Two-color diffuse in vivo flow cytometer.

Area of Science:

• Biomedical Engineering
• Cancer Research
• Flow Cytometry

Background:

• Hematogenous metastasis relies on circulating tumor cells (CTCs) and CTC clusters (CTCCs).
• Diffuse in vivo flow cytometry (DiFC) was previously developed to detect single fluorescent protein (FP) expressing CTCs in small animals.
• Simultaneous detection of two FPs would enable concurrent study of distinct CTC sub-populations or heterogeneous CTCCs.

Purpose of the Study:

• To develop and validate a two-color DiFC system for non-invasive detection of circulating cells expressing two distinct FPs.
• To enable simultaneous monitoring of different CTC populations in vivo.
• To facilitate research on tumor development and therapeutic responses in heterogeneous CTC environments.

Main Methods:

• Designed and constructed a DiFC instrument capable of detecting cells expressing either green FP (GFP) or tdTomato.
• Tested the instrument in vitro using tissue-mimicking flow phantoms.
• Validated the system in vivo in multiple myeloma-bearing mice.

Main Results:

• Accurate differentiation of GFP+ and tdTomato+ CTCs and CTCCs was achieved in vitro phantoms.
• In vivo studies showed an increase in CTCs expressing both FPs during disease progression.
• Most CTCCs (86.5%) expressed single FPs, while the remainder expressed both, supported by hyperspectral cryo-imaging.

Conclusions:

• Two-color DiFC successfully enables concurrent detection of two distinct CTC and CTCC populations.
• This technology facilitates the study of tumor development and treatment responses in different CTC sub-populations within the same animal.
• The developed system offers a novel approach for in vivo analysis of circulating tumor cell heterogeneity.