Fig 2: HCV induces the expression of the ErbB3 ligands NRG1 and Epiregulin.(A) Total RNA was isolated from Huh9-13 replicon cells or Huh7 control cells. Transcript levels of NRG1, NRG2 and Epiregulin were quantified by rtPCR as outlined in figure legend to Fig 1 and the Material and Methods section using primer pairs described in the supporting information (Table A in S1 File). (B) Supernatants from cell cultures of Huh9-13 cells comprising the subgenomic HCV replicon or Huh7 cells for control were analysed for production and release of soluble NRG1 using ELISA specifically recognizing human NRG1. (C) Huh7.5 cells were infected with the HCVcc strain JC1. Total RNA was prepared 72 hours after infection and NRG1 was determined by rtPCR. For (A) to (C) the results are expressed as fractions of the normalized value of the control which was set to 1; data are presented as mean + SEM of at least three independent experiments.

Fig 3: HER3 inhibition with CDX-3379 in syngeneic HNSCC model remodels the tumor immune microenvironment.a 4MOSC1 cells were treated with different concentrations of CDX-3379 for 1 h and cell lysates were analyzed by western blot analysis as indicated. Data shown are representative blots of results from three independent experiments with similar results. b Left, single cell suspension of 4MOSC1 tumors was stained with CD45 and HER3 antibodies and analyzed by flow cytometry. Shown is a representative flow cytometry plot of the frequency of tumor cells (CD45−) and immune cells (CD45+) expressing HER3 (n = 4). Right, in mice with 4MOSC1 tumors, HER3 expression was measured in CD90.2+ T cells, CD90.2+ CD4+ T, and CD90.2+ CD8+ T cells in both the cervical lymph node (LN) and splenic compartment (the relative HER3 MFI of Cal27 and 4MOSC1 cells from culture serve as a positive control). Data were reported as relative mean fluorescence intensity (MFI) as compared to FMO (n = 3 for Cal27 and 4MOSC1; n = 5 for lymph nodes and spleen); Relative MFI was calculated by subtracting MFI-HER3 from MFI-FMO. c Immunofluorescent staining of CD8, HER3, and CK5 in slides of 4MOSC1 tumors to show HER3 co-expressed with cancer cells (CK5 positive), but not with CD8+ T cells. d Left, C57Bl/6 mice with 4MOSC1 tumors were treated IP with isotype control antibody and CDX-3379 (20 mg/kg) (n = 6). Biological populations identified via CyTOF were categorized in tSNE map. FlowSom depiction of different cell lineages within in the tumor micronvironment (colored) were identified by lineage-specific markers. Right, count of M2 macrophages (M2Φ) and M-MDSC cells. Data were reported as mean ± SEM; two-sided Student’s t-test. e Total count of major immune cell population within live cells from panel (d). f Chemokine and cytokine protein expression profile in 4MOSC1 tumors. Tongue tumors were established and treated as in panel d. Tumor lysates (total protein concentration 2 mg/ml) were used for mouse cytokine array/chemokine array analysis. Significantly altered molecules and the pg/mL range with HER3i are shown (n = 3). Data were reported as mean ± SEM; two-sided Student’s t-test. Source data are provided as a Source Data file.

Fig 4: Representative images of HER2, HER3, HER4, and GATA4 immunohistochemical staining and HER2 silver in situ hybridization in adult GCTs. A GCT sample with (A) high-level expression and (B) low-level expression of HER2 on the cell membrane. High-level expression of HER3 localized into the nucleus and cytoplasm (C). In the majority of GCTs HER3 immunostaining was negligible (D). HER4 was localized into the cell membrane in both (E) high-level and (F) low-level expressing GCTs. Eight GCTs showed small-scale amplification of the HER2 gene (G), while gene copy number was normal in most tumors (H). High-level nuclear expression of the transcription factor GATA4 (I) and a GCT with low-level GATA4 expression (J). The magnification of (A–F, I, and J) is 20× and the scale bar represents 50 μm. The magnification of (G and H) is 100× and the scale bar represents 10 μm.

Fig 5: Anti-ERBB3 mAbs inhibit ligand-mediated ErbB3 signaling.A) Serum-starved BT-474, NCI-N87 and ACHN cells were treated with appropriate antibodies or PBS for 30 minutes followed by HRGβ stimulation for 10 minutes. B) NCI-N87 cells were treated with either the A5/F4 oligoclonal or PBS and stimulated with HRGβ, as appropriate, over a 60 minute time course. Cell lysates were analyzed by western blot and probed with anti-pAKT(S473) as a marker for ligand-induced signaling. Membranes were probed with anti-beta actin mAb that served as loading control.