In vivo quantitation of circulating tumor cells based on custom-built high-speed confocal microscopy for cancer metastasis research

Abstract

Although the major cause of approximately 90% of death is the result of vital organ failure by the cancer metastasis in the solid cancer patients, it is difficult to diagnose this cancer micro-metastasis at the early point of cancer progress. During the progress of cancer metastasis, cancer cells are disseminated from the primary tumor into the bloodstream and extravasated into the distant organs. In this cancer metastasis cascade, the cancer cells circulating in the bloodstream, which are called circulating tumor cells (CTCs). The quantitation of CTCs can provide significant information for predicting cancer metastasis, evaluating anti-cancer treatment, also potentially for early diagnosis of cancer recurrence. At first, we implemented a custom-design high-speed laser-scanning confocal microscopy system incapable of direct visualization of fast flowing CTC in the great saphenous vein (GSV) of a live animal model, in vivo. Continuous acquisition of video-rate images at GSV revealed the highly dynamic time-dependent changes in the number of intravenously injected CTCs. By extracting a calibration factor through the hemocytometric analysis of intravenously injected long-circulating red blood cells, we established a novel quantitation method for CTC in whole body blood, in vivo. In addition, we observed the dynamic changes of CTCs in spontaneous metastasis models by long-term GSV imaging. For monitoring the level of CTCs in the blood circulation, we estimated the detection volume of intravital GSV imaging. By repeatedly quantitating the CTCs of metastatic breast tumor model in a long time, we confirmed that the number of CTCs at the early point was associated with the formation of lung metastatic colonies at the late stage of the tumor. Finally, we designed the bloodstream mimicking circulation system for the real-time measurement of nanoparticle targeting efficacy. By image-based analysis of target cells, the cell-targeting mechanism was analyzed. Changing of various parameters, we obtained time-dependent targeting kinetics between CTCs and targeting nanoparticle during circulation condition.