Detection of Apoptotic Circulating Tumor Cells Using in vivo Fluorescence Flow Cytometry

Abstract

Most cancer patients die from metastatic disease as a result of a circulating tumor cell (CTC) spreading from a primary tumor through the blood circulation to distant organs. Many studies have demonstrated the tremendous potential of using CTC counts as prognostic markers of metastatic development and therapeutic efficacy. However, it is only the viable CTCs capable of surviving in the blood circulation that can create distant metastasis. To date, little progress has been made in understanding what proportion of CTCs is viable and what proportion is in an apoptotic state. Here, we introduce a novel approach toward in situ characterization of CTC apoptosis status using a multicolor in vivo flow cytometry platform with fluorescent detection for the real-time identification and enumeration of such cells directly in blood flow. The proof of concept was demonstrated with two-color fluorescence flow cytometry (FFC) using breast cancer cells MDA-MB-231 expressing green fluorescein protein (GFP), staurosporine as an activator of apoptosis, Annexin-V apoptotic kit with orange dye color, and a mouse model. The future application of this new platform for real-time monitoring of antitumor drug efficiency is discussed.

Circulating Tumor Cells from Enumeration to Analysis: Current Challenges and Future Opportunities

“…In this regard, direct detection of CTCs in patients without an invasive procedure is an ideal approach in clinical settings. Nolan et al demonstrated that GFP + CTCs can be directly detected by using in vivo flow cytometry (IVFC) in animal models [ 92 ]. Moreover, the recent development of Cytophone technology (University of Arkansas for Medical Science, AR, USA) shows direct detection of CTCs in patients with melanoma, based on the detection of melanin-bearing CTCs by using an in vivo photoacoustic flow cytometry [ 93 ].…”

<p>Development and Clinical Prospects of Techniques to Separate Circulating Tumor Cells from Peripheral Blood</p>

“…74 Another study has used IVFC to observe the interactions between circulating breast cancer cells and dendritic cells. Mixed fluorescence and photoacoustic IFVC was also used successfully to detect GFP + breast cancer cells 75 and to identify apoptotic CTC in vivo. After androgen deprivation therapy, patients with prostate cancer were monitored using IVFC, revealing that the therapy reduced CTC count in peripheral blood.…”

Real‐Time Monitoring of Bacteria Clearance From Blood in a Murine Model

“…In an effort to detect circulating bacteria cells in the bloodstream of nu/nu mice, we used our custom built in vivo FFC set up, described elsewhere. (25,39) In brief, nu/nu mice are anesthetized using isoflurane and placed on a warmed microscope stage. Mice were anesthetized throughout the entirety of each experiment.…”