁸F-AlF-NEB/EB and the skin was removed.