I am trying to establish drug-resistant BT474 colonies prior to sequencing analysis. These HER2-dependent breast cancer cells appear to have difficulty in form colony when very less cells survive drug treatment, even though the surviving cells don't die but they are not proliferating either (at least in drug-containing media). Someone suggested to seed these cells on a layer of feeder cells. Anyone had related experience please share your highly valued insights, thanks heaps!
https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis
Article Blebbishields, the Emergency Program for Cancer Stem Cells: ...
Hi, after selection in media+drugs you could also withdraw the drug for a short period (3-4 days) and see if they re-grow. Also you should check how is their state with drug (quiescent?senescent?) these should be useful information too. indeed maybe some cytokines should help or some conditioned media from highly proliferating cells..in general, single cell clonogenic always takes long time, even with normally high proliferating cells, so ...good luck!
thanks Michela for your useful tips. do you have experience with testing cell quiescence or senescence while they are undergoing drug treatment please?
at single cell level any test should be tricky, you should search specific literature about. while testing drugs you can monitor cell cycle distribution and for senescnece you could test b-gal expression, H2AX expression, you could also do WB for p21, p27 and other cell cycle indicators, isolating or not different population (CSC vs non CSC). with single cells, i guess you should do some IF staining, but to have consistent and reliable data you would need many cells. I have no specific protocol, but those are general standard assay, check on Bioprotocol online journal (nice and detailed proyocol) or in papers!good luck
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