Intrinsic temporal variations of ABCB1 and ABCB8 gene expression in human melanoma cell lines during spheroid formation

Melanoma, which is known for its aggressiveness and high degree of metastasis, poses therapeutic challenges due to drug resistance. Given that transporter proteins of the ATP-binding cassette (ABC) family play critical roles in drug resistance mechanisms, our aim was to explore the intrinsic expression of ABCB1, ABCB5, and ABCB8 using real-time quantitative reverse transcription‒polymerase chain reaction (RT‒qPCR) in a three-dimensional (3D) in vitro model. We utilized two human melanoma cell lines (A375 and SK-MEL-37) and compared three commercial methods for forming spheroids, emphasizing the challenges encountered rather than direct method comparisons. Immunohistochemistry, transmission electron microscopy (TEM), and lactate dehydrogenase (LDH) activity assessments were performed using the selected methods. Our findings revealed increased proportions of S-100+ and caspase-3+ cells, as well as reduced percentages of HMB-45+ and Ki-67+ cells, compared with those in monolayer cultures. Intercellular tight junctions were observed within the spheroid structure using TEM. Increased LDH activity suggested the occurrence of concurrent apoptotic and necrotic processes during spheroid growth. ABCB5 exhibited negligible gene expression in both cell lines, whereas ABCB1 and ABCB8 showed distinct temporal variations in the two cell lines over different days of incubation throughout spheroid formation. This presents a significant challenge because studies employing spheroids as a drug evaluation tool typically begin drug incubation after spheroid formation is deemed complete. Whether the same occurs with other types of tumor cell lines remains to be studied. General significance: Temporal fluctuations in the gene expression of ABCB1 and ABCB8 drug efflux transporters observed during spheroid formation in human melanoma cell lines suggest potential implications for future drug resistance and screening studies using this 3D cell culture model.