Imaging flow cytometry of tumoroids: A new method for studying GPCR expression

Area of Science:

• Cell Biology
• Molecular Biology
• Cancer Research

Background:

• Confocal microscopy is standard for analyzing G protein-coupled receptors (GPCRs) expression and localization.
• Traditional microscopy methods are time-consuming and limit sample size.
• Imaging flow cytometry (IFC) combines microscopy and flow cytometry for enhanced analysis of cellular processes.

Purpose of the Study:

• To compare fluorescence confocal microscopy and IFC for studying orexin receptor (OXR) expression in human digestive cancers.
• To investigate the anti-tumoral OX1R expression using orexin A (OxA) ligand coupled to rhodamine (OxA-rho).
• To adapt IFC for analyzing adherent cancer cells and 3D tumoroids.

Main Methods:

• Comparison of confocal microscopy and IFC techniques.
• Utilized OxA-rho to study OX1R expression in HT-29 colon cancer cells (2D and 3D cultures).
• Assessed OX1R internalization and colocalization with early endosomes.

Main Results:

• IFC enables analysis of large cell populations and visualization of cell integrity and marker localization.
• In 2D HT-29 cultures, 19% ± 3% of OX1R internalized and colocalized with early endosomes.
• In 3D HT-29 cultures, OX1R internalization peaked at 60 min, with 30.7% ± 6.4% colocalizing with early endosomes.

Conclusions:

• IFC is a powerful tool for studying native GPCR expression in cancer cells.
• This study demonstrates the first application of IFC for analyzing native OX1R expression in 2D and 3D cancer cell cultures.
• IFC provides a more efficient and comprehensive approach for GPCR research in oncology.