Abstract
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Objectives: Lung cancer remains the leading cause of cancer related death worldwide, accounting for more than 13% of cancers diagnosed each year. The development of new strategies for treatment and imaging of lung cancer may decrease the lethality of the disease. For this reason, new tools are needed for imaging malignancies, monitoring therapeutic response, and selecting patients that may benefit from targeted therapies. Previously, CD146 was identified as an excellent biomarker in lung cancer and its overexpression in solid tumors has been linked to disease progression, invasion, and metastasis. In this study, we investigate 64Cu-labeled YY146, an antibody targeting human CD146, for imaging of CD146 expression in two orthotopic models of lung cancer.
Methods: CD146 expression and binding was evaluated in H460 (CD146+) and H358 (CD146-) lung cancer cell lines by flow cytometry. Binding assays were also performed in each cell line. For PET, the antibody (YY146) was conjugated with NOTA and radiolabeled with Cu-64 (64Cu-NOTA-YY146). Mice were intravenously injected with two million tumor cells. CT imaging was used after four weeks to determine anatomic location of the lungs and corresponding metastatic nodules. The radiolabeled antibody was then injected into the orthotopic tumor-bearing mice and animals were subjected to PET-CT imaging at 4, 24, and 48 h post-injection. PET signal was quantified as both whole lung signal and maximum lung signal, as there were several small lung nodules throughout the whole organ volume. Image analysis was verified through ex vivo biodistribution by organ harvesting and gamma counting of organ activity. Further validation was accomplished through immunofluorescence imaging of excised tumors with CD31 and CD146.
Results: Flow cytometry revealed strong binding of YY146 to H460 cells due to the high expression of CD146, which was previously determined through Western blot analysis. Both YY146 and NOTA-YY146 showed similar binding, suggesting that conjugation of the antibody had no ill effects on its binding affinity. Binding assays revealed a Kd of 2.84 ± 0.53 nM for H460 cells. Imaging with 64Cu-NOTA-YY146 revealed rapid, persistent, and highly specific accumulation in CD146-expressing tumors. The whole lung signal was calculated for H460 and H358 as 7.42 ± 0.38 %ID/g and 3.95 ± 0.47 %ID/g at 48 h post-injection, respectively (n=3, p<0.05), while the maximum lung signal was determined to be 13.85 ± 1.07 (H460) and 6.08 ± 0.73 %ID/g (H358) at the same time point (n=3, p<0.05). Ex vivo biodistribution validated these findings with lung tumor signal being two-fold higher for H460 as compared to H358. Lastly, confocal imaging of excised lung tumor sections in the H460 group showed high CD146 expression on both vasculature and surrounding cells, which was much lower in the H358 group.
Conclusion: The novel tracer targeting CD146 (64Cu-NOTA-YY146) was successfully employed for imaging of orthotopic lung cancer in mice implanted with H460 (CD146+) or H358 (CD146-) cell lines. This study provides the initial evidence supporting the future clinical utilization of 64Cu-NOTA-YY146 for enhanced patient stratification or monitoring of therapeutic response. Research Support: NIH, American Cancer Society, and the University of Wisconsin - Madison