Cancer cell growth has been shown to affect receptor expression and organelle morphology. Therefore, differences in mitochondrial morphology and function in cancer cells are of key importance for the successful implementation of targeted drug therapy in the clinic. To improve our understanding of the heterogeneity of mitochondrial morphology in breast cancer cells, we have analyzed the morphology of mitochondria in a comparative manner across 2D-culture breast cancer cell systems. Moreover, we used machine learning methods to develop a classification method that can assign each 3D rendered mitochondrial object to their respective breast cancer cell type with good accuracy.
KEYWORDS: Near infrared, Luminescence, Fluorescence resonance energy transfer, Receptors, Microscopy, In vivo imaging, Cancer, In vitro testing, Proteins, Therapeutic antibodies
Monitoring the binding of protein ligands and therapeutic antibodies to their respective receptors (target engagement) is crucial to compound prioritization in anti-cancer targeted drug screening. However, current in vivo optical imaging techniques cannot distinguish between co-localization and actual receptor-ligand binding at the tumor region. Since transferrin receptor (TfR) level is significantly elevated in cancer cells compared to non-cancerous cells, transferrin (Tf) has been successfully used in molecular imaging and targeted anti-cancer drug delivery. The homodimeric nature of TfR allows for measuring fluorescence lifetime FRET (FLI-FRET) to quantitate the TfR-Tf binding and internalization into cancer cells, based on the reduction of donor fluorophore lifetime. Near infrared (NIR) FLI-FRET has been used to directly visualize and quantitate TfR-Tf binding and internalization by providing the fraction of donor-labeled entity that is interacting with its respective receptor. NIR FLI-FRET has been validated at multiscale, using both in vitro microscopy as well as in vivo macroscopy whole-body deep imaging assays using different NIR FRET pairs. Accuracy of NIR FLI-FRET quantitation has been compared between fluorescence intensity and lifetime measurements using both microscopy and macroscopy fluorescence imaging. NIR FLI-FRET employs well-characterized quantitative lifetime-based metrics, standard in FRET microscopy, but with the additional benefit of a seamless multiscale technological platform. In summary, we have successfully demonstrated quantitative imaging of receptor-mediated binding and uptake of Tf using NIR FLI-FRET microscopy and macroscopy imaging in vitro and in vivo, respectively. This novel approach can be extended to other receptors, currently targeted in oncology. Hence, NIR FLI-FRET can find numerous applications in pre-clinical drug delivery and targeted therapy assessment and optimization.
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