CRRC for motile cells

Scientists at York University, Toronto, led by Prof. Sergey Krylov have developed a methodology that permits the tracking of motile cells for the measurement of Cytometry of Reaction Rate Constant (CRRC). 

In essence, CRRC is a valuable tool in understanding tumour cell biology.  One can measure the difference in a chemical reaction's rate between individual cells in a tumour population.  This can act as a parameter to aid description of the relative proportions of bulk tumour cells and tumour-initiating cells for their reaction kinetics in resistance to chemotherapy, for instance, that might be modelled by clearing or metabolising a drug-like small molecule as the reporter.  Typically, this reporter is fluorescent e.g. fluorescein.

Amongst the challenges that arise are how to robustly segment cells to be measured in a time-lapse fashion, when fluorescence alone might at some point not be sufficiently detectable to describe the cell boundary and, perhaps more significantly, when cells are motile and the "masked" boundary no longer accords with the cell at a later time point.  

To address these concerns the authors (Yosief et al.) tested different transmitted light methods to determine the cell boundaries and settled upon brightfield (BF) as the one of choice.  However, this brought a further potential issue due to the difference in the focal planes of BF and fluorescence (ca. 10 µm) that might impact on the fluorescence quantitation of increasingly out of focus emitted light.  Helpfully, however, this difference accords closely with a typical cell diameter and they embarked on tests to calculate the signal loss at distances away from coincident focal planes.  As a label of fluorescence, cells were incubated with the far-red fluorescent cytoplasmic probe DRAQ9™ and the emitted signal captured for the coincident and non-coincident (+/-) focal planes of fluorescence and transmitted light (BF).  It transpired that the 10 µm offset had a minor impact on the quantitation of fluorescence.

DRAQ9™ staining of cells was simple.  DRAQ9™ was applied to the cells at a final concentration of 2 µM for 30 minutes before excess was removed by washing (3x) and then imaged using epifluorescence microscopy.  DRAQ9™ was detected using a Cy5 cube.

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Reference:

Yosief, Robel, Giammarco Nebbioso, Vasilij Koshkin, Yumin Qiu, Chun Peng, Vadim Elisseev, and Sergey Krylov. "Making Cytometry of Reaction Rate Constant (CRRC) Applicable to Motile Cells." (2022). Preprint on ChemRxiv, 21 April 2022. DOI:10.26434/chemrxiv-2022-gtgzf

Future prospects: 

DRAQ9™ has been shown to be non-toxic over many days exposure to cells at 2 µM both for tracking growth of spheroid microtissues and in scratch-wound motility assays (manuscript in preparation).  Thus, this may present the opportunity to combine it with spectrally-compatible, fluorescently-tagged reporting targets of aggressive tumour cells' metabolism e.g. fluorescein (used in this work), FITC-conjugates, naturally fluorescing pharmacophores (topotecan, hoechst, etc.) as indicators of multi-drug-resistance, and so on.  Indeed, in theory, it should be possible to multiplex reporters for cellular response to combination chemotherapy due to the spectral space afforded by DRAQ9's far-red fluorescence.  This use of DRAQ9™ to demark the cell boundary with fluorescence rather than BF would further reduce any impact of focal plane offset since all measurements - reporter(s) and cell boundary - would be of fluorescence emissions.  This may become more important in cell models where artefacts due to the production of ECM are confounding to automated cell segmentation using BF.