In vivo quantitation of rare circulating tumor cells by multiphoton intravital flow cytometry

Abstract

Quantitation of circulating tumor cells (CTCs) constitutes an emerging tool for the diagnosis and staging of cancer, assessment of response to therapy, and evaluation of residual disease after surgery. Unfortunately, no existing technology has the sensitivity to measure the low numbers of tumor cells (<1 CTC per ml of whole blood) that characterize minimal levels of disease. We present a method, intravital flow cytometry, that noninvasively counts rare CTCs in vivo as they flow through the peripheral vasculature. The method involves i.v. injection of a tumor-specific fluorescent ligand followed by multiphoton fluorescence imaging of superficial blood vessels to quantitate the flowing CTCs. Studies in mice with metastatic tumors demonstrate that CTCs can be quantitated weeks before metastatic disease is detected by other means. Analysis of whole blood samples from cancer patients further establishes that human CTCs can be selectively labeled and quantitated when present at approximately 2 CTCs per ml, opening opportunities for earlier assessment of metastatic disease.

Fig. 1.

Optimization of in vivo detection of fluorescent cells in circulation. (a) Dot plot of signal and background intensities of individual fluorescent DiIC₁₈ (3)-labeled RBCs flowing through the vasculature in the ear of a live mouse, as determined by multiphoton (red, n = 107), confocal (green, n = 55), and nonconfocal (blue, n = 100) microscopy. (b) Kinetics of the clearance of folate-rhodamine from circulation in anesthetized mice. Red arrow marks the injection point. (c) Overlay image of three consecutive frames spanning 1 s showing an in vivo folate-rhodamine-labeled L1210A cell traveling in a blood vessel. (d) Specificity of in vivo labeling of L1210A tumor cells with folate-FITC demonstrated by the overlap (yellow) of folate-FITC (green) and DiD (red) fluorescence. (e) Visualization of digitized signals of fluorescent L1210A cells labeled in vivo with folate-FITC and detected/analyzed by one-dimensional line scanning using software developed on MATLAB platform. (Scale bars: c and d, 10 μm.) Dotted lines outline blood vessel walls identified by transillumination in c and d.

Fig. 2.

Quantitation of CTCs by both in vivo imaging of animal models and ex vivo flow cytometry of peripheral blood samples from ovarian cancer patients. (a) Change in CTCs as a function of time after M109 tumor cell implantation. CTCs were quantitated by measuring the number of fluorescent tumor cells per minute flowing through a blood vessel in the ear of each mouse. Red bars represent mean values. (b) Method of image processing. One thousand line scans, spanning a 2-s time period, across a blood vessel are combined sequentially by using MATLAB software to yield the displayed image. (c) CTC counts in peripheral blood samples from 12 ovarian cancer patients with different pathologies. Red bars represent mean values from multiple independent measurements. (d) Confocal images of single CTC in blood smears from an ovarian cancer patient. Overlap of green (folate-AlexaFluor 488) with red (rhodamine-X-labeled anti-cytokeratin antibody) fluorescence is displayed as yellow fluorescence. (Scale bar: 10 μm.) (e) Comparison of the labeling intensities of folate-FITC (green) and three different polyclonal anti-FR antibodies conjugated with FITC: PU9 (red), PU10 (yellow), and PU17 (navy). Unlabeled KB cells (black) and KB cells incubated with 10 μM folic acid plus 100 nM folate-FITC (orange, totally competed) serve as negative controls.