Figure 7 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>Birinapant and fulvestrant enhance antigen presentation, T-cell migration, and T-cell–mediated cytotoxicity. <b>A,</b> mRNA expression levels of IFNγ and antigen presentation–related genes in MCF7 cells that were treated with vehicle control (DMSO), fulvestrant (10 nmol/L), birinapant (100 nmol/L), and the combination of fulvestrant and birinapant for 3 days and stimulated with IFNγ for the last 24 hours. <b>B–E,</b> Median fluorescence intensity (MFI) quantification of MHC-I and PD-L1 levels assessed by flow cytometry in MCF7 cells grown in HD or in the presence of E2 (<b>B</b> and <b>C</b>) or treated with fulvestrant (10 nmol/L; <b>D</b> and <b>E</b>), and treated with the addition of doses of birinapant for 3 days. Cells were stimulated with IFNγ (10 ng/mL) for the last 24 hours. MFI levels are relative to no-IFNγ conditions (*, <i>P</i> < 0.05; ***, <i>P</i>< 0.001; n.s., not significant). Error bars, mean ± SD of at least two replicates. <b>F,</b> Whole-cell lysates immunoblots of ER, NF-KB subunits, and IFNγ target genes in MCF7 cells grown treated with vehicle control (DMSO), fulvestrant (10 nmol/L), birinapant (100 nmol/L), and the combination with and without IFNγ (10 ng/mL) stimulation for 24 hours. <b>G,</b> Representative immunofluorescence images from T-cell migration assay after treatment with vehicle control (DMSO), fulvestrant (10 nmol/L), birinapant (100 nmol/L), and the combination MCF7 cells were pretreated and seeded in the AIM 3D cell culture chips. Primary CD8<sup>+</sup> T cells stained with CellTrace Red Stain were seeded on the lateral channels. <b>H,</b> Quantification of migration to the matrix was measured after 5 days (*, <i>P</i> < 0.05. Error bars are mean ± SD of at least 5 replicates. Two-way ANOVA). <b>I,</b> Representative figures of immunofluorescent stains of cocultured MCF7_NYESO1 and T cells transduced with NYESO1-specific TCR. Immunofluorescent stains include MHC-I (red), granzyme B (green), actin (yellow), DAPI for nuclear staining (blue), and a merged image. <b>J,</b> MCF7 cells expressing specific NYESO1 antigen were pretreated with vehicle (DMSO), fulvestrant (10 nmol/L), birinapant (100 nmol/L), and the combination of both drugs and cocultured with primary NYESO1 TCR<sup>+</sup> T cells for 16 hours. The number of cancer cells alive was measured, and data are relative to MCF7_NYESO1 grown without T cells. *, <i>P</i> < 0.05; ***, <i>P</i> < 0.001; n.s., not significant. Error bars are mean ± SD of at least three replicates. Two-way ANOVA.</p>