Journal: Scientific Reports

Article Title: EGFR and HER3 expression in circulating tumor cells and tumor tissue from non-small cell lung cancer patients

doi: 10.1038/s41598-019-43678-6

Figure Lengend Snippet: Protein expression and frequency distribution in primary and metastatic lung cancer tissue. EGFR protein immunohistochemistry ( A ) and HER3 protein immunohistochemistry ( B ) was performed on tissue microarray from primary NSCLC tumors and brain metastases. Representative strong (left), intermediate (center) and negative (right) staining are shown in 200x magnification (Zeiss Axiovision).

Article Snippet: For HER3 we tested two different antibodies; the biotinylated human anti-HER3 antibody (clone REA508; Miltenyi Biotec, Bergisch Gladbach, Germany) and the biotinylated human anti-HER-3 antibody (clone 1B4C3; BioLegend, San Diego, California).

Techniques: Expressing, Immunohistochemistry, Microarray, Staining

Journal: Scientific Reports

Article Title: EGFR and HER3 expression in circulating tumor cells and tumor tissue from non-small cell lung cancer patients

doi: 10.1038/s41598-019-43678-6

Figure Lengend Snippet: EGFR ( A ) and HER3 ( B ) expression distribution in primary lung, lymph node and brain metastatic tissue and association between HER3 protein expression in primary and clinicopathological parameters ( C ). HER3 is more frequently expressed in brain metastasis of oligo-brain metastatic patients (p = 0.028) compared to patients with other metastatic sites. HER3 expression in primary NSCLC tumors is significantly associated with a decreased time to metastatic progression (p = 0.006; log-rank test) ( D ) and decreased relapse-free survival time (p = 0.013; log-rank test) ( E ).

Article Snippet: For HER3 we tested two different antibodies; the biotinylated human anti-HER3 antibody (clone REA508; Miltenyi Biotec, Bergisch Gladbach, Germany) and the biotinylated human anti-HER-3 antibody (clone 1B4C3; BioLegend, San Diego, California).

Techniques: Expressing

Journal: Scientific Reports

Article Title: EGFR and HER3 expression in circulating tumor cells and tumor tissue from non-small cell lung cancer patients

doi: 10.1038/s41598-019-43678-6

Figure Lengend Snippet: CTC positivity rates in NSCLC patients (n = 45). ( A ) Significantly higher detection rates when combining the methods in comparison to either single use CellSearch (p = 0.0023) or magnetic cell separation with EGFR/HER3 (p = 0.0109). ( B ) CTC enumeration detected by CellSearch and MACS.

Article Snippet: For HER3 we tested two different antibodies; the biotinylated human anti-HER3 antibody (clone REA508; Miltenyi Biotec, Bergisch Gladbach, Germany) and the biotinylated human anti-HER-3 antibody (clone 1B4C3; BioLegend, San Diego, California).

Techniques: Comparison, Magnetic Cell Separation

Journal: Scientific Reports

Article Title: EGFR and HER3 expression in circulating tumor cells and tumor tissue from non-small cell lung cancer patients

doi: 10.1038/s41598-019-43678-6

Figure Lengend Snippet: Representative images of two Circulating Tumor Cell cluster (DAPI + /CD45 − /CK + ) isolated via magnetic cell separation with HER3-protein antibody in one NSCLC patient. Heterogeneous MET expression was found within the cluster as well as CD45-positive leukocytes.

Article Snippet: For HER3 we tested two different antibodies; the biotinylated human anti-HER3 antibody (clone REA508; Miltenyi Biotec, Bergisch Gladbach, Germany) and the biotinylated human anti-HER-3 antibody (clone 1B4C3; BioLegend, San Diego, California).

Techniques: Isolation, Magnetic Cell Separation, Expressing

Journal: bioRxiv

Article Title: A DNA nanoassembly-based approach to map membrane protein nanoenvironments

doi: 10.1101/836049

Figure Lengend Snippet: a , A 1:1 mixture of ECD-Her2 and ECD-Her3 was covalently attached to the SPR surfaces of two separate flow cells. Her2-NanoCombs were injected and binding to the anchored ECD-Her2 was detected by an increase in the sensorgram signal. Anti-Her3 affibody-oligo conjugates, previously hybridized with a blocking strand, were added to flow cell 1 (on the left) but not to flow cell 2 (on the right). In flow cell 1, a first invading strand (invading strand 1) was injected, promoting unblocking of the anti-Her3 affibody-oligo conjugates and hybridization to the detection prongs of the NanoComb. The sequences of the anti-Her2 conjugates preloaded on the reference prong contained an added 7-nt at 3’ end in this experiment. This allowed for a second toehold exchange reaction that promoted the displacement of anti-Her2 conjugates from the reference prong. This caused a larger decrease in the SPR signal in flow cell 2, where binding of the NanoComb to the surface is mediated only by the reference prong (bottom-right), than in cell flow 1, where the NanoComb remains bound to the surface through the interaction of the detection prongs with the anti-Her3 conjugates (bottom-left). b , EMSA using native PAGE (13%) was performed on samples that underwent blocking/unblocking reactions in solution. The hybridization of blocking strand to affibody-oligo conjugate was visualized by a shift of the conjugate band (the red line indicates the level without blocking). Displacement of the blocking strand by the invading strand was visualised by a shift in the conjugate band and the presence of the released dsDNA fragment consisting of the blocking strand hybridized to the invading strand. We observed a further shift in the conjugate band when adding the complementary prong.

Article Snippet: The coding sequences of anti-Her2 (Z Her2:342 ), anti-Her3 (Z 08699 ) and anti-EGFR (Z EGFR:2377 ) affibodies were synthetized (BioCat) and cloned (XhoI/BamHI) at the C-terminal of the VirD2 protein connected via a flexible linker (GGGGS) in the expression plasmid pET-16-b and the sequences were validated by sequencing.

Techniques: Injection, Binding Assay, Blocking Assay, Hybridization, Clear Native PAGE

Journal: bioRxiv

Article Title: A DNA nanoassembly-based approach to map membrane protein nanoenvironments

doi: 10.1101/836049

Figure Lengend Snippet: a , Schematic representation of the SPR assay; equivalent amounts of E CD-Her3 were covalently attached to two SPR flow cells. Anti-Her3 affibody-oligo conjugates bound first to the target proteins and then hybridized with the NanoCombs. To promote the release of the barcoded dsDNA sequences, T4 polymerase DNA elongation and restriction enzyme cleavage were performed. The enzymatic reactions were carried out only on flow cell 1 and flow cell 2 was used as a negative control. Finally, streptavidin that is able to bind to the desthiobiotin on 3’ end of NanoComb backbone, was injected over the two flow cells to detect the residual amounts of NanoCombs remaining on the surface. b , SPR signals of the conjugates and NanoCombs were comparable on the two flow cells. The binding of streptavidin was significantly reduced in flow cell 1 compared to flow cell 2, demonstrating the efficiency of the enzymatic reactions. c , Barcoded dsDNA sequences visualized on native PAGE (13%) after PCR amplification were recovered from flow cell 1 but not from the negative control, flow cell 2.

Article Snippet: The coding sequences of anti-Her2 (Z Her2:342 ), anti-Her3 (Z 08699 ) and anti-EGFR (Z EGFR:2377 ) affibodies were synthetized (BioCat) and cloned (XhoI/BamHI) at the C-terminal of the VirD2 protein connected via a flexible linker (GGGGS) in the expression plasmid pET-16-b and the sequences were validated by sequencing.

Techniques: SPR Assay, Negative Control, Injection, Binding Assay, Clear Native PAGE, Amplification

Journal: bioRxiv

Article Title: A DNA nanoassembly-based approach to map membrane protein nanoenvironments

doi: 10.1101/836049

Figure Lengend Snippet: a , 1:1 mixtures of ECD-Her2 and ECD-Her3 were covalently attached to the SPR surfaces of two sensor chips at two different surface densities. NanoDeep was performed by first flowing Her2-NanoCombs and then binder libraries consisting of anti-Her2 and anti-Her3 conjugates. Binding to the anchored proteins was monitored by sensorgram signals, which reflect the amount of anchored proteins. NGS analyses were performed in duplicate and presented as mean values in the heatmap. Barcode reads from the detection sequences were scaled to the reference sequence reads. b , NanoDeep was performed on SPR surfaces presenting different compositions of EGFR family receptors: Her2-Her3 (top), Her2-EGFR (middle) and a 1:1:1 mixture of Her2-Her2, Her2-Her3 and Her2-EGFR (bottom). NGS analyses were performed in duplicate and presented as mean values in the heatmap.

Article Snippet: The coding sequences of anti-Her2 (Z Her2:342 ), anti-Her3 (Z 08699 ) and anti-EGFR (Z EGFR:2377 ) affibodies were synthetized (BioCat) and cloned (XhoI/BamHI) at the C-terminal of the VirD2 protein connected via a flexible linker (GGGGS) in the expression plasmid pET-16-b and the sequences were validated by sequencing.

Techniques: Binding Assay, Sequencing

Journal: bioRxiv

Article Title: A DNA nanoassembly-based approach to map membrane protein nanoenvironments

doi: 10.1101/836049

Figure Lengend Snippet: a , NGS analysis revealed a linear correlation between NGS reads and the concentration of Her2-NanoCombs. SKBR3 cells (400,000 cells/sample) were treated with three different concentrations of Her2-NanoCombs: 0.5 μg/ml, 5 μg/ml and 30 μg/ml. NGS reads are presented in heatmaps (left panel) and as a plot showing a linear correlation between the number of NGS reads of reference sequences and NanoComb concentrations (right panel). b , SKBR3 cells, Her2-overexpressing, and MCF7 cells, which show basal levels of expression of Her2, were grown to similar densities and analyzed by NanoDeep using Her2-NanoCombs and anti-Her2, -Her3 and -EGFR binder libraries. All measurements were performed in triplicate and presented as mean values in two types of heatmaps, showing the reads from the reference sequences (top) and detection sequences (bottom). c , SKBR3 cells were starved for 24 h and then stimulated for 15 min with HRG-β 1, followed by NanoDeep assay. Untreated cells were used as controls. For each position of the NanoComb, the number of reads of detection sequences was plotted versus the protein identity barcodes, for HRG-β1 treated cells (orange dots) and control cells (black dots). Dots represent three independent experiments and lines represent means of each condition. *p≤0.05; **p≤0.01.

Article Snippet: The coding sequences of anti-Her2 (Z Her2:342 ), anti-Her3 (Z 08699 ) and anti-EGFR (Z EGFR:2377 ) affibodies were synthetized (BioCat) and cloned (XhoI/BamHI) at the C-terminal of the VirD2 protein connected via a flexible linker (GGGGS) in the expression plasmid pET-16-b and the sequences were validated by sequencing.

Techniques: Concentration Assay, Expressing

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: 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, Control, 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: 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: 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: Control, 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: 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, Control, Expressing, Immunoprecipitation