Quantify Cell Aggregation

I am trying to quantify the aggregation of THP-1 macrophage cells to various cancer cell lines. I have used DeltaVision to take images of certain positions within the petri dish every 2 minutes for 3 hours - creating a total of 91 time points. Example shown below:

Is there any way I can use Fiji/ImageJ in order to quantify the aggregation of macrophage cells towards the cancer cells? This way I can quantify the changes in aggregation between different cancer cell lines.

Thanks in advance!

Hi @hgriffiths

Welcome to the Forum.

I would start by clarifying how you will define an aggregation of macs with cancer cells. Will that definition be based on cell centroid distances? Will that definition include a certain threshold of the number of either/both macs/cancer cells? etc. First define what you will call an aggregate (you could even have different ‘classes/stages’ of aggregates if it makes more biological sense…).

You can use Fiji/ImageJ to answer these questions… but the first step is to clearly define the question you have (ie - how to define an aggregation). That will help lead to an answer.

Hope this helps a bit to get you started.

eta :slight_smile:


Hi @etadobson

Thanks for your response! Yes, the definition was going to be based on the central point of a macrophage cell at each time point and I was going to measure a maximum of 30 macrophages for each position.

I’ve been able to use manual tracking in Fiji, clicking at the centre of each macrophage for each time point, giving me (x,y) position results. But I’m stuck with where to move forward with quantification from here. I am a complete newcomer to Fiji! Also, this doesn’t take into account the position of the cancer cells in relation to the macrophages either.

Is there anything else you’d suggest?


ok. well @hgriffiths … this might be getting a bit beyond my current skill set. It seems that what you’ll want to do is some type of cluster analysis… Take a look at this old Forum post and see if it gives you a start in the right direction.

I hope others here on the Forum can give you better insight/help!


Thanks again @etadobson

If you’ve got a simpler method of quantifying aggregation I’d be really keen to hear it! I’m looking for the simplest way possible if I’m honest. The above was a suggestion of one of my supervisors.

All the best,

I believe the Manual Tracking plugin has a directionality feature – you can define the reference point to be the cancer cell that is nearest to the macrophage at the last timepoint.

Then you can get the % of macrophages that are moving towards a cancer cell (though with the high density you might have some false positives there).

Other simple measures that come to mind:

  • average distance between the cancer cell and the 5 (or some suitable number of) closest macrophages
  • number of macrophages within a certain radius of the cell – calculate the difference between that number at the end vs. at the beginning of the time-lapse.

You could get all these values by recording the cancer cell coordinates using the Tracking plugin or the point tool, and calculating distances in a spreadsheet or math application.

I’d recommend trying a few different methods on positive and negative controls, and get an idea of what measure best captures the actual behavior.

Hope this helps.


A post was split to a new topic: How to quantify cytoplasmic aggregations