Cell viability and chemotherapeutic resistance across melanoma lines from various stages of progression
Authors: Reed Madison , Hyde Erin , Baer Robert 
Published: May 2024

Background: The melanoma stem cell (MSC) hypothesis holds that a subpopulation of cells with stem-cell like features are important for tumor progression and chemoresistance. We examined four V600E BRAF-mutated melanoma lines at various stages of melanoma progression. Within each cell line, we assessed the expression of putative MSC markers: CD20, CD44, CD133, CD186, and CD271. Subsequently, we assessed cell viability in the four lines during mono- or combination chemotherapy relative to surface marker expression. Methods: We examined two primary melanoma lines: WM35 (radial growth phase) and WM278 (vertical growth phase). Additionally, we analyzed two metastatic melanoma lines from brain metastases: LM-Mel-71 and LM-Mel-45, exhibiting epithelial and fibroblastic morphologies, respectively. MSC surface marker expression was determined using a Biorad S3e cell sorter to collect 25,000 cells each into one of 4 chemotherapeutic treatments on a 96-well plate. The treatments included: control, dabrafenib (1.25 μM, BRAF inhibitor), trametinib (0.125 μM, MEK inhibitor), and a combination of dabrafenib (1.25 μM) and trametinib (0.125 μM). Following 72 hours of incubation, we analyzed cell viability using an MTT assay. Statistical analysis was performed using ANOVA and Tukey posthoc testing. Results: All cell lines exhibited over 99% CD44+ cells. The only surface markers detected were CD44 and CD271; the remaining markers were undetectable across the four cell lines. LM-Mel-71 and WM35 cells presented both CD44+/CD271+ (14% and 31%, respectively) population and a larger CD44+/CD271- population. LM-Mel-45 and WM278 cells displayed a CD44+/CD271- population. LM-Mel-45 consistently exhibited higher viability than LM-Mel-71 (p<0.002) or WM271 (p<0.03) under both control and the three chemotherapeutic regimens. For LM-Mel-71, cell viability did not differ with CD271 expression in any treatment condition (p=0.64). For WM35 cells, viability was significantly higher in CD271+ compared to CD271- cells under all treatment conditions. Based on cell viability, monotherapy with trametinib or combination therapy appeared more effective than monotherapy with dabrafenib alone across all melanoma lines. Conclusions: The results suggest that there is not a simple relationship between CD271 expression and the origin of the cell line. While CD44+CD271+ expression may correlate with increased cell viability — as observed in WM35 radial growth phase melanoma — such an association is not universal, as CD44+/CD271+ and CD44+/CD271- cells demonstrated similar viability under each chemotherapeutic condition. Additional factors beyond the examined surface markers likely modulate melanoma progression and BRAF/MEK chemotherapy effcacy. Supported by A T Still Univeristy Student Research Fund Grant 560-950 to EH and MR. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.