Abstract 1697: Signaling pathways involved in melanoma cell functions and dedifferentiation induced by inflammatory cytokines

• Published in: Cancer research (Chicago, Ill.) Ill.), 2020-08, Vol.80 (16_Supplement), p.1697-1697
• Main Authors: Cerniglia, Michael, Campbell, Katie M., Schwerdtfeger, Julianne, Martignier, Christophe, Gilfillan, Connie B., Schaeuble, Karin, Cheung-Lau, Gardenia, Teper, Yaroslav, Maneiro, David Berger, Guemes-Aragon, Miriam, Garcia, Alejandro J., Speiser, Daniel E., Ribas, Antoni, Comin-Anduix, Begoña
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2020-1697

INTRODUCTION: Melanoma-acquired resistance against T cell-based immunotherapy may be mediated by alterations of their responses to inflammatory cytokines. However, the cytokine signaling pathways in cancer cells remain poorly understood. METHODS: 8 melanoma cell lines were exposed to interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) over a time course. Signaling molecules and their phosphorylated state were subsequently analyzed using mass cytometry. Briefly, cells were incubated with IFNγ (220 IU/mL), TNFα (1000 IU/mL), and both cytokines (220 IU/mL IFNγ + 200 IU/mL TNFα) for up to 48 hours. Staining was done with antibodies specific for (1) phosphorylated - STAT1(Y701), STAT3(Y707), AKT(S437), ERK1/2(T202/Y204), IKK(S176/180), NK-kbetap65 (S536), JNK (T183/T22), P38 (T180/Y182), Rb(S807/Y182), cMYC (S62), S6 (S235/S236), Histone3 (S28) and (2) non-phosphorylated - cPARP, β-catenin, CyclinB1, Ki67. Cells were read on a Helios Mass cytometer and de-barcoded using PREMESSA (R package). FlowJo was used to normalize time and eliminate calibration beads. Viable cell files were introduced to Cytobank where fold change (FC) with respect to time 0 of each protein was calculated with median intensity. The FC was exported to R for final analysis. RESULTS: All cell lines were sensitive to IFNγ/TNFα, with detectable (phosphorylated) signaling molecules. No synergistic effect was observed at any dose for IFNγ+TNFα. For the following data, numbers contained within parentheses represent the mean range of FC control vs treatment or mean standard error. pSTAT1 was significantly activated at all time points for IFNγ ([0.8, 1.0] vs [1.8, 3.6] p