Katheryne E. Wilson1, Sunitha V. Bachawal1, Lotfi Abou-Elkacem1, Kristen Jensen2, Steven Machtaler1, Lu Tian3, and Jürgen K. Willmann1
- Department of Radiology, Molecular Imaging Program at Stanford, Stanford University, School of Medicine, Stanford, California, USA;
- Departments of Pathology, Stanford University, School of Medicine, Stanford, California, USA;
- Department of Health Research and Policy, Stanford University, School of Medicine, Stanford, California, USA.
Purpose: Breast cancer imaging methods lack diagnostic accuracy, in particular for patients with dense breast tissue, and improved techniques are critically needed. The purpose of this study was to evaluate antibody-indocyanine green (ICG) conjugates, which undergo dynamic absorption spectrum shifts after cellular endocytosis and degradation, and spectroscopic photoacoustic (sPA) imaging to differentiate normal breast tissue from breast cancer by imaging B7-H3, a novel breast cancer associated molecular target.
Methods: Quantitative immunohistochemical staining of endothelial and epithelial B7-H3 expression was assessed in 279 human breast tissue samples, including normal (n=53), benign lesions (11 subtypes, n=129), and breast cancers (4 subtypes, n=97). After absorption spectra of intracellular and degraded B7-H3-ICG and Isotype control-ICG (Iso-ICG) were characterized, sPA imaging in a transgenic murine breast cancer model (FVB/N-Tg(MMTVPyMT)634Mul) was performed and compared to imaging of control conditions [B7-H3-ICG in tumor negative animals (n=60), Iso-ICG (n=30), blocking B7-H3+B7-H3-ICG (n=20), and free ICG (n=20)] and validated with ex vivo histological analysis.
Results: Immunostaining showed differential B7-H3 expression on both the endothelium and tumor epithelium in human breast cancer with an area under the ROC curve of 0.93 to differentiate breast cancer vs non-cancer. Combined in vitro/in vivo imaging showed that sPA allowed specific B7-H3-ICG detection down to the 13 nM concentration and differentiation from Iso-ICG. sPA molecular imaging of B7-H3-ICG showed a 3.01-fold (P<0.01) increase in molecular B7-H3-ICG signal in tumors compared to control conditions.
Conclusions: B7-H3 is a promising target for both vascular and epithelial sPA imaging of breast cancer. Leveraging antibody-ICG contrast agents and their dynamic optical absorption spectra allows for highly specific sPA imaging of breast cancer.
Keywords: Photoacoustic Imaging, Breast Cancer, Molecular Imaging, Spectroscopic, Indocyanine Green
CovalX Technology Used (Click each option to learn more)
To determine the dye/Ab binding ratio, a mass spectrometer that had been modified with a CovalX high mass detection system was used. Before analysis, the B7-H3 antibody and conjugated B7-H3-ICG were purified using pipette tip resin that had been activated with HPLC-grade acetonitrile and then equilibrated using 0.1% Trifluoroacetic acid (TFA) in three washes. 10 μg of the samples were placed in 20 μl of 0.1% TFA and passed through the resin 8 times. To desalt and purify the samples after binding, the resin was washed three times with 0.1% TFA. The samples were then extracted into 2 μl sinapic acid and spotted onto a plate. By dividing the difference in peak averages by the mass of one ICG-NHS dye molecule (810 Da), the average number of dyes bound to an antibody can be determined. The average number was found to be approximately 7-8 ICG dye molecules per antibody.