I want to study the effect of a chemical on breast cancer cell lines .For example MDA-MB 231. I found one chemical , When I use that chemical I can see less proliferation of cells with increasing concentration of the chemical. Probably apoptosis at higher concentration. Before giving it for micro array. i want to know whether it can also decrease migration,adhesion, invasion , apoptosis or has other effect and at what concentration of the chemical . I have made cdna at different concentration of chemical after 48 hours and now I want to see its effect via different marker before going further for study. please suggest markers.
One way to measure migration without biomarkers is just to do a scratch assay, where you take a pipet tip and make a scratch across the plate where the cells are nearly 100% confluent. you then can measure migration by the amount of space left after a few days. To have proper controls you need multiple pates, or wells in a six well plate to view how much migration occurs. The paper you can find below has the procedure and some data showing how to do a scratch assay.
While its not quite what you are looking for this is one way to measure the migration response. This paper may also suggest some proteins you could potentially look at as well.
Article HER2/HER3 regulates extracellular acidification and cell mig...
Hi Varun,
Your question is too broad. I work with triple negative cell lines too. For all of the points that you have asked for..there are several markers that you can go after. But in my opinion, the best way to start out is, to look for structurally similar compounds that have been reported in the literature and choose your target. If nothing that is out there matches with what you are trying to test, then you have to probably start out with the question/pathway that appeals to you the most. Be it cell proliferation or migration or invasion. Starting with basic assays such as colony formation, scratch assay would be a good idea. Based on your results, choose the question that appeals to you the most and then narrow down on associated signalling pathways. Good luck!
HI Harini, Thanks for your reply.I am new to this field.Can you give me more specific answer about which markers and signalling pathways should i look. Also, is it ok if I dont want to see the mechanism. I am thinking of just using different breast cancer cell lines to show the effect of the compound and not exact mechanism.
I think you need to do some basic biology before looking for markers - there are only a few breast cancer cell lines that will invade for example. You also need to know which time point and concentration of chemical to use in these assays. I would start with an SRB assay - compares proliferation by cell numbers - if you compare the effect of your chemical with a day 0 value, it will tell you if you have a death effect or just an anti-proliferative effect.
You can see loss of adhesion down a microscope (fairly simple), but dead cells don't adhere - so you will have to take that into account. A scratch assay (as above) will tell you if your cells will invade/migrate, but again you need to make sure a your cells aren't just dying.
hi, you mean to distinguish between antiproliferative effect and cytotoxicity ,only way to look under the microscope.
Varun,
Carol brings out an important point. Since you are saying that you are not interested in going after the mechanism, you should first do some basic analysis such as establishing the IC50, figuring out if cell death is due to apoptosis or autophagy etc. Also, since you mentioned that you are going to test the compound on multiple TNBC cell lines , I would advice you to start with one cell line first and establish as much basic information as you can. Then expand your study to include other cell lines. You should be aware that within TNBC cell lines, several sub types such as luminal, basal etc exist. Here are some papers that might guide you.
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