Figure 7 from Breast Cancer Stem Cell–Derived Tumors Escape from γδ T-cell Immunosurveillance <i>In Vivo</i> by Modulating γδ T-cell Ligands
Authors: Raute Katrin , Strietz Juliane , Parigiani Maria Alejandra , Andrieux Geoffroy , Thomas Oliver S. , Kistner Klaus M. , Zintchenko Marina , Aichele Peter , Hofmann Maike , Zhou Houjiang , Weber Wilfried , Boerries Melanie , Swamy Mahima , Maurer Jochen , Minguet Susana 
Published: June 2023

<p>Zoledronate sensitizes xenograft-derived tumor cells to γδ T cell–mediated killing. <b>A,</b> HMGCR expression levels determined by mass spectrometry. Protein intensities (log<sub>2</sub>) are shown as means ± SEM for culture and xenograft-derived tumor cells (vehicle). Unpaired <i>t</i> test, two tailed. <b>B,</b> HMGCR expression levels determined by IP and Western blot analysis. Quantification shows mean ± SEM for xenograft-derived tumor cells (vehicle). Unpaired <i>t</i> test, two tailed. <b>C,</b> HMGCR activity on total cellular lysates, purified HMGCR is used as positive control and purified HMGCR incubated with pravastatin, as negative control. Means ± SEM are shown. One-way ANOVA followed by Dunnett <i>post hoc</i> test. <b>D,</b><i>In vitro</i> killing of <sup>51</sup>Cr-labeled tumor cells by (MACS)-separated Vδ2<sup>+</sup> T cells. Target cells were pretreated with 10 μmol/L zoledronate or 25 μmol/L mevastatin for 2 hours. Cells were cocultured at an E:T ratio of 10:1 for 20 hours in the presence of the indicated drug. Results for 3 healthy donors of γδ T cells from four independent experiments were pooled (means ± SEM). Killing of BCSC5 culture cells in the presence of zoledronate was set to 100% for normalization. Two-way ANOVA followed by Tukey <i>post hoc</i> test comparing all groups with each other. <b>E,</b> Cox regression of progression-free survival for patients with TNBC sorted by high (top quartile) and low (bottom quartile) average expression of both proteins BTN2A1 and BTN3A1. β Cox coefficient was −0.8 and the Cox <i>P</i> value was 0.008. <b>F,</b> Cox regression of progression-free survival for patients sorted by high (top quartile) and low (bottom quartile) average clustered expression of BTN2A1, BTN3A1, Fas, MICB, and ICAM-1. β Cox coefficient was −0.6 and the Cox <i>P</i> value was 0.02. <b>G,</b> IFNγ secretion by (MACS)-separated Vδ2<sup>+</sup> T cells in response to BCSC5 culture cells or xenograft-derived tumor cells was determined. Target cells were pretreated with 20 μmol/L zoledronate for 2 hours before coculture with γδ T cells at a ratio of 1:1 in the presence of 10 μmol/L zoledronate. The culture supernatant after 24 hours was analyzed for secreted IFNγ using ELISA. Results were baseline corrected by the corresponding amounts of IFNγ secreted by γδ T cells alone. Results for 3 healthy donors of γδ T cells from two experiments testing eight individual tumors were pooled (means ± SEM). Two-way ANOVA followed by Sidak <i>post hoc</i> test comparing treatment with respective untreated groups. <b>H,</b> IFNγ secretion by (MACS)-separated Vδ2<sup>+</sup> T cells in response to BCSC5 culture cells or xenograft-derived tumor cells was determined. Tumor cells were pretreated with 10<sup>3</sup> U or 10<sup>4</sup> U IFNα2B for 1 hour before coculture with γδ T cells at a ratio of 1:1 in the presence of IFNα2B. The culture supernatant after 24 hours was analyzed for secreted IFNγ using ELISA. Results were baseline corrected by the corresponding amounts of IFNγ secreted by γδ T cells alone. Results for 2 healthy donors of γδ T cells from one experiment testing three individual tumors were pooled (means ± SEM). Two-way ANOVA followed by Dunnett <i>post hoc</i> test comparing treatment to respective untreated groups. <b>I,</b><i>In vitro</i> killing of <sup>51</sup>Cr-labeled tumor cells by (MACS)-separated Vδ2<sup>+</sup> T cells. Target cells were pretreated with 10<sup>4</sup> U IFNα2B for 1 hour and then cocultured with γδ T cells in the presence of IFNα2B at an E:T ratio of 10:1 for 20 hours. Representative results for one healthy donor of γδ T cells is shown. Indicated are technical replicates (means ± SEM). *, <i>P</i> ≤ 0.05; **, <i>P</i> ≤ 0.01; ***, <i>P</i> ≤ 0.001; ****, <i>P</i> ≤ 0.0001.</p>