br In vivo fluorescence image analysis br CTCs in the
2.6. In vivo fluorescence image analysis
CTCs in the bloodstream were localized by in vivo fluorescence imaging after injecting 1 × 106 CFSE-labeled CT26 cells via the lateral
tail vein of BALB/c mice. After 1 h the mice were anesthetized and imaged using a PhotonIMAGER system (Biospace Lab, France). The mice were then sacrificed by cervical dislocation after euthanizing with isoflurane (3%). The heart, liver, spleen, lung, kidney, large intestine and small intestine were removed and imaged with the PhotonIMAGER system.
2.7. Organ-specific metastasis analysis in mice
Tumor cells were harvested and grown in vitro 5–7 days, then wa-shed thrice in PBS and resuspended in PBS at a density of 2.5 × 105 cells per ml, the actual number of cells was determined in an automated cell counter (Adam, NanoEntek, South Korea). Single-cell suspensions of greater than 95% viability were used for the study of organ-specific metastasis in mice. B16F10 and LLC were derived from C57BL/6 mice, CT26 and 4T1 were derived from BALB/c mice. Tumor cells (5 ×104 cells in 200 μl PBS) were injected into the lateral tail vein of syngeneic mice. Two or four weeks after the inoculation the mice were sacrificed by cervical dislocation after euthanizing with isoflurane (3%). The heart, liver, spleen, lung, kidney, large intestine and small intestine were removed and fixed in Bouin's solution (75 ml saturated picric DCFH-DA solution, 25 ml 40% formaldehyde and 5 ml glacial acetic acid). The metastases were qualitatively evaluated by locating the tumor nodules on the specific organ surface under a dissecting microscope.
3.1. The dynamics of CTCs survival in the bloodstream
To track the proliferation and fate of CTCs in the bloodstream, CT26 cells were labeled with CellTrace™ CFSE reagent, injected into the tail vein of mice and detected by flow cytometry . As shown in Fig. 1, the discrete peaks in the histograms represent successive generations of CT26 cells after culture in vitro. Furthermore, CFSE-labeled CT26 cells formed clear metastatic nodules in the lungs of BALB/c mice four weeks after the tail vein injection (data not shown), indicating that CFSE did not affect the metastatic capacity of the CT26 cells. Fig. 2A shows the experimental design; CFSE-labeled CT26 cells were injected into a lat-eral tail vein of BALB/c mice and after the indicated time period, blood samples were collected by cardiac puncture. To investigate the re-lationship between time and the number of CTCs in the bloodstream, blood samples (500 μl per mouse) were collected by cardiac puncture at 0, 1, 5, 30, 60, 120, 240, 360 and 480 min. After lysis of the blood cells the remaining cells were analyzed by flow cytometry. The gating strategy for the detection of CTCs is presented in Fig. 2B. When 1.25 × 104 CFSE-labeled CT26 cells were spiked into 500 μl naïve
BALB/c mouse blood, the average recovery of the spiked cells was 53.2%, which is consistent with our previous studies . As shown in Fig. 2C and D, there was a very rapid time-dependent decrease in the number of CTCs in the bloodstream. One minute after injection of CTCs, the number of detected CTCs was reduced by 50%, compared to the zero (input) time point. Surprisingly, no CTCs were detected or survived two hours after injection into the bloodstream.
3.2. The number of CTCs in the bloodstream is a dependent on the number of blood circulation cycles
The results in Fig. 2D shows a very rapid initial CTC decay rate followed by a slower rate of decay. This bi-phasic decay has a corre-lation coefficient of 0.9929 (Fig. 3A). The initial wave of decay of the CTCs in the bloodstream occurred with a half-life of about one minute (0.9042 min) followed by a second wave with a longer half-life of 12.10 min. This bi-phasic decay was consistent with the data obtained from the flow cytometry data.
Based on the above result, we hypothesized that there may be an association between the number of CTCs and the number of blood circulation cycles. For the purposes of this study, one circulation cycle is defined as the time it takes for blood to emerge from the left ventricle, pass through the systemic circulation to return to the left ventricle. The time for one cycle of blood circulation therefore is equal to the total volume of blood/ (heart rate × ventricular contraction). In healthy adults, the total volume of blood in the body is about 4000 ml, the heart rate is 60–100 beats/ minute and the volume of blood pumped from the ventricles during each contraction is about 70 ml. Using the above formula, one full cycle time in humans is about 1 min. In order to fa-cilitate the calculation and simulation, we extrapolate that one cycle of blood circulation in mice to be similarly one minute. The two-phase decay model was also used to analyze the relationship between the number of CTCs and the number of blood circulation cycles. As shown in Fig. 3B, after one circulation cycle in mice, the number of CTCs was cut down to 50%. The fast half-life of the CTCs in the bloodstream was 1 cycle, and slow half-life of the CTCs in the bloodstream was 12 cycles. This result indicates that the number of CTCs in bloodstream is closely related with the number of blood circulation cycles.