Monitoring circulating tumor cells in vivo by a confocal microscopy system

Abstract

Circulating tumor cells (CTCs) play a key role in cancer metastasis but are very difficult to detect. in vivo monitoring CTCs has been recognized as an important technique for cancer research and clinical diagnosis. Recently, a noninvasive method, in vivo flow cytometry (IVFC) has been developed to enable continuous, real-time, and long-duration detection of CTCs in animal models by detecting CTC fluorescence in blood vessels excited by lasers. In this study, we present a simple optical scheme for direct noninvasive CTC detection using confocal microscopes. We demonstrate that line scanning of confocal microscopy can provide effective and quantitative CTC detection in live mice during cancer development. Rare CTC signals can be acquired at the early stage of the tumor development after implantation of subcutaneous tumor and monitored continuously to the end. Signals from CTC clusters can also be acquired and distinguished from single CTCs. Our results suggest confocal microscopy is a simple and reliable method for biologists and doctors to use for cancer research.CIRCULATING tumor cells (CTCs) have been recognized as an important role in cancer metastasis (1). The existence of CTCs in the circulating system brings the risk that CTCs may migrate into an organ and develop a new tumor (2). Generally, in practical biological and clinical research, the in vitro assays including immunomagnetic platforms and microchips, are the most commonly used for the CTC study (3,4). The CTCs can be isolated from blood or lymph and captured by those chips. An in vivo method to capture CTCs in blood vessels was developed in 2012 by using structured medical wires (5,6). The recent observation of CTC clusters in vitro has challenged previous understandings and presented new insights on the mechanism of metastasis by CTCs (7,8). They were found with high risk of developing new tumors. The ratio of CTC clusters in total CTCs was estimated as only less than 9% in those studies but CTC clusters were believed to have higher metastatic potential than individual CTCs up to 50-fold.It is difficult to detect rare single CTCs and CTC clusters by isolation of CTCs from blood in vitro at the early stage of cancer. Moreover, it is impossible to monitor single CTCs and CTC clusters in real time by in vitro methods. Particularly, CTC clusters may be broken into single CTCs in the purification process of blood samples. In 2004, the concept of in vivo flow cytometry (IVFC) was proposed for CTC detection (9,10). The blood flow was considered as a fluidic system of cytometer and lasers were focused into the blood vessels noninvasively to excite fluorescently-labeled CTCs. The fluorescence of CTCs that passed through the laser focus could then be excited, collected and detected in vivo. Great progresses have been made by this method in cancer research (11,12). Recently, the proportion of

Recent progress of nanostructure-based enrichment of circulating tumor cells and downstream analysis

“…10A). 23 If the diameter of the vein (−80 μm) is too large compared to the CTC size (diameter: 10-20 μm), the CTCs may flow through the optical part of the scan line in the vessel. Therefore, such confocal line scans may miss those out-offocus signals, resulting in reduced sensitivity of CTCs in whole vessels.…”

High counting of circulating tumor cells in blood is not directly related to metastasis

“…We then used a homemade inverted confocal microscope to monitor CTCs in the PC3 tumor mouse model in vivo [21]. The blood vessels were at first fluorescently labeled by tail intravenous injection of the vascular endothelial antibody, PE-CD105.…”

Constructing an In Vitro and In Vivo Flow Cytometry by Fast Line Scanning of Confocal Microscopy

“…Although the information of sample morphology is theoretically embedded in the fluorescence signal, i.e., the width of the fluorescence pulse given the fluidic speed is a constant, in practice, the morphology is extracted from the scattering light, even indirectly, to eliminate the error of fluctuation of the fluidic speed and the fluorescence labeling. Only the amplitude of fluorescence signals is counted as the indication of molecules [ 5 ].…”