Near infrared fluorescent optical imaging for nodal staging

Abstract

Current techniques to assess lymph node metastases in cancer patients include lymphoscintigraphy after administration of a nonspecific radiocolloid in order to locate and resect lymph nodes for pathological examination of harbored cancer cells. Clinical trials involving intradermal or subcutaneous injection of antibody-based nuclear imaging agents have demonstrated the feasibility for target-specific, molecular imaging of cancer-positive lymph nodes. The basis for employing near-infrared (NIR) optical imaging for assessing disease is evidenced by recent work showing functional lymph imaging in mice, swine, and humans. We review antibody-based immunolymphoscintigraphy with an emphasis on the use of trastuzumab (or Herceptin) to target human epidermal growth factor receptor-2 (HER2) overexpressed in some breast cancers. Specifically, we review in vitro and preclinical imaging data from our laboratory that show how the dual-labeled agent ((111)In-DTPA)(n)-trastuzumab-(IRDye800)(m) utilizes the high photon count provided by an NIR fluorescent dye, IRDye 800CW, and the radioactive signal from a gamma emitter, Indium-111, for possible detection of HER2 metastasis in lymph nodes. We show that the accumulation and clearance of ((111)In-DTPA)(n)-trastuzumab-(IRDye800)(m) from the axillary nodes of mice occurs 48 h after intradermal injection into the dorsal aspect of the foot. The requirement for long clearance times from normal, cancer-negative nodes presents challenges for nuclear imaging agents with limited half-lives but does not hamper NIR optical imaging.

Fig. 1

(a) shows the while light image of a right breast from a patient who has been injected with IC-Green (100 μg) around the areola region. The injection sites have been covered to prevent oversaturation of the camera. (b) is the corresponding optical image, showing trafficking into the axillary lymph node. (c) is an overlay of optical imaging over the white light image of a swine’s hind limb, showing IC-Green (32 μM) transiting to the middle iliac node after i.d. delivery. (d) shows the lymph trafficking of IC-Green (1.3 mM) to the axillary node of a mouse after i.d. injection into the footpad. All images have been reproduced with permission from Refs. –.

Fig. 2

Schematic of (¹¹¹In-DTPA)_n-trastuzumab-(IRDye800)_m.

Fig. 3

Confocal image of one SKBr3 cell after incubation with (DTPA)_n-trastuzumab-(IRDye800)_m. Nuclear staining is by sytox green and is pseudo-colored in green, while fluorescence from IRDye 800 is represented in red. (Color online only.)

Fig. 4

(a) and (b) are representative white light and whole-body fluorescence images of an athymic nude mouse inoculated with SKBr3-luc xenografts, taken 48 h after i.v. administration of (¹¹¹In-DTPA)_n-trastuzumab-(IRDye800)_m. High agent uptake is visible in the left flank, which is consistent with the tumor region. Similar uptake is observed in nuclear imaging obtained from ¹¹¹In, as represented by planar scintigraphy (c) and SPECT/CT (d). Reproduced from Ref. .

Fig. 5

Plot of nuclear versus optical tumor-to-muscle ratios (TMRs), normalized per gram of tissue. No statistical differences have been observed, but optical imaging with smaller error bars indicates higher signal-to-noise ratios (SNRs). Reproduced from Ref. .

Fig. 6

Intradermal injection trastuzumab-(IRDye800)_m in the footpads of mice show trafficking to the axillary nodes (a) and popliteal nodes (b). The injection site has been covered in (b). A higher magnification of the region around the popliteal nodes shows lymphatic channels that drain into the node (c).

Fig. 7

Representative white light (a) and optical imaging (b) of accumulation and clearance of (In-DTPA)_n-trastuzumab-(IRDye800)_m (1 μg, 5.4 pmole) in the axillary node after administration into the forepaw footpad. Quantitative analysis of lymph node trafficking in five mice is represented in (c).