Two-color diffuse in vivo flow cytometer.

Significance: Hematogenous metastasis is mediated by circulating tumor cells (CTCs) and CTC clusters (CTCCs). We recently developed "diffuse in vivo flow cytometry" (DiFC) to detect fluorescent protein (FP) expressing CTCs in small animals. Extending DiFC to allow detection of two FPs simultaneously would allow concurrent study of different CTC sub-populations or heterogeneous CTCCs in the same animal.

Aim: The goal of this work was to develop and validate a two-color DiFC system capable of non-invasively detecting circulating cells expressing two distinct FPs.

Approach: A DiFC instrument was designed and built to detect cells expressing either green FP (GFP) or tdTomato. We tested the instrument in tissue-mimicking flow phantoms in vitro and in multiple myeloma bearing mice in vivo.

Results: In phantoms, we could accurately differentiate GFP+ and tdTomato+ CTCs and CTCCs. In tumor-bearing mice, CTC numbers expressing both FPs increased during disease. Most CTCCs (86.5%) expressed single FPs with the remainder both FPs. These data were supported by whole-body hyperspectral fluorescence cryo-imaging of the mice.

Conclusions: We showed that two-color DiFC can detect two populations of CTCs and CTCCs concurrently. This instrument could allow study of tumor development and response to therapies for different sub-populations in the same animal.