Fluorescence Lifetime Imaging of Apoptosis

Area of Science:

• Biomedical Imaging
• Cancer Research
• Cell Biology

Background:

• Genetically-encoded fluorescence resonance energy transfer (FRET) reporters are valuable for analyzing cell signaling in 2D cultures.
• Applying FRET reporters to 3D environments is challenging due to light absorption complexities.
• Quantifying apoptosis in complex 3D settings requires advanced imaging techniques.

Purpose of the Study:

• To establish fluorescence lifetime imaging microscopy (FLIM) with phasor analysis as a method for quantifying apoptosis in breast cancer.
• To assess the efficacy of a combined metabolic therapy targeting glycolysis and glutamine pathways.
• To investigate the role of bone marrow stromal cells in mediating chemotherapy resistance.

Main Methods:

• Utilized genetically-encoded FRET reporters in breast cancer cells.
• Employed fluorescence lifetime imaging microscopy (FLIM) with phasor analysis for real-time apoptosis quantification.
• Applied the imaging technique across 2D cultures, 3D spheroids, and orthotopic xenograft mouse models.

Main Results:

• FLIM with phasor analysis successfully quantified apoptosis in diverse 3D environments, including living mice.
• Combined metabolic therapy significantly reduced breast cancer metabolism and induced apoptosis.
• Identified distinct bone marrow stromal cell subpopulations influencing breast cancer cell chemotherapy resistance.

Conclusions:

• FLIM with phasor analysis is a robust tool for real-time apoptosis assessment in cell-based assays and in vivo models.
• The study demonstrates the potential of combined metabolic therapy for breast cancer treatment.
• Revealed heterogeneity in treatment response attributed to bone marrow stromal cell interactions.