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Determining the fate of cells with Flow Cytometry

The ability to measure multiple forms of cell death simultaneously represents a significant development for such techniques. I have been using antibodies and more specific forms of dyes to identify mitochondrial activity and reactive oxygen in roughly fifty populations, whereas normally it would only be able to measure one at a time. I will be discussing this work at the Flow Cytometry Congress, and it could prove enormously beneficial to drug and immunotherapy development.

Using Flow Cytometry to determine the fate of cells

Shikonin induced necroptosis (RIP3High+ve/Caspase-3-ve), classic apoptosis (RIP3-ve/Caspase-3+ve), RIP1-dependent apoptosis (RIP3+ve/Caspase-3+ve) and Double Negative cells (DN) after gating on Live/apoptotic and dead cells (Cell Viability+ve/Caspase-3±ve). This up-regulation of RIP3 was blocked by necrostatin-1 (blocks the phosphorylation of RIP1). While zVAD a pan-caspase blocker abrogated apoptosis after shikonin treatment.

Chloroquine initiates autophagy and a small increase in apoptosis. This was detected by intracellular labelling of autophagosomes with anti-LC3B.

Further markers for DNA Damage (H2AX) and PARP allowed the determination of the degree of DNA damage, cleaved PARP, hyper-activation of PARP (H2AX+ve/PARP+ve) in apoptotic cells and parthanatos in necrotic or caspase-3-ve cells.

It has always been assumed that autophagy protects cells, but following the Flow Cytometric work done last year in my lab, we were able to demonstrate that there is more DNA repair occurring in cells when autophagy occurs. Previously, this had only been concluded by inference from other results, but I actually measured directly how cell health improves via autophagy. As far as I know, this has not previously been demonstrated in a single experiment.

Cell death can frequently be discussed without any knowledge of how the cell(s) died. This is a long-standing issue regarding cell death. It is classified as ‘Accidental cell death’ and usually ignored as being of little interest because there has been no identified pathway that causes it. However, it may be linked directly to some forms of programmed necrosis more than was previously thought.

What I discovered from these cells undergoing this form of necrosis is that it can be divided into sixteen different compartments rather than just one. RIP3 and Caspase-3 intracellular labelling with a fixable live/dead probe allowed us to detect necroptosis, apoptosis and RIP-1 dependent apoptosis using Flow Cytometry. Oncosis phenotypes were modulated with zVAD and Necrostain-1, which suggests that there must be a pathway involved somewhere.

Further labelling with LC3B allowed us to also detect autophagic cells. DNA damage was identified through labelling with H2AX and PARP, and autophagy was shown to reduce DNA damage and therefore protect cells.

Developing the current method
  1. In developing a flow cytometric assay for necroptosis based on up-regulation of RIP3 we added anti-active-caspase-3 antibody to identify apoptotic cells. Then realized that you could identify necroptosis, classic apoptosis and RIP1-dependent apoptosis in both live and dead populations.
  2. Double Negative cells from such an assay showed no markers of cell death, so added H2AX and PARP to identify DNA Damage, cleaved PARP and hyper-activation of PARP in apoptotic cells and parthanatos in necrotic cells (caspase-3 negative cells).
  3. Also added markers for autophagy (LC3B) and ER stress (PERK).

Data pertaining to mitochondrial activity and the generation of reactive oxygen has not yet been analysed. I expect that the next interesting area of investigation will present itself once this is done.

In the meantime, the following present themselves as an area of possible improvement.

1. Improving the detection necroptosis

The methodology isn’t currently specific for this as a phosphorylated antibody for this is currently not available: the whole genesis of this project was the availability of antibodies for microscopy work which could translate to Flow Cytometry very easily. Using MLKL to improve the assay has been tried but didn’t prove to be useable.

2. Measuring other outstanding forms of cell death

There are still some outstanding forms of cell death that I haven’t measured, most of which are site-specific in the body or polymorphs which display Netosis.

3. Analysing Necrosis in more detail

So far I have only analysed one way of inducing cell death by metabolic poison. There are other possibilities such as heat shock, pH change, or the breaking of the plasma membrane. It would be possible to see whether there is any difference between the various types.

Immunotherapeutic benefits: testing drug effectiveness

At our institute, we’ve been using the technique to identify how drugs kill certain cells. This has been very useful as it has been assumed that a drug will cause cell via one mechanism only. However, we found that a drug will induce multiple forms of cell death, which has been very revealing in regards to the mechanism of immunotherapeutic drugs.

A lot of drugs will seek to resolve cancer by eradicating it, i.e. inducing some form of cell death in the tumour. It has previously been difficult to measure the mode of action for these drugs because it was necessary to do a western blot to measure each form of cell death separately. This would require a million cells, which is a lot to get from a patient. This inefficiency is mitigated using my technique, which uses far fewer cells and will measure eight different forms of cell death simultaneously.

As mentioned previously, I am still analysing the data from the work done in 2018 and hoping to get a few more papers out on top of those already published. My hope is that my methodology will be taken up and improvements made to the assays which I haven’t been able to do on my own. With increased interest and allocation of resources, the technique could be taken further and be extremely useful to drug and immunotherapy treatment in the future.

 

Gary Warnes is Flow Cytometry Core Facility Manager at Queen Mary London University. Currently, the Facility offers under and post-graduate (MSc) student places for projects studying autophagy by flow cytometry.

 

Join us at the Flow Cytometry Congress: Europe to get the latest insights on flow control and advances in hydrodynamic and acoustic-assisted focussing. Download the agenda and see the full list of speakers and topics.

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