Figure 3 from Endocrine Therapy Synergizes with SMAC Mimetics to Potentiate Antigen Presentation and Tumor Regression in Hormone Receptor–Positive Breast Cancer
Authors: Hermida-Prado Francisco , Xie Yingtian , Sherman Shira , Nagy Zsuzsanna , Russo Douglas , Akhshi Tara , Chu Zhengtao , Feit Avery , Campisi Marco , Chen Minyue , Nardone Agostina , Guarducci Cristina , Lim Klothilda , Font-Tello Alba , Lee Irene , García-Pedrero Juana , Cañadas Israel , Agudo Judith , Huang Ying , Sella Tal , Jin Qingchun , Tayob Nabihah , Mittendorf Elizabeth A. , Tolaney Sara M. , Qiu Xintao , Long Henry , Symmans William F. , Lin Jia-Ren , Santagata Sandro , Bedrosian Isabelle , Yardley Denise A. , Mayer Ingrid A. , Richardson Edward T. , Oliveira Giacomo , Wu Catherine J. , Schuster Eugene F. , Dowsett Mitch , Welm Alana L. , Barbie David , Metzger Otto , Jeselsohn Rinath 
Published: October 2023

<p>E2 modulates response to IFNγ stimulation. <b>A,</b> Histogram of MHC-I levels assessed by flow cytometry following 3 days of E2 stimulation or HD in the presence or absence of IFNγ (10 ng/mL) for the last 24 hours in MCF7 cells. <b>B,</b> Quantification of median fluorescence intensity (MFI) of MHC-I from <b>A</b>. Values are normalized to E2 stimulation with vehicle control (no IFNγ) cells. <b>C–E,</b> MHC-I levels assessed by flow cytometry following 3 days of E2 stimulation or HD in the presence or absence of IFNγ (10 ug/mL) for the last 24 hours in ER<sup>+</sup> T47D cells (<b>C</b>), CAMA1 cells (<b>D</b>), and ZR75.1 (<b>E</b>). <b>F</b> and <b>G,</b> Histograms (<b>F</b>) and MFI quantification (<b>G</b>) of MHC-I levels following 3 days of E2 stimulation or HD in the presence or absence of IFNγ (10 ng/mL) for the last 24 hours in MCF7 cells expressing the ESR1 Y537S mutation induced by DOX treatment cells. Two-way ANOVA. <b>H,</b> Histogram of MHC-I levels assessed by flow cytometry following 3 days of vehicle or fulvestrant (10 nmol/L) treatment in the presence or absence of IFNγ (10 ng/mL) for the last 24 hours in MCF7 cells. <b>I,</b> MFI quantification of MHC-I from <b>H</b>. Values are normalized to vehicle control (no IFNγ) cells. Error bars, mean ± SD of at least two replicates. *, <i>P</i> < 0.05, paired <i>t</i> test. <b>J,</b> Histogram of MHC-I levels assessed by flow cytometry following 3 days of E2 stimulation or HD in the presence or absence of IFNγ (10 ng/mL) for the last 24 hours in ER-negative MDA-MB-231 cells. <b>K,</b> MFI quantification of MHC-I from <b>J</b> values is normalized to E2 stimulation (no IFNγ) cells. <b>L,</b> MFI quantification of MHC-I levels assessed by flow cytometry of cells grown in HD, E2 conditions, or treated with fulvestrant or DMSO for 72 hours in the absence of IFNγ (no IFNγ) or with a 15-minute treatment of IFNγ (10 ng/mL) 24 hours prior flow analysis (Pulse) or for the last 24 hours (continuous, cont) prior to flow cytometry analysis. <b>M,</b> PD-L1 levels assessed by flow cytometry following 3 days of E2 stimulation or HD in the presence or absence of IFNγ (10 ng/mL) ×24 hours in MCF7 cells. <b>N,</b> MFI quantification of MHC-I from <b>M</b>. <b>O,</b> PD-L1 levels assessed by flow cytometry following 3 days of vehicle or fulvestrant (10 nmol/L) treatment in the presence or absence of IFNγ (10 ng/mL) ×24 hours in MCF7 cells. <b>P,</b> MFI quantification of MHC-I from <b>O</b>. *, <i>P</i> < 0.05. <b>Q,</b> MFI quantification of PD-L1 levels assessed by flow cytometry after treatment for 72 hours in the absence of IFNγ (no IFNγ), with a 15-minute treatment of IFNγ (10 ng/mL) 24 hours prior to flow cytometry analysis (Pulse) or for the last 24 hours (cont) prior to flow cytometry analysis. Statistics for the panel, if not mentioned differently, are ***, <i>P</i> < 0.001; n.s., not significant. Error bars, mean ± SD of at least two replicates. Two-way ANOVA.</p>