Fig 1: HE4 blockade exerts therapeutic efficacy across multiple mouse tumor models(A–C) Mice bearing subcutaneous LLC tumors were treated intraperitoneally with isotype immunoglobulin G (IgG) or anti-mHE4 mAbs (clones #1 or #117). Treatment scheme (A), tumor growth curves (B), and representative tumors with weights (C) are shown.(D and E) Anti-mHE4 (clone #117) treatment suppressed urethane-induced lung tumorigenesis, shown by representative H&E staining and tumor quantification. Scale bar, 1,000 μm.(F and G) HE4 blockade attenuated ID8 intraperitoneal tumor progression, shown by representative abdominal images and ascites volume.(H and I) Therapeutic efficacy of anti-mHE4 (clone #117) in an orthotopic HGS-1 ovarian tumor model, shown by treatment scheme and representative tumors with weights.(J) Serum-free HE4 levels measured by ELISA in LLC-tumor-bearing mice one day after the final antibody treatment.(K and L) Anti-mHE4 (clone #117) reduced PD-L1 expression on tumor-associated macrophages in the LLC subcutaneous and ID8 intraperitoneal models.(M and N) Anti-mHE4 (clone #117) failed to suppress LLC tumor growth in Cd274-deficient mice.(O) Combination therapy with anti-mHE4 and anti-mCTLA-4 enhanced tumor suppression in the LLC subcutaneous model.(P) Combination therapy with anti-mHE4 and paclitaxel (PTX) enhanced antitumor efficacy in the LLC subcutaneous model.(Q) Serum cytokines and clinical chemistry parameters following treatment with isotype IgG, anti-mHE4 mAb, or anti-mPD-1 mAb.(R and S) Safety assessment showing representative H&E images and incidence of inflammation in heart, liver, lung, and colon after anti-mHE4 or anti-mPD-1 treatment.Statistical analyses were performed using two-way ANOVA (B and N–P), one-way ANOVA (C, K, and N–Q), two-tailed unpaired t tests (E, G–J, and L), and chi-squared test (S). Data in (A–P) are pooled from two independent experiments. Scale bars, 100 μm.
Fig 2: HE4 neutralization exerts therapeutic activity in human cancer models(A–C) PMA-differentiated THP-1 macrophages were stimulated with Fc or hHE4-Fc, and PD-L1 expression was assessed by flow cytometry (A), immunoblotting (B), and RT-qPCR (C); a commercial hHE4-Fc was included as an independent control.(D) Binding of hHE4 to PMA-differentiated THP-1 cells assessed by flow cytometry.(E and F) PMA-differentiated THP-1 cells were pretreated with ruxolitinib, fludarabine, or Stattic, followed by hHE4-Fc stimulation; PD-L1 was quantified by RT-qPCR (E) and flow cytometry (F).(G and H) Anti-hHE4 monoclonal antibodies inhibited hHE4-induced PD-L1 upregulation in PMA-differentiated THP-1 cells, assessed by RT-qPCR (G) and flow cytometry (H).(I) Anti-hHE4 mAb clone #10 blocked hHE4 binding to PMA-differentiated THP-1 cells.(J) Binding of wild-type or epitope-mutant hHE4-Fc to anti-hHE4 mAb clone #10 was quantified by ELISA.(K) Pharmacokinetic analysis of anti-hHE4 mAb clone #10 in C57BL/6 mice following intravenous administration.(L–O) Fresh human LUAD tumor cell suspensions were treated with anti-hHE4 mAb clone #10, followed by flow cytometric analysis of PD-L1 and ELISA measurement of IFN-γ and granzyme B. (P) Recombinant HE4 suppressed IFN-γ production in human LUAD tumor cell suspensions.(Q–T) HE4 blockade enhanced PBMC-mediated antitumor activity in humanized C-NKG mice bearing OVCAR3 or NCI-H358 tumors, shown by treatment scheme, representative tumors, and tumor weights.Schematics (L and Q) were created using BioRender. Statistical analyses were performed using one-way ANOVA (C, E, and G), paired t tests (M–P), or unpaired t tests (S and T). Data in (A–J) are representative of three independent experiments; data in (Q–T) are pooled from two independent experiments.
Fig 3: Elevated HE4 expression in human cancers correlates with improved response to immune checkpoint inhibitors(A) Pan-cancer transcriptional profiling of WFDC2 (HE4) expression across TCGA tumors and GTEx normal tissues (log2[TPM+1]). ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.(B and C) HE4 protein levels in paired LUAD tumor (LT) and adjacent non-tumor (LN) tissues (n = 16) analyzed by immunoblotting, with densitometric quantification.(D) Immunohistochemical analysis of HE4 expression in LUAD tumors (LT) and paracancerous tissues (LN) (n = 103), with representative images and quantitative scoring.(E–G) HE4 expression in tumor samples from immunotherapy-sensitive versus resistant patients across two independent cohorts, assessed by immunohistochemistry.(H) Correlation between HE4 expression and immunotherapy response in an online NSCLC cohort.(I) Working model illustrating that tumor-derived HE4 engages IFN-γR on myeloid cells, biases signaling toward JAK-STAT3, induces PD-L1 transcription, and that HE4 neutralization restores antitumor immunity.Statistical analyses were performed using two-tailed unpaired (A, E, G, and H) or paired (C and D) Student’s t tests. Scale bars, 100 μm.
Fig 4: HE4 blockade promotes antitumor immunity in the tumor microenvironment(A–C) scRNA-seq analysis of LLC tumors from mice treated with control IgG or anti-HE4 antibody (n = 3 per group), showing UMAP clustering of 26 cell populations (A), relative abundance of each cluster (B), and aggregated cell types (C). ∗p < 0.05(D and E) HE4 neutralization reduced Cd274 (PD-L1) expression in myeloid compartments. UMAP feature plots show Cd274 expression in macrophage/monocyte and epithelial/malignant populations (D), with paired comparison across macrophage clusters (E).(F) UMAP visualization of nine intratumoral T cell subclusters in control IgG– and anti-HE4–treated tumors.(G) In the LLC subcutaneous model, intratumoral IFN-γ+ and CD69+ CD8+ T cells were quantified by flow cytometry.(H) In the ID8 intraperitoneal model, IFN-γ+ CD8+ T cells were quantified by flow cytometry.(I–K) HE4 neutralization failed to suppress LLC tumor growth in Rag1−/− mice, shown by treatment scheme and tumor growth/endpoint analyses.(L–O) CD8+ T cell depletion abrogated the antitumor efficacy of HE4 blockade, with treatment scheme, tumor growth/endpoint measurements, and confirmation of depletion efficiency by flow cytometry.(P–S) Macrophage depletion using anti-CSF1R diminished the antitumor efficacy of HE4 neutralization, with tumor growth/endpoint measurements and confirmation of depletion efficiency by flow cytometry.Statistical analyses were performed using unpaired t tests (B, C, H, and K), paired t test (E), one-way ANOVA (G, N, O, R, and S), and two-way ANOVA (J, M, and Q). Data in (G, H, and L–S) are pooled from two independent experiments.
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