In Vivo Monitoring of Fluorescently Labeled Cancer Cells

Whole animal in vivo imaging has contributed significantly to the detection of disease progression and drug efficacy for the past several years (1). With the introduction of sensitive imaging instruments, applications in fluorescent imaging have expanded to monitoring tumor cell growth and gene expression. The new fluorescent proteins have improved brightness, photostability and better tissue penetration of signals at a far red wavelength. In addition, there has been an explosion of innovation in a variety of fluorescent proteins developed from existing red fluorescent proteins. Previously, we developed brighter bioluminescent cancer cell lines using enhanced firefly luciferase 2 (luc2) (2). Because 4T1-luc2 cells are so bright, we were able to detect a single bioluminescent cell in vivo. To expand the applications of genetically labeled fluorescent tumor cells, we have developed single and dual labeled 4T1, PC3M and MDA-MB-231 cell lines using tdTomato fluorescent protein and luc2. First, we engineered a vector encoding the tdTomato protein in which expression is under the control of human ubiquitin C promoter (3). Parental cells were stably transfected with the tdTomato vector and antibiotic resistant cells were selected. In parallel, luc2 labeled cells were transfected with the tdTomato vector to develop dual labeled cell lines (4T1-luc2-tdTomato, PC3M-luc2-tdTomato and MDA-MB-231-luc2-tdTomato). Our initial analyses showed that the expression level of tdTomato protein was unstable over time in both single and dual labeled cells. Therefore, we attempted single cell cloning and isolated individual clones of both single and dual labeled cells. Positive clones were subjected to fluorescent activated cell sorting (FACS). Once cells were isolated, luciferase expression was characterized in the dual labeled cell lines. Cultures were maintained to test the stability of tdTomato and luc2 expression levels over a 4 week time period. The results indicated that the cloned populations were stably expressing both reporters. We confirmed that the growth rates of both single and dually labeled cells were comparable to those of the parental cell line. To monitor tumor growth in vivo, cells were implanted subcutaneously and orthotopically into nu/nu mice. Tumor progressions were monitored non-invasively and in real time using an enclosed imaging system containing a cooled CCD camera. Our data demonstrates that we could follow primary tumor growth and metastases in vivo using fluorescent and bioluminescent imaging. These dual reporter systems enable the monitoring of pathway specific signaling in vivo with exquisite sensitivity using both bioluminescence and fluorescence. Furthermore, histological sections can later be interrogated with fluorescent detection.