Background/Aim: Circulating tumor cells (CTCs) may initiate metastasis. Some studies show that the number of CTCs and existence of CTC clusters can be prognostic. In the present study, our color-coded imaging nude mouse model of metastatic lymphoma was utilized to investigate the microenvironment of CTC clusters using fluorescent-protein imaging. Materials and Methods: EL-4 mouse lymphoma cells expressing red fluorescent protein (RFP) were injected into the spleen of transgenic C57B/6-green fluorescent protein (GFP) mice. Three weeks later, the number of CTCs and CTC clusters both in heart blood and portal blood were quantified and characterized using confocal microscopy for color-coded imaging. Results: There was no significant difference in the number of CTCs between heart and portal blood. CTC clusters comprised 8.8% of CTCs, determined by color-coded imaging. Heterotypic CTC clusters containing other types of cells were distinguishable from homotypic CTCs. Heterotypic CTC clusters comprising cancer cells and fibroblasts were more rare than homotypic ones. Heterotypic CTC clusters with fibroblasts were observed only in portal blood, not in heart blood. Conclusion: CTCs can have variable properties depending on the blood source. CTCs can form clusters, which may contain fibroblast that may play a role in promoting CTC metastasis. Our results demonstrate the concept of the CTC microenvironment (CME), which may play a critical role in CTC behavior, including of metastasis.
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