Figure 8.

Analysis of Immune Checkpoint and B7-Class Inhibitory Molecule Expression in MDA-MB-231 and MDA-MB-436 Models

(A) BCC lines were stained with antibodies directed against the indicated cell-surface proteins known to constitute one part of an immune checkpoint axis. Analysis was by flow cytometry using (left) FMO controls to categorize cells into populations positive or negative for the indicated molecule, while (right) obtained mean fluorescence intensities (MFI; median) values were compared between cell lines. (B) Analysis as in (A) but for indicated B7-class inhibitory molecules. (A and B) Notably, additional molecules were analyzed but if no differences were found between MDA-MB-231 and MDA-MB-436 cell lines, then data are shown in the Supplemental Information. For representative corresponding histograms see Supplemental Information. Error bars represent SD; n ≥ 3 different experiments. (C) Immunofluorescence staining for human PD-L1 in tumors established from the indicated cell lines. (Top) Representative micrographs are shown; scale bars are 100 μm. (Bottom) Cumulative quantitative intensity analysis of tumor tissues stained for anti-human PD-L1. Error bars are SD; n = 6 different tumors; all comparisons are significant (p < 0.0001 by ANOVA with Tukey’s multiple comparison test) except for controls (no primary and secondary antibody only; p = 0.6128), which was expected. Data demonstrated that in vitro differences in PD-L1 expression in these cell lines were retained in orthotopic in vivo tumor models.