Toward complete laser ablation of melanoma contaminant cells in a co-culture outgrowth model via image cytometry.
BACKGROUND
Contaminant cancer cells in autologous transplant tissue can cause relapse and the rates are unknown. A method capable of removing all contaminant cells with a high probability detected by cytomic analyses would be useful. Neither 100% cell purging nor techniques for measuring the probability of success have been developed. Here, we report a method for removing 100% of the cells under ideal staining conditions and quantify the probability of success.


METHODS
Laser ablation was combined with previously reported automated microscopy to purge contaminant cells and evaluate 100% ablation in a co-culture model of prestained mouse melanoma cells mixed with mouse NIH-3T3 cells. Melanoma passage efficiency was measured by: (1) micropipetting single cells into microtiter wells and (2) ablating all but one melanoma cell in co-cultures.


RESULTS
(74 +/- 5)% of single melanoma cells pipetted into microtiter plate wells divided at least once. With ablation of all but one contaminant cell in co-cultures, melanoma dominated in (62 +/- 8)% cultures in 21 days. With 100% ablation in six additional experiments, no melanoma outgrowth was observed, giving a >99.1% probability that all contaminant melanoma cells were purged.


CONCLUSIONS
We successfully demonstrated a model for complete ablation within a defined probability using automated high-content image cytometry with ideal staining conditions. The results show that the instrumentation is capable of delivering 100% ablation at a defined probability and establishes the basis for further studies with clinical models wherein pretherapeutic cytomic analyses of unique cellular expression and/or morphological characteristics will be key for contaminant cancer cell identification.
Shen
Feimo
F
Department of Pharmacology, University of North Carolina at Chapel Hill, 27599, USA.
Price
Jeffrey H
JH
D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002999:Clone Cells; D018920:Coculture Techniques / Q000295:instrumentation / Q000379:methods; D005456:Fluorescent Dyes; D019044:Image Cytometry / Q000295:instrumentation* / Q000379:methods*; D007834:Lasers; D008543:Melanins / Q000378:metabolism; D008546:Melanoma, Experimental / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D041681:NIH 3T3 Cells
D005456:Fluorescent Dyes; D008543:Melanins