Fig 2: Human B7-H4 gene expression is a prognostic factor in HER2+ BCs and limits the therapeutic efficacy of trastuzumab. (A) Quantitation of CD68 and B7-H4 IHC staining in paired samples of HER2+ BC patients before and after neoadjuvant trastuzumab therapy or neoadjuvant chemotherapy without trastuzumab (n = 50 in the trastuzumab group; n = 58 in the chemotherapy group). ***P < 0.001, Student’s t test. (B) Representative images of CD68, B7-H4, CD8, and CD56 in serial sections of the paired clinical samples from HER2+ breast cancer patients before and after neoadjuvant therapy. Scale bar, 50 µm. (C) Kaplan–Meier survival curve for BC patient data and stratified into low and high groups based on the average expression of B7-H4 in BC-infiltrating macrophages. (D) Correlation of infiltrations (number per view field) of B7-H4+ macrophages and NK cells or CD8+ T cells in clinical samples from BC patients after neoadjuvant trastuzumab therapy [n = 50, Pearson’s correlation coefficient (R and P are shown)].

Fig 3: B7-H4 blockade enhanced trastuzumab efficacy in a humanized HER2+ BC mouse model. (A) Four treatment arms were established: control IgG (200 µg weekly; n = 15); B7-H4 blockade (100 µg weekly; n = 15); trastuzumab (200 µg weekly; n = 15); and trastuzumab combined with B7-H4 neutralizing Ab (n = 15). Individual animals were consecutively enrolled into a specific treatment arm as soon as palpable BCs were detected (~200 mm3). The total tumor burden per mouse is shown. Animals were euthanized when their total TV reached >2000 mm3. (B) Survival of mice in each treatment arm. Time of start was on the day of palpable tumor detection and treatment enrollment. The log rank (Mantel–Cox) test was used for survival analysis. ****P < 0.0001, treatment vs. control group; ##P < 0.01, difference observed between the trastuzumab and trastuzumab + anti-B7-H4 groups. (C) Humanized mice bearing palpable xenografts were administrated with the indicated treatments. Representative IHC images of immune cell infiltration are shown (scale bar, 50 µm). (D–F) Immune cell populations per high-power microscope field in tumor tissues (n = 5 mice/group).*P < 0.05; **P < 0.001;***P < 0.0001 according to a paired t test.

Fig 4: B7-H4 neutralization enhances trastuzumab efficacy in immunocompetent mice harboring BC tumors. (A) Tumor-growth experiment involving a B7-H4 neutralizing Ab (100 µg/mouse) alone or combined with 4D5-IgG2a (200 µg). (B) Macrophage populations were analyzed by FACS after tumor volume reached >1000 mm3. Data represent the mean ± SEM (n = 8–10). (C and D) In vivo ADCP experiment. MM3MG-HER2?16 cells were labeled with Vybrant DiD dye and implanted (1 × 106 cells) into the mammary fat pads of BALB/c mice. After the TV reached ~1000 mm3, mice were treated with a control Ab, 4D5-IgG2A (200 µg), or 4D5-IgG2A in combination with the B7-H4 neutralizing Ab (100 µg). Tumors were harvested, and tumor-phagocytic macrophages were quantified by FACS (D). Representative FACS plots and graphical summary showing the frequency of macrophages (CD11b+F4/80+LY6G-LY6C-) that have phagocytosed DiD-labeled BC cells.