Journal: bioRxiv

Article Title: Ligand-independent role of ErbB3 in endocytic recycling

doi: 10.1101/575449

Figure Lengend Snippet: a-d Confocal immunofluorescence imaging of traced surface labelled integrin β1 and ErbB3: MCF7 cells were labelled on ice with an Alexa488-conjugated anti-integrin β1 antibody prior to incubation for 30 minutes at 37°C to allow integrin β1 internalisation, and subsequent cell-fixation and immunolabelling of ErbB3 (red) and counterstained with DAPI (blue). c Histogram of fluorescence intensities along dotted lines indicated in ( b ). d Analysis of colocalization of integrin β1 and ErbB3. The enrichment of integrin β1 in ErbB3-positive intracellular structures (0.5-2 μm diameter) was determined by the formula (a-b)/b where a is the integrin β1 intensity at ErbB3 positive structures, and b the adjacent intensity (background) for each structure. Average intensity projections of all analysed structures are shown on the right-hand side. e Schematic outline of the recycling assays conducted in ( f-k ) in the absence of growth factors: Briefly, the surface-pool of integrin β1 was labelled with an Alexa488-conjugated antibody and allowed to endocytose. Fluorophore label remaining on the cell surface was quenched with an anti-Alexa488 antibody, prior to visualisation of traced integrin β1 re-emerging on the cell surface by live-cell TIRF microscopy. The recycling assays were conducted after prior transfection with non-targeting control siRNA or siRNA targeting indicated proteins. f Represenative TIRF microscopy images of integrin β1 from peripheral areas of MCF10A cells. g Quantifications of recycled integrin β1 performed on indicated number of cells (outside of brackets on the right-hand side of graphs), from three independent experiments and shown as Alexa488 intensity normalized between 0-1, with the control as reference where F norm =((F max -F min )/(F-F min )). h Representative TIRF microscopy images of integrin β1 from prHMEC cells. i Quantifications of recycled integrin β1 in prHMECs performed as described in ( g ). j , k . Quantified recycling of integrin β1, after prior siRNA-mediated depletion of either EGFR ( j ) or ErbB2 ( k ). Data are presented as mean values ± s.e.m. and P-values determined by two-tailed paired student’s t-test. ns=non significant. Scale bar: 10 μm, except figure 1d (scale bar:1 μm)

Article Snippet: The following primary antibodies were used: anti-ErbB3 (clone 2F12; Upstate Cell Signaling Solutions) for IP; anti-ErbB3 (clone D22C5; Cell Signaling) for western blotting; anti-integrin β1 (monoclonal, ab52971 from Abcam); anti-Rabaptin5 (monoclonal, sc-271069 from Santa Cruz Biotechnology); anti-GGA3 (clone 8; BD Transduction Laboratories); anti-Arf6 (clone 3A-1, Santa Cruz Biotech.); anti-phospho-ErbB3 Tyr1289 (#4791, Cell Signalling Technology); anti-EGFR (#2232, Cell Signalling Technology); anti-ErbB2 (06-562, Millipore); anti-phospho-AKT Thr308 (#2965, Cell Signalling Technology); anti-AKT (#9272, Cell Signalling Technology); anti-phospho ERK1/2 (#9101, Cell Signalling Technology); anti-ERK1/2 (#9102.

Techniques: Immunofluorescence, Imaging, Incubation, Fluorescence, Microscopy, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: Ligand-independent role of ErbB3 in endocytic recycling

doi: 10.1101/575449

Figure Lengend Snippet: a-d Confocal immunofluorescence imaging of surface-labelled integrin β1 (green) or actin (blue/black) on confluent sheets of MCF10A cells at 0h or 1h after labelling, as outlined in ( a ). Note in ( b ) that depletion of ErbB3 abrogates integrin β1 localisation at the leading front. c Enrichment of integrin β1 determined as ((a-b)/b) where a= mean fluorescence intensity (integrin β1) at a defined area of the leading edge ( c ) or cell-cell contact ( d ) and b=mean intensity of adjacent cytoplasm of same area. Data are presented as mean values (>74 cells per data point) ± s.e.m., n=3 independent experiments. e Scratch closure assay of control or ErbB3-depleted MCF10A cells, cultured in serum-containing but growth factor-deprived media in the presence or absence of the EGFR/ErbB2 inhibitor Lapatinib. Wound area highlighted in yellow. f , g Quantification of scratch aperture ( f ) or area under curve, AUC, ( g ) of samples treated as in ( e ). Data are presented as mean values ± s.e.m., n-values indicated in parenthesis. h Quantification of cell proliferation as incorporation of EdU for indicated times in control or ErbB3 siRNA-transfected cells in the presence or absence of 1 μM lapatinib. Data are presented as mean values ± s.e.m., n=3 independent experiments.

Article Snippet: The following primary antibodies were used: anti-ErbB3 (clone 2F12; Upstate Cell Signaling Solutions) for IP; anti-ErbB3 (clone D22C5; Cell Signaling) for western blotting; anti-integrin β1 (monoclonal, ab52971 from Abcam); anti-Rabaptin5 (monoclonal, sc-271069 from Santa Cruz Biotechnology); anti-GGA3 (clone 8; BD Transduction Laboratories); anti-Arf6 (clone 3A-1, Santa Cruz Biotech.); anti-phospho-ErbB3 Tyr1289 (#4791, Cell Signalling Technology); anti-EGFR (#2232, Cell Signalling Technology); anti-ErbB2 (06-562, Millipore); anti-phospho-AKT Thr308 (#2965, Cell Signalling Technology); anti-AKT (#9272, Cell Signalling Technology); anti-phospho ERK1/2 (#9101, Cell Signalling Technology); anti-ERK1/2 (#9102.

Techniques: Immunofluorescence, Imaging, Fluorescence, Cell Culture, Transfection

Journal: bioRxiv

Article Title: Ligand-independent role of ErbB3 in endocytic recycling

doi: 10.1101/575449

Figure Lengend Snippet: a Confocal imaging of Alexa594-conjucated transferrin chased with unlabelled holo-transferrin for indicated times in MCF7 cells. Note that siRNA-mediated depletion of ErbB3 caused prolonged intracellular retention of transferrin. b Quantification of Alexa594 fluorescence intensity in cells treated as in a (n>17 cells for each data point from three experiments) normalised against the control siRNA treated, 0 hour timepoint of each independent experiment. b , d , f Data are presented as mean values ± s.e.m. P values determined by two-tailed paired student’s t-test. Scale bar: 10 μm. c experimental outline of the VSVG trafficking experiments ( d and e). d , e Western blot analysis of the surface pool of VSV-G-ts45-GFP (pulldown of surface-biotinylated VSV-G-ts45-GFP), after its release from the endoplasmic reticulum (ER) at permissive temperature for indicated times. Note that ErbB3-depletion did not influence secretive trafficking of VSVG from the ER. e quantification of normalised levels of VSV-G-GFP in biotin-pulldowns as determined by immunoblot band intensities (n=3 independent experiments). Data are presented as mean values ± s.e.m. P determined by two-tailed paired student’s t-test. ns=non significant.

Article Snippet: The following primary antibodies were used: anti-ErbB3 (clone 2F12; Upstate Cell Signaling Solutions) for IP; anti-ErbB3 (clone D22C5; Cell Signaling) for western blotting; anti-integrin β1 (monoclonal, ab52971 from Abcam); anti-Rabaptin5 (monoclonal, sc-271069 from Santa Cruz Biotechnology); anti-GGA3 (clone 8; BD Transduction Laboratories); anti-Arf6 (clone 3A-1, Santa Cruz Biotech.); anti-phospho-ErbB3 Tyr1289 (#4791, Cell Signalling Technology); anti-EGFR (#2232, Cell Signalling Technology); anti-ErbB2 (06-562, Millipore); anti-phospho-AKT Thr308 (#2965, Cell Signalling Technology); anti-AKT (#9272, Cell Signalling Technology); anti-phospho ERK1/2 (#9101, Cell Signalling Technology); anti-ERK1/2 (#9102.

Techniques: Imaging, Fluorescence, Two Tailed Test, Western Blot

Journal: bioRxiv

Article Title: Ligand-independent role of ErbB3 in endocytic recycling

doi: 10.1101/575449

Figure Lengend Snippet: a , b Confocal immunofluorescence imaging of traced internalised integrin β1: The MCF10A cells were transfected with control or ErbB3 siRNA and assay performed in growth factor deprived media. c Quantification of immunofluorescence intensity of internalised integrin β1 traced for indicated times (n>32 cells per data point from 5 independent experiments). d Determination of integrin β1 turnover by pulse-chase metabolic labelling: Control or ErbB3 siRNA-transfected MCF10A cells were pulse-chase labelled with radioactive ( S) methionine and cysteine. Radiolabelled integrin β was visualised by radiography of immunoprecipitates (upper panel). Cell lysates and immunoprecipitates were analysed by immunoblotting. e Quantification of pulse chased 35S-labelled integrin β1, as in ( d ) (n=4 independent experiments). f , g Confocal immunofluorescence imaging of surface-labelled integrin β1 (using an Alexa488-conjugated anti-integrin β1 antibody), prior to (0 hours) or after tracing at 37°C for 1.5 hours. A scratch was inflicted prior to antibody incubation. Note that application of the lysosome inhibitor chloroquine caused accumulation of integrin β1 in intracellular vesicular compartments both in control of ErbB3-depleted cells. h Quantification of integrin β1 fluorescence intensity in cells bordering the migratory front in samples treated as in g , showing that chloroquine restored levels of integrin β1 in ErbB3-depleted cells. Data presented as mean values ± s.e.m., n=19-27 cells per data point from 3 independent experiments. P values determined by two-tailed paired student’s t-test. ns=non-significant.

Article Snippet: The following primary antibodies were used: anti-ErbB3 (clone 2F12; Upstate Cell Signaling Solutions) for IP; anti-ErbB3 (clone D22C5; Cell Signaling) for western blotting; anti-integrin β1 (monoclonal, ab52971 from Abcam); anti-Rabaptin5 (monoclonal, sc-271069 from Santa Cruz Biotechnology); anti-GGA3 (clone 8; BD Transduction Laboratories); anti-Arf6 (clone 3A-1, Santa Cruz Biotech.); anti-phospho-ErbB3 Tyr1289 (#4791, Cell Signalling Technology); anti-EGFR (#2232, Cell Signalling Technology); anti-ErbB2 (06-562, Millipore); anti-phospho-AKT Thr308 (#2965, Cell Signalling Technology); anti-AKT (#9272, Cell Signalling Technology); anti-phospho ERK1/2 (#9101, Cell Signalling Technology); anti-ERK1/2 (#9102.

Techniques: Immunofluorescence, Imaging, Transfection, Pulse Chase, Metabolic Labelling, Western Blot, Incubation, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Ligand-independent role of ErbB3 in endocytic recycling

doi: 10.1101/575449

Figure Lengend Snippet: a Confocal imaging of ErbB3-mCherry and indicated Rab marker expressed in MCF7 cells, with or without prior treatment with the recycling inhibitor primaquine (PQ). b Analysis of colocalization of ErbB3-mCherry and Rab4 or Rab11. The relative enrichment of ErbB3 at the Rab-positive structures was determined by the formula (a-b)/b where a is the ErbB3-mCherry intensity of the center of Rab4 structures, and b the adjacent volume (background) for each structure. Each data point represents the average of a minimum of 20 structures in one cell. P-values were determined using unpaired, 2-tailed Student’s t-test. c Average projections of all analysed (indicated number) of GFP-Rab4 or GFP-Rab11 positive structures from indicated number of cells (3 independent experiments).

Article Snippet: The following primary antibodies were used: anti-ErbB3 (clone 2F12; Upstate Cell Signaling Solutions) for IP; anti-ErbB3 (clone D22C5; Cell Signaling) for western blotting; anti-integrin β1 (monoclonal, ab52971 from Abcam); anti-Rabaptin5 (monoclonal, sc-271069 from Santa Cruz Biotechnology); anti-GGA3 (clone 8; BD Transduction Laboratories); anti-Arf6 (clone 3A-1, Santa Cruz Biotech.); anti-phospho-ErbB3 Tyr1289 (#4791, Cell Signalling Technology); anti-EGFR (#2232, Cell Signalling Technology); anti-ErbB2 (06-562, Millipore); anti-phospho-AKT Thr308 (#2965, Cell Signalling Technology); anti-AKT (#9272, Cell Signalling Technology); anti-phospho ERK1/2 (#9101, Cell Signalling Technology); anti-ERK1/2 (#9102.

Techniques: Imaging, Marker

Journal: bioRxiv

Article Title: Ligand-independent role of ErbB3 in endocytic recycling

doi: 10.1101/575449

Figure Lengend Snippet: a Immunoblotting of ErbB3 immunoprecipitates or input cell lysates, after 30 minutes treatment with primaquine (PQ), showing endogenous binding of ErbB3 with GGA3 and Rabaptin5 that increases upon PQ treatment, and the presumed accumulation of recycling endosomes (representative of 3 independent experiments). b Immunoblotting of Arf6 immunoprecipitates or input cell lysates, following control or ErbB3 siRNA transfection and 10 minutes treatment with PQ or vehicle. Note that endogenous co-precipitation of Arf6 with both GGA3 and rabaptin5 is reduced in the absence of ErbB3. c Quantification of GGA3 and Rabaptin5 protein levels (by western blotting) and mRNA levels (by quantitative RT-PCR) in ErbB3 siRNA-transfected MCF10A cells relative to control-transfected cells (n=4 experiments for protein and n=3 for mRNA). Data are presented as mean values ± s.e.m. and P values (one sample student’s t-test). d Structural model highlighting the putative GGA3-binding motif 864-DxxLL-867 in the ErbB3 kinase domain. e Immunoblotting of ErbB3 immunoprecipitates or input cell lysates, after ectopic expression of ErbB3 or the ErbB3 LL866/867AA mutant with GGA3 in HEK293T cells. Note that the LL866/867AA mutant ErbB3 fails to co-precipitate with GGA3. f The LL866/867AA mutation compromises the ability of ErbB3 to promote assembly of the Arf6-GGA3-Rabatin5 sorting complex: Immunoblotting of Arf6 immunoprecipitates or input cell lysates, following ectopic expression of Arf6, GGA3 and Rabaptin5 (Rbtn5), with or without ErbB3 or ErbB3-LL866/867AA.

Article Snippet: The following primary antibodies were used: anti-ErbB3 (clone 2F12; Upstate Cell Signaling Solutions) for IP; anti-ErbB3 (clone D22C5; Cell Signaling) for western blotting; anti-integrin β1 (monoclonal, ab52971 from Abcam); anti-Rabaptin5 (monoclonal, sc-271069 from Santa Cruz Biotechnology); anti-GGA3 (clone 8; BD Transduction Laboratories); anti-Arf6 (clone 3A-1, Santa Cruz Biotech.); anti-phospho-ErbB3 Tyr1289 (#4791, Cell Signalling Technology); anti-EGFR (#2232, Cell Signalling Technology); anti-ErbB2 (06-562, Millipore); anti-phospho-AKT Thr308 (#2965, Cell Signalling Technology); anti-AKT (#9272, Cell Signalling Technology); anti-phospho ERK1/2 (#9101, Cell Signalling Technology); anti-ERK1/2 (#9102.

Techniques: Western Blot, Binding Assay, Transfection, Quantitative RT-PCR, Expressing, Mutagenesis

Journal: NPJ Breast Cancer

Article Title: FOXA1 and adaptive response determinants to HER2 targeted therapy in TBCRC 036

doi: 10.1038/s41523-021-00258-0

Figure Lengend Snippet: a Schematic overview of LCCC1214 (Clinical trials identifier NCT01875666) for the analysis of adaptive responses to HER2 targeted therapy. b Normalized RNAseq data from matched patient samples was used to generate log2 fold changes for expressed genes (post-treatment vs. pre-treatment). The fold change in expression was used for the principal component analysis (PCA). Labels indicate patient number and dot color indicates treatment arm. The blue dashed line indicates distinct matched samples (from patients 116, 119, and 123) determined to be strongly responsive based on their distinct expression changes. c Log 2 fold change for FOXA1 for matched patient samples is plotted. The blue dashed line indicates the strongly responsive sample pairs. d Log 2 fold changes of the top 5000 differentially expressed genes for matched patient samples were used for unsupervised hierarchical clustering and nearest neighbor analysis was performed for the FOXA1 . The top 10 genes by Pearson correlation are indicated, as well as select genes identified from HER2+ cell line SE analysis. e The breast invasive carcinoma (TCGA, provisional) data set was used to identify genes most significantly co-expressed with HER3 expression in patient tumors. The top genes were XBP1 (not shown) and FOXA1 . The dashed line indicates regression line. Table contains legend for mutation status, if known. f Pathway analysis of gene expression data from matched patient samples was performed and the pathway scores differences was used for marker selection comparing the strongly responsive sample pairs (blue dashed line) to all other sample pairs. The top 25 pathways (increased and decreased) are shown (10% FDR cutoff). Representative significant pathways are listed with associated reference.

Article Snippet: Equal amounts of extracted protein (determined by Bradford assay) were separated by SDS-PAGE, transferred to nitrocellulose and probed with anti-BRD4 (Bethyl Laboratories, Cat. A301-985A100, 1:1000), anti-ERK2 (Santa Cruz Cat. sc-1647, 1:2000), anti-FOXA1 (Abcam, Cat. ab5089, 1:1000) or the following antibodies (all with Cell Signaling Cat. numbers and used at 1:1000): anti-FOXO1 (2880), anti-FOXO3 (2497), anti-phospho-HER2/ErbB2 (Tyr1221/1222) (2243), anti-HER2 (2242), anti-HER3 (4754), anti-phospho-AKT (Thr308) (4056), anti-phospho-AKT (Ser473) (4060), anti-phospho-ERK1/2 (4370).

Techniques: Expressing, Mutagenesis, Marker, Selection

Journal: NPJ Breast Cancer

Article Title: FOXA1 and adaptive response determinants to HER2 targeted therapy in TBCRC 036

doi: 10.1038/s41523-021-00258-0

Figure Lengend Snippet: a All post-treatment samples with RNAseq data were used to filter for expressed kinases. The normalized kinome expression data was used for principal component analysis (PCA). Patient number is indicated in red for each dot and color indicates LCCC1214 treatment arm. Dashed line indicates previously identified patients 116, 119, and 123 and neighboring samples 109, 118, and 121. b RNAseq expression levels of strongly responsive post-treatment samples were compared to the less-responsive post-treatment samples by DESeq2 to identify differentially expressed kinases. The volcano plot indicates the magnitude and the significance of the identified expression differences. Dashed line indicates FDR 5%. c Log 2 normalized gene expression data (GSE76360) of the kinome was analyzed from the 03-311 clinical trial (Clinical trials identifier NCT00148668), in which patient samples were obtained pre-treatment and after 10–14 days of trastuzumab. Post-treatment gene expression data of the kinome was used for unsupervised hierarchical clustering and a segregated cluster of 7 post-treatment patient samples (of 50 total) was identified. Marker selection was performed in Morpheus (Broad Institute) to identify the top 20 kinases significantly down in the strongly responsive post-treatment samples for 03-311 trial data and from DESeq2 analysis of the LCCC1214 trial from ( b ). Venn diagram shows the 6 kinases common to both data sets. Asterisks indicate that more than one gene probe was identified by marker selection for the 03-311 data set (Illumina expression beadchip). d DESeq2 analysis of differentially expressed transcription factors in the strongly responsive versus weakly responsive post-treatment LCCC1214 samples. The volcano plot indicates the magnitude and the significance of the identified expression differences. e Marker selection was performed in Morpheus (Broad Institute) to identify the top 20 transcription factors with decreased expression in the strongly responsive post-treatment samples for 03-311 trial and compared with the DESeq2 analysis for LCCC1214 from ( a ). The Venn diagram shows the 5 transcription factors common to both clinical data sets. f The post-treatment/pre-treatment log 2 fold change of HER2 , HER3 , and PTK6 expression is shown for paired patient samples. g Paired post-treatment and pre-treatment samples from the LCCC1214 study were processed for kinome analysis by MIB/MS. The log 2 MIB binding changes (post/pre) were plotted as the sum of strongly molecular responsive samples (patients 109, 116, 119, and 121) versus the average difference for weakly-responsive matched pairs and the top 25 differences in both directions are shown. h – j Log 2 fold changes for the indicated matched patient samples as determined by MIB binding ( x -axis) and RNAseq ( y -axis).

Article Snippet: Equal amounts of extracted protein (determined by Bradford assay) were separated by SDS-PAGE, transferred to nitrocellulose and probed with anti-BRD4 (Bethyl Laboratories, Cat. A301-985A100, 1:1000), anti-ERK2 (Santa Cruz Cat. sc-1647, 1:2000), anti-FOXA1 (Abcam, Cat. ab5089, 1:1000) or the following antibodies (all with Cell Signaling Cat. numbers and used at 1:1000): anti-FOXO1 (2880), anti-FOXO3 (2497), anti-phospho-HER2/ErbB2 (Tyr1221/1222) (2243), anti-HER2 (2242), anti-HER3 (4754), anti-phospho-AKT (Thr308) (4056), anti-phospho-AKT (Ser473) (4060), anti-phospho-ERK1/2 (4370).

Techniques: Expressing, Marker, Selection, Binding Assay

Journal: mAbs

Article Title: Targeting HER3 using mono- and bispecific antibodies or alternative scaffolds

doi: 10.1080/19420862.2016.1212147

Figure Lengend Snippet: Schematic illustration of the epidermal growth factor receptors EGFR, HER2, HER3 and HER4. The receptors consist of an extracellular unit, divided into 4 domains (I-IV), a transmembrane region, a cytoplasmic tyrosine kinase domain as well as a C-terminal tail with phosphotyrosine residues. Whereas inactive EGFR, HER3 and HER4 adopt a tethered extracellular conformation, HER2 is constantly in an untethered conformation available for dimerization. Binding of the ligands NRG1 or NRG2 to the catalytically inactive HER3 results in exposure of a dimerization arm on domain II and subsequent receptor dimerization, here exemplified by the HER2/HER3 heterodimer. Extracellular dimerization induces intracellular signaling pathways such as the MAPK and PtdIns-3K pathways, mainly resulting in increased proliferation and survival.

Article Snippet: Millegen S.A. has generated HER3-specific antibodies targeting either domain 1 or 3 of the receptor by phage display or lymphocyte hybridization.

Techniques: Binding Assay, Protein-Protein interactions

Journal: mAbs

Article Title: Targeting HER3 using mono- and bispecific antibodies or alternative scaffolds

doi: 10.1080/19420862.2016.1212147

Figure Lengend Snippet: Overview of protein-based HER3-targeting agents in development.

Article Snippet: Millegen S.A. has generated HER3-specific antibodies targeting either domain 1 or 3 of the receptor by phage display or lymphocyte hybridization.

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-based PET Imaging

doi: 10.1158/1078-0432.CCR-17-2754

Figure Lengend Snippet: (A) Scheme illustrating the radiolabeling reaction of DFO-conjugated ZHER3:8698 affibody molecule with the positron emitter 89Zr. (B) Saturation binding of 89Zr-DFO-ZHER3:8698 to MCF-7 cells. The data are expressed as the mean values ± SEM (n = 3 independent experiments). (C) HER3 expression in a panel of breast cancer cell lines. Representative Western blot from whole cell lysates, with GAPDH used as the loading control. (D) In vitro binding specificity of 89Zr-DFO-ZHER3:8698 in breast cancer cells and specific blocking using 100-fold molar excess of either unlabeled ZHER3:8698 or the natural HER3 ligand HRG. The data are expressed as the mean values ± SEM (n = 3 independent experiments). *P = 0.0357; *P = 0.0446; for MDA-MB468, **P = 0.0087; *P = 0.015 for MCF-7, and **P = 0.009; **P = 0.0097 for BT-474 cells.

Article Snippet: Immunoprecipitation Tissue lysates (300 μg) and whole cell lysates (200 μg) were incubated overnight in a thermomixer at 4°C and 650 rpm, with 10 μg of anti-HER3 mouse mAb (Merck Millipore, UK).

Techniques: Radioactivity, Binding Assay, Expressing, Western Blot, In Vitro, Blocking Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-based PET Imaging

doi: 10.1158/1078-0432.CCR-17-2754

Figure Lengend Snippet: (A) Representative 15 min coronal fused PET/CT images of mice bearing MCF-7, MDA-MB-468, or MDA-MB-231 xenografts. The mice received ~ 8 MBq of either 89Zr-DFO-ZHER3:8698 or 89Zr-DFO-ZTAQ via tail vein injection, with image acquisition taking place 3 h after injection. The arrowheads indicate the tumors and the kidneys. (B) Ex vivo biodistribution at 3 h after injection of the radioconjugates. Data are expressed as the mean values ± SD (n = 3 animals). *P = 0.0136; ***P = 0.0002; ****P < 0.0001. (C) Representative Western blot of whole tumor tissue lysates evaluating HER3, HER2 and EGFR expression in the indicated xenograft models. (D) Histopathological analysis of HER3 expression in MCF-7, MDA-MB-468, and MDA-MB-231 xenografts displaying the highest HER3 expression in MCF-7 xenografts and the lowest in MDA-MB-231. (E) Representative ex vivo autoradiography sections taken 3 h after injection of 89Zr-DFO-ZHER3:8698. (F) Autoradiography quantification from panel E as the intensity per region of interest area (A.U./cm2). Data are expressed as the mean values ± SD (n = 10 sections). **P = 0.0028; ****P < 0.0001. (G) Representative segmented xenografts following PET/CT image acquisition 3 h after 89Zr-DFO-ZHER3:8698 injection. These images highlight the greater radioactivity accumulation observed in MCF-7 xenografts, and the heterogeneity of uptake across the tumor burden. Color map defined within the tumor volume only.

Article Snippet: Immunoprecipitation Tissue lysates (300 μg) and whole cell lysates (200 μg) were incubated overnight in a thermomixer at 4°C and 650 rpm, with 10 μg of anti-HER3 mouse mAb (Merck Millipore, UK).

Techniques: Positron Emission Tomography-Computed Tomography, Injection, Ex Vivo, Western Blot, Expressing, Autoradiography, Radioactivity

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-based PET Imaging

doi: 10.1158/1078-0432.CCR-17-2754

Figure Lengend Snippet: MCF-7 xenografts were randomized into two groups: control and treatment. The treatment group received 40 mg/kg of AUY922 i.p. every second day for a period of two weeks. (A) Representative axial fused PET/CT images of mice bearing MCF-7 tumors. Each mouse received 7.2-8.1 MBq of 89Zr-DFO-ZHER3:8698 via tail vein injection, with image acquisition taking place at 3 h after injection. The mice were imaged before initiating AUY922 treatment (day 0), and following administration of the last treatment dose (day 14). The %ID/g ratios were determined by dividing the %ID/g on day 14 by that obtained on day 0. (B) Scatter plot of the %ID/g ratios for both control (n = 6) and AUY922-treated mice (n = 7). The horizontal lines indicate the mean for each group. *P = 0.0131. (C) Scatter plot of the ex vivo tumor biodistribution at 3 h after injection of the radioconjugate on day 14, for both control (n = 4) and AUY922-treated mice (n = 6). The horizontal lines indicate the mean per group. **P = 0.0036. (D) Histopathological analysis of control and AUY922-treated MCF-7 xenografts. Tumor sections were stained with hematoxylin and eosin (H&E), HER3, or CD31.

Article Snippet: Immunoprecipitation Tissue lysates (300 μg) and whole cell lysates (200 μg) were incubated overnight in a thermomixer at 4°C and 650 rpm, with 10 μg of anti-HER3 mouse mAb (Merck Millipore, UK).

Techniques: Positron Emission Tomography-Computed Tomography, Injection, Ex Vivo, Staining

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-based PET Imaging

doi: 10.1158/1078-0432.CCR-17-2754

Figure Lengend Snippet: (A) HER receptors, IGF-1Rβ, and PI3K/AKT pathway activation were monitored by Western blot using whole tissue lysates from all control and AUY922-treated mice. Hsp70/72 expression was used as a surrogate for AUY922 treatment efficacy, and GAPDH as a loading control. (B) Minimum to maximum box & whiskers plot of the quantified protein expression represented in A. Data are expressed as the normalized protein expression per antibody for control and AUY922 groups. The black lines represent the median value. *P = 0.0306; **P = 0.0022; ****P < 0.0001. (C, D) Correlation between HER3/IGF-1Rβ and Hsp70/72 protein expression per mouse. The dashed lines represent the ninety-five percent confidence levels. (E) Correlation between %ID/g ratios obtained from 89Zr-DFO-ZHER3:8698 PET images and Hsp70/72 protein expression per mouse. The dashed lines represent the ninety-five percent confidence levels. (F) BT-474 and MCF-7 cells were treated with 32 nM of AUY922 for 48 h. Equal amounts of whole cell lysates were immunoprecipitated with a mouse anti-HER3 antibody followed by Western blot analysis of HER3 and IGF-1Rβ. Whole tissue lysates from control mouse C5 and AUY922-treated mouse A6 were also immunoprecipitated against HER3 and analyzed by Western blot. Ten percent of the input lysates were used as loading controls.

Article Snippet: Immunoprecipitation Tissue lysates (300 μg) and whole cell lysates (200 μg) were incubated overnight in a thermomixer at 4°C and 650 rpm, with 10 μg of anti-HER3 mouse mAb (Merck Millipore, UK).

Techniques: Activation Assay, Western Blot, Expressing, Immunoprecipitation

Journal: Journal of Hematology & Oncology

Article Title: Characterization and validation of potential therapeutic targets based on the molecular signature of patient-derived xenografts in gastric cancer

doi: 10.1186/s13045-018-0563-y

Figure Lengend Snippet: Expressions of several critical molecules in GC PDX models. a EGFR expression was evaluated with scores of: 0, 1+, 2+, and 3+. For the FISH assay, the red and green signals represented EGFR and CEN7, respectively. Scale bar represents 100 μm. b HER3 expression was evaluated with scores of: 0, 1+, 2+, and 3+. Scale bar represents 100 μm. c MET expression was evaluated with H-scores. Scale bar represents 100 μm. d EBV infection status was detected by EBV-encoded RNA in situ hybridization. EBER-positive cells were observed in 20% or more of the tumor cells. Scale bar represents 100 μm. e PD-L1 expression was detected in the placenta (positive control), normal gastric tissue (negative control), and PDX models. Patterns with ≥ 5% positive tumor cells or immune cells was considered to be PDL-1 positive. Scale bar represents 100 μm

Article Snippet: Candidate targets, including EGFR, HER3, MET, and PD-L1, were stained via immunohistochemistry (IHC) using anti-EGFR antibody (#4267, Cell Signaling Technology, Danvers, MA, USA), anti-HER3 antibody (#2708, Cell Signaling Technology), anti-MET antibody (#790-4430, Ventana Medical Systems, Tucson, AZ, USA), and anti-PD-L1 antibody (#M4420, Spring Bioscience Corp., Pleasanton, CA, USA) according to the manufacturers’ instructions.

Techniques: Expressing, Infection, RNA In Situ Hybridization, Positive Control, Negative Control