Overexpression of B7-H4 in the EMT6 murine model induced resistance to anti-PD-L1 ICI. A, EMT6 cells were virally transduced with the pBabe-B7-H4 retroviral vector and overexpress exogenous murine B7-H4. B, EMT6 B7-H4+ tumors maintain high B7-H4 expression in vivo assessed by IHC. C, Animals were orthotopically injected with EMT6 cells ± B7-H4 and treated 1x per week with anti-PD-L1 at 200 µg (first dose) or 100 µg (subsequent doses) for 4 weeks, after tumors reached 100 mm3. D, EMT6-B7-H4+ tumors are significantly resistant to anti-PD-L1 immunotherapy compared with control tumors. Data were analyzed by one-way ANOVA of individual AUC values with Tukey post hoc test for multiple comparisons between EMT6 anti-PD-L1 and EMT6-B7-H4+ anti-PD-L1–treated groups (P = 0.0174, EMT6 Isotype n = 21, EMT6 anti-PD-L1 n = 23, EMT6-B7-H4+ Isotype n = 21, EMT6-B7-H4+ anti-PD-L1 n = 23. Data were collected from a total of three independent experiments). E and F, When tumor response is categorized into three groups, EMT6-B7-H4+ tumors have overall greater intrinsic resistance to treatment and reduced complete response compared with EMT6 control tumors (P = 0.035, χ2 = 6.683, df = 2, n = 23 mice for EMT6 parental tumors and n = 31 mice for B7-H4 tumors). G, EMT6-B7-H4+ tumors were stained by mIF. B7-H4 is expressed on CD45− tumor cells in vivo. Representative image shown. Scale bar 10 µm. H, Quantification of G, n = 3 mice. Data analyzed by unpaired t test. Data were analyzed in GraphPad Prism v10.
ARTICLE ABSTRACT
Combinations of immune checkpoint inhibitors (ICI, including anti-PD-1/PD-L1) and chemotherapy have been FDA approved for metastatic and early-stage triple-negative breast cancer (TNBC), but most patients do not benefit. B7-H4 is a B7 family ligand with proposed immunosuppressive functions being explored as a cancer immunotherapy target and may be associated with anti-PD-L1 resistance. However, little is known about its regulation and effect on immune cell function in breast cancers. We assessed murine and human breast cancer cells to identify regulation mechanisms of B7-H4 in vitro. We used an immunocompetent anti-PD-L1–sensitive orthotopic mammary cancer model and induced ectopic expression of B7-H4. We assessed therapy response and transcriptional changes at baseline and under treatment with anti-PD-L1. We observed B7-H4 was highly associated with epithelial cell status and transcription factors and found to be regulated by PI3K activity. EMT6 tumors with cell-surface B7-H4 expression were more resistant to immunotherapy. In addition, tumor-infiltrating immune cells had reduced immune activation signaling based on transcriptomic analysis. Paradoxically, in human breast cancer, B7-H4 expression was associated with survival benefit for patients with metastatic TNBC treated with carboplatin plus anti-PD-L1 and was associated with no change in response or survival for patients with early breast cancer receiving chemotherapy plus anti-PD-1. While B7-H4 induces tumor resistance to anti-PD-L1 in murine models, there are alternative mechanisms of signaling and function in human cancers. In addition, the strong correlation of B7-H4 to epithelial cell markers suggests a potential regulatory mechanism of B7-H4 independent of PD-L1.
This translational study confirms the association of B7-H4 expression with a cold immune microenvironment in breast cancer and offers preclinical studies demonstrating a potential role for B7-H4 in suppressing response to checkpoint therapy. However, analysis of two clinical trials with checkpoint inhibitors in the early and metastatic settings argue against B7-H4 as being a mechanism of clinical resistance to checkpoints, with clear implications for its candidacy as a therapeutic target.
