ImmunoPET imaging of CD146, a novel epithelial-mesenchymal transition (EMT) marker

Objectives Aberrant activation of EMT is tightly associated with tumor aggressiveness, however few studies have imaged this process in vivo. Our goal is to establish CD146, a proposed EMT activator, as an in vivo marker for EMT in a glioblastoma (GBM) model.

Methods A fast immunization approach was used to generate murine antibodies against human CD146. ELISA was performed to select 5 hybridoma clones secreting monoclonal antibodies (mAb), and the best clone called YY146 was selected for in vivo studies. Western blot, flow cytometry (FACS), and staining were conducted in >10 cell lines to determine the CD146 expression level. YY146 was conjugated to NOTA for ⁶⁴Cu-labeling, which maintained CD146 specificity/avidity based on FACS. Subcutaneous and orthotopic U87MG (CD146+) and PC3 (CD146-) tumor models were selected to evaluate ⁶⁴Cu-NOTA-YY146 in vivo. Progression of orthotopic U87MG tumor was monitored by T2-weighted MRI. Sequential PET scans, blocking, histologic, and biodistribution studies were carried out to confirm the specificity of ⁶⁴Cu-NOTA-YY146 for CD146.

Results We developed a new and efficient mAb production method, and produced large amount (~100 mg) of anti-CD146 mAbs for in vivo studies. In vitro studies showed that YY146 was the best clone. ⁶⁴Cu-labeling of NOTA-YY146 was achieved with good yield (>60%) and specific activity (~1 Ci/μmol). MicroPET studies revealed high and persistent uptake of ⁶⁴Cu-NOTA-YY146 in CD146+ U87MG tumor (4.6±0.2, 13.2±0.6, and 13.7±0.7 %ID/g at 4, 24, and 48h post-injection;n=3), significantly higher than that in CD146- PC3 tumor (< 5%ID/g). Blocking and histology studies both confirmed CD146 specificity of the tracer in vivo. Importantly, ⁶⁴Cu-NOTA-YY146 PET in a more clinically relevant orthotopic U87MG brain tumor model also showed high tumor uptake, comparable to the xenograft model.

Conclusions We produced a mAb that strongly binds to CD146, a key player in EMT, and report the first in vivo imaging of CD146. Successful PET imaging of CD146 with ⁶⁴Cu-NOTA-YY146 in GBM envisages the potential of this novel mAb for cancer diagnosis/therapy.