Journal: bioRxiv

Article Title: Integrating extracellular vesicle and circulating cell-free DNA analysis on a single plasma aliquot from breast cancer patients improves the detection of HER2 positivity

doi: 10.1101/2023.03.02.530645

Figure Lengend Snippet: a. Scheme of ONCE ( ON e Aliquot for C irculating E lements) protocol for multi-analyte liquid biopsies in BrCa. An aliquot of whole blood was collected from n=44 early-stage non metastatic BrCa patients into K2EDTA tubes and the extracted plasma (1.5ml) was processed by ONCE protocol combined with the CB method for the isolation of two diverse circulating analytes: EVs and cfDNA. EVs and cfDNA are sources of circulating nucleic acids for investigating tumor biomarkers by RNA-Seq/DNA-Seq and /or ultra-sensitive ddPCR. The dilution of plasma with PBS1x is an essential step to reduce plasma viscosity, thereby facilitating the binding of the beads to the EVs and guaranteeing the performance of the CB method. b. Quantification of EVs by TRPS (Tunable Resistive Pulse Sensing) measurements. EVs were isolated from early-stage breast cancer patients (n=44) of 2 different subtypes: HER2+ and HER2-. n.s.: not significant differences by t-test. c. Western Blot assay showing the EV-enriched proteins Tsg101, Flotillin 2, Flotillin 1, CD9 and the contaminant proteins Apolipoprotein A1 (ApoA1) and Albumin in representative n=3 HER2+ and n=3HER2- EV samples isolated from BrCa patients (PMB). EVs were isolated by CB isolation method as shown on panel a . d. Detection of CD8, CD63, and CD81 tetraspanins by MACSPlex exosome assay on EVs isolated by CB method throughout ONCE protocol. X axis plots the detection signal (Median Intensity level on APC channel) in log10+1 scale. Y axis reports BrCa patient code. Data are from n= 8 HER2+ and n=6 HER2- BrCa patients. e. Quantification of EV-RNA extracted from EVs after isolation by CB method. EV-RNA was quantitated by Agilent RNA pico assay. EV-RNA samples were derived from n=17 HER2+ and n=20 HER2- early-stage BrCa patients. n.s.: not significant differences by t-test. f. Quantification of cfDNA by Qubit dsDNA HS Assay (Thermo Fisher Scientific). cfDNA was extracted from plasma leftovers, after EVs enrichment, according with ONCE protocol. Data are from n=23 HER2+ and n=21 HER2- early-stage BrCa. n.s.: not significant differences by One–way Anova. g. Scatter plot of levels of cfDNA (ng/mL of plasma) and EVs (number of EVs/ mL of plasma) recovered by ONCE from n=44 BrCa patients. Data are stratified by HER2 status (HER2+ vs HER2-) based on histopathological classification. The IHC score, derived from tissue biopsies, is shown with symbols. cfDN A: cell free DNA; EVs : Extracellular Vesicles; EV-RNA : RNA extracted from EVs. BrCa : Breast Cancer; HER2+ : HER2 positive, HER2- : HER2 negative; IHC : Immunohistochemistry. See also .

Article Snippet: Isolated EV samples (10 10 -10 11 particles/mL) were resuspended in 1x Phosphate-Buffered Saline (1xPBS) (Gibco) and incubated overnight at 4C with HER2/ErbB2 (29D8) – PE-conjugated antibody (1:50) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) (Cell Signaling Technology).

Techniques: Isolation, RNA Sequencing Assay, DNA Sequencing, Binding Assay, Tunable Resistive Pulse Sensing, Western Blot, Derivative Assay, Immunohistochemistry

Journal: bioRxiv

Article Title: Integrating extracellular vesicle and circulating cell-free DNA analysis on a single plasma aliquot from breast cancer patients improves the detection of HER2 positivity

doi: 10.1101/2023.03.02.530645

Figure Lengend Snippet: a. Circos plots of cfDNA analyzed by whole-exome DNA-Seq (WES) of n=4 representative BrCa patients. Squares outline the ERBB2 gene locus (Chr.17q12) shown as linear visualization in the inset; ERBB2 gene amplification is detectable on samples PMB2.8 and PMB2.36, (cfDNA isolated from HER2+ BrCa patients); no ERBB2 amplification is detectable in cfDNA samples PMB 2.30 and PMB2.26 (cfDNA isolated from HER2- BrCa patients). Purity indicates the circulating tumor content. b. Heatmap showing the detection of relevant breast cancer biomarkers in n=4 patients: PMB2.8 and PMB2.36, (HER2+), PMB2.30 and PMB2.26 (HER2-). Transcripts were detected by RNA-Seq from EV-RNA from liquid biopsies. Proteins were detected by IHC on tissue biopsies. DNA amplification of ERBB2 gene was detected by FISH on tissue biopsies. ERBB2 (HER2), ESR1 (Estrogen Receptor alpha) and MKI67 (Marker of Proliferation Ki-67). c. Representative plots and quantification of HER2+ EVs subpopulations by imaging flow cytometry (Amnis Imagestream x MK II). Plots show CellMask staining utilized to visualize the bulk of lipid nanoparticles on x-axis (Intensity_MC_Ch_11) and PE-HER2 Antibody staining on y-axis (Intensity_MC_Ch_03). Data are from technical replicates of measurements performed on EVs samples isolated from n=3 HER2- and n=3 HER2+ BrCa patients (PMB). ** p=0.0041 by t-test. cfDN A: cell free DNA; EVs : Extracellular Vesicles; EV-RNA : RNA extracted from EVs; BrCa : Breast Cancer; HER2+ : HER2 positive, HER2- : HER2 negative; IHC : Immunohistochemistry. See also

Article Snippet: Isolated EV samples (10 10 -10 11 particles/mL) were resuspended in 1x Phosphate-Buffered Saline (1xPBS) (Gibco) and incubated overnight at 4C with HER2/ErbB2 (29D8) – PE-conjugated antibody (1:50) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) (Cell Signaling Technology).

Techniques: DNA Sequencing, Amplification, Isolation, RNA Sequencing Assay, Marker, Imaging, Flow Cytometry, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: Integrating extracellular vesicle and circulating cell-free DNA analysis on a single plasma aliquot from breast cancer patients improves the detection of HER2 positivity

doi: 10.1101/2023.03.02.530645

Figure Lengend Snippet: a. Western Blot assay showing the breast tissue associated proteins HER2, CD44, EpCaM and EV-enriched proteins Flotillin 1, Flotillin 2 and CD9 in representative n=2 protein extracts of EV samples isolated by CB from conditioned medium of SKBR3 (HER2+) and MDAMB231 (HER2-) human breast cancer cell lines. b. Quantification of PE+ signal detected by imaging flow cytometry (Amnis Imagestreamx MK II). EV samples were stained with the IgG isotype control (PE-Conjugated) for the anti-HER2 antibody utilized in . As expected, only minimal and comparable percentages of fluorescent particles are detected on both HER2- and HER2+ samples. Data are from technical replicates of measurements performed on EVs samples isolated from n=3 HER2- and n=3 HER2+ BrCa patients.

Article Snippet: Isolated EV samples (10 10 -10 11 particles/mL) were resuspended in 1x Phosphate-Buffered Saline (1xPBS) (Gibco) and incubated overnight at 4C with HER2/ErbB2 (29D8) – PE-conjugated antibody (1:50) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) (Cell Signaling Technology).

Techniques: Western Blot, Isolation, Imaging, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Integrating extracellular vesicle and circulating cell-free DNA analysis on a single plasma aliquot from breast cancer patients improves the detection of HER2 positivity

doi: 10.1101/2023.03.02.530645

Figure Lengend Snippet: a. Concomitant ddPCR of ERBB2 gene on cfDNA and transcript on EV-RNA. Red dots: HER2+ BrCa (n=24); blue dots: HER2- BrCa (n=14) (stratification based on tissue biopsies data); grey dots: HDs (n=7). Marginal boxplots show distributions for each group of samples. Labeled samples are shown in panel b. Colored areas show the area that contains all the points of a specific class of samples (HDs, HER2+ or HER2-). Grey lines mark thresholds based on HDs distributions for HER2 positivity prediction (maximum level of RNA and DNA detected in HDs is set as threshold). Measurements from EV-RNA of SKBR3 (HER2+) and MDAMB231 (HER2-) breast cancer cell lines (n=3 replicates) are indicated on the Y axis by red and blue lines, respectively. b. Fluorescent In Situ Hybridization (FISH) pictures of breast tumor tissue biopsies show HER2 gene amplification. Quantification of amplification was based on HER2 (red) and CEP17 (green) ratio according with the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines. Data are associated to BrCa patients: PMB2.89 (HER2/CEP17 ratio: 3.0), PMB2.70 (HER2/CEP17 ratio: 5.8), PMB2.19 (HER2/CEP17 ratio: 2.9). c. Performance of HER2 status classifiers: combining cfDNA and EV-RNA data increases the performance of liquid biopsy biomarker detection. Liquid biopsy classifier (violet) is obtained using data from panel a. Classifier for Tissue biopsies (yellow) uses tissue data from . Symbol shape indicates if the classifier uses only DNA or RNA data or a combination of both. Samples with values higher than the thresholds are classified as HER2+ based on one of the following rules: 1. only RNA: only the signal of RNA reaches the threshold; 2. only DNA: only the signal of DNA reaches the threshold; 3. Combo AND: both DNA and RNA signals reach the threshold; 4. Combo OR: signal for any DNA or RNA reaches the threshold. Precision is the ratio between predicted true positives and the total number of predicted positives. The recall is the ratio between predicted true positives and the total number of positives in the population. Lines in the background show the F1 score (harmonic mean between recall and precision). The F1 score is a score of measurements’ performance and ranges from 0 (lowest recall; lowest precision) to 1 (highest recall; highest precision). cfDN A: cell-free DNA; EVs : Extracellular Vesicles; EV-RNA : RNA extracted from EVs. ddPCR : droplet digital PCR; HDs : Healthy Donors, BrCa : Breast Cancer; HER2+ : HER2 positive, HER2 -: HER2 negative; HDs : healthy donors. See also .

Article Snippet: Isolated EV samples (10 10 -10 11 particles/mL) were resuspended in 1x Phosphate-Buffered Saline (1xPBS) (Gibco) and incubated overnight at 4C with HER2/ErbB2 (29D8) – PE-conjugated antibody (1:50) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) (Cell Signaling Technology).

Techniques: Labeling, In Situ Hybridization, Amplification, Biomarker Assay, Digital PCR

Journal: Cancer Cell International

Article Title: MiRNA-125a-5p: a regulator and predictor of gefitinib’s effect on nasopharyngeal carcinoma

doi: 10.1186/1475-2867-14-24

Figure Lengend Snippet: miR-125a-5p mediated the expression of p53 and Her2 proteins in NPC cells. Western blot analysis confirmed that p53 protein expression was lower in HNE-1 cells than in HK-1 cells. After transfection with oligo-miR-125a-5p mimic, p53 protein expression was increased in HNE-1 cells compared with the control group. Her2 protein expressions were decreased in both cell lines following transfection with oligo-miR-125a-5p mimic in HNE-1 and HK-1 cells. Her2 protein expressions in HK-1 cells were increased after transfection with oligo-miR-125a-5p inhibitor.

Article Snippet: Membranes were incubated in blocking buffer (1× Tris-buffered saline [TBS], 0.1% Tween-20, and 5% w/v dry nonfat milk) for 1 h at room temperature, and then membranes were incubated with anti-p53 DO-1 (1:300, Santa Cruz, Dallas, Tex, USA), or anti-Her2 29D8 (1:300, Cell Signaling Technology, Beverly, MA, USA), or anti-β-actin (sc-47778, 1:1000, Santa Cruz) for overnight at 4°C, followed by incubation with an appropriate peroxidase-conjugated secondary antibody at room temperature for 1 h. Reactive bands were detected by enhanced chemiluminescence (Millipore).

Techniques: Expressing, Western Blot, Transfection

Journal: Breast Cancer Research : BCR

Article Title: Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells

doi: 10.1186/bcr3204

Figure Lengend Snippet: Characterization of vaccine-induced anti-HER2 antibodies by flow cytometry and western blot analysis . (A) Recognition of cell-surface HER2 by vaccine-induced anti-HER2 antibodies (HER2-VIA). HCC1569 cells were incubated with a mouse anti-human-HER2 mAb (HER2-phycoerythrin (PE)) or isotype control (Becton Dickinson (BD), San Jose, CA, USA), or with diluted (1:200) mouse serum antibodies (HER2-VIA or LacZ-VIA) for 1 hour at 4°C. Samples were then analyzed by BD LSRII flow cytometry, with results represented as histograms. (B) Recognition of total HER2 but not epidermal growth factor receptor (EGFR)-GFP by HER2-VIA. HER293 cells expressing vector (lane 1), HER2-FLAG (lane 2) or EGFR-GFP (lane 3) as well as SK-BR-3 cells lysates were western blotted with HER2-VIA (top panel), anti-FLAG antibody (upper middle panel), and anti-GFP antibody (lower middle panel). β-actin was used as a loading control (bottom panel). IB, immunoblot.

Article Snippet: The cell lysate was then spun at 18,000 g for 10 minutes to remove the beads, and the supernatant was incubated overnight with 1 µl anti-HER2 antibody 29D8 (Cell Signaling).

Techniques: Flow Cytometry, Western Blot, Incubation, Expressing, Plasmid Preparation

Journal: Breast Cancer Research : BCR

Article Title: Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells

doi: 10.1186/bcr3204

Figure Lengend Snippet: Vaccine-induced anti-HER2 antibody treatment of established human breast tumor xenografts in mice suppresses tumor growth . (A) Schema for the passive transfer of vaccine-induced antibodies to treat established BT474M1 human breast tumor xenografts in NOD.CB17- Prkdc scid /J mice. (B) Mean tumor volume ± standard error (mm 3 ) for AdGFP-VIA-treated (solid squares) and AdHER2-VIA-treated (open squares) mice is presented as a time course from day 14 (initiation of HER2-VIA or GFP-VIA injections) through day 39 (tumor harvest). * P <0.05, ** P <0.001. (C) Schema for the passive transfer of vaccine-induced antibodies to treat established HCC1569 human breast tumor xenografts in NOD.CB17- Prkdc scid /J mice. (D) Mean fold-change in tumor growth of implanted HCC1569 cells for the mice treated with HER2-VIA or PBS for days 8 through 20. ** P <0.001. (E) Effect of vaccine-induced anti-HER2 antibodies (HER2-VIA) and trastuzumab on human breast cancer cell proliferation. HER2-positive cells (BT474 or SKBR3) were treated with 3 µl pooled mice crude serum in 100 µl culture medium for 3 days and cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide assay. Trastuzumab (Herceptin) (10 µg/ml) was used as a positive control and sera from mice receiving Ad-LacZ vaccine (VIA-SHAM) or saline (untreated) were used as negative controls. Proliferation is plotted relative to the growth of cells in the untreated condition. Error bars represent standard deviation. Data are representative of four experiments. P <0.001.

Article Snippet: The cell lysate was then spun at 18,000 g for 10 minutes to remove the beads, and the supernatant was incubated overnight with 1 µl anti-HER2 antibody 29D8 (Cell Signaling).

Techniques: Positive Control, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells

doi: 10.1186/bcr3204

Figure Lengend Snippet: Activation of multiple signaling molecules by vaccine-induced anti-HER2 antibodies and inhibition of activation by lapatinib . SK-BR-3 cells were left untreated (lanes 1 to 5) or were pretreated with 2 µM lapatinib for 3 hours (lanes 6 to 10) followed by 100 µg/ml vaccine-induced anti-HER2 antibodies (HER2-VIA) stimulation at 37ºC for the indicated time. After treatment, cells were washed and then lysed in 2 × SDS sample buffer followed by sonication. Equal amounts of protein from each sample were used to visualize the indicated molecules by immunoblotting. ERK, extracellular signal-regulated protein kinase.

Article Snippet: The cell lysate was then spun at 18,000 g for 10 minutes to remove the beads, and the supernatant was incubated overnight with 1 µl anti-HER2 antibody 29D8 (Cell Signaling).

Techniques: Activation Assay, Inhibition, Sonication, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells

doi: 10.1186/bcr3204

Figure Lengend Snippet: Effects of vaccine-induced anti-HER2 antibodies on HER2 internalization . (A) Internalization of HER2-YFP by vaccine-induced anti-HER2 antibodies (HER2-VIA). HEK 293 cells transiently expressing HER2-YFP were stimulated with (a, b) HER2-VIA, (c, d) LacZ-VIA, or (e, f) trastuzumab, respectively for 1 hour. Confocal images from the same cells were taken before and after antibody incubation. Formation of vesicles in the cells indicated receptor internalization. The experiment was repeated three times. NS, non-stimulated. (B) . Imaging endogenous HER2 internalization by HER2-VIA. SK-BR-3 and HCC1569 cells were allowed to grow for 24 hours and were treated as described in Materials and methods. Confocal images of cells that (a, e) were left untreated, or were treated with (b, f) HER2-VIA, (c, g) LacZ-VIA or (d, h) trastuzumab. (C) Endogenous HER2 internalization by HER2-VIA as assessed by cell surface Biotin-labeling. Upper panel: immunoblot (IB) for protected (internalized) biotin-labeled HER2 in cells treated with indicated agents/conditions. Lower panel: β-actin serves as loading control to ensure equal amounts of cell lysate. EGF, epidermal growth factor. (D) Effect of lapatinib on HER2-YFP internalization induced by HER2-VIA. Confocal images of HEK293 cells expressing HER2-YFP that (a) were left untreated, or were treated with (b) HER2-VIA, (c) lapatinib or (d) lapatinib then HER2-VIA for 1 hour. (E) The same transfected cells were immunoblotted (IB) for HER2 tyrosine phosphorylation (upper panel) and total HER2 expression (lower panel).

Article Snippet: The cell lysate was then spun at 18,000 g for 10 minutes to remove the beads, and the supernatant was incubated overnight with 1 µl anti-HER2 antibody 29D8 (Cell Signaling).

Techniques: Expressing, Incubation, Imaging, Labeling, Western Blot, Transfection

Journal: Breast Cancer Research : BCR

Article Title: Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells

doi: 10.1186/bcr3204

Figure Lengend Snippet: HER2 internalizes through a clathrin-coated mechanism . (A) Effect of HER2 and epidermal growth factor receptor (EGFR) trafficking by vaccine-induced anti-HER2 antibodies (HER2-VIA) and epidermal growth factor (EGF). HEK 293 cells transiently expressing HER2-RFP and EGFR-GFP were (a to c, g to i, m to o) left untreated, or were stimulated with (d to f) HER2-VIA, (j to l) EGF and (p to r) HER2-VIA/EGF at 37°C for 30 minutes. Confocal images from the same cells were taken before and after HER2-VIA and/or EGF ligand stimulation. Arrowheads indicate internalized receptors. Representative images from three experiments are presented. (B) Co-localization of HER2-VIA-stimulated HER2-YFP with the β 2 -adrenergic receptor (β 2 AR). Confocal images of unstimulated HEK293 cells expressing both (a) HER2-YFP and (b) β 2 AR-RFP. (c) Merged image of (a) and (b). ( d to f) Confocal images of cells simultaneously stimulated with 0.1 μM isoproterenol (Iso) and 100 μg/ml HER2-VIA at 37°C for 1 hour. (f) Merged image of (d) and (e). Arrowheads indicate co-localized vesicles. (C) Co-localization of HER2-VIA-stimulated HER2-YFP with transferrin. ( a to c) Confocal images of cells expressing the HER2-YFP and treated simultaneously with Alexa-546 transferrin (Tf) (100 μg/ml) and HER2-VIA (100 µg/ml) for 1 hour at 37°C. (c) Merged picture. Arrowheads indicate co-localized vesicles.

Article Snippet: The cell lysate was then spun at 18,000 g for 10 minutes to remove the beads, and the supernatant was incubated overnight with 1 µl anti-HER2 antibody 29D8 (Cell Signaling).

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells

doi: 10.1186/bcr3204

Figure Lengend Snippet: HER2 ubiquitination, degradation, and fragmentation induced by vaccine-induced anti-HER2 antibodies . (A) Vaccine-induced anti-HER2 antibodies (HER2-VIA) stimulation led to HER2 ubiquitination. SK-BR-3 cells expressing ubiquitin-Myc were pretreated with MG132 for 2 hours and then treated with HER2-VIA for the indicated time. The ubiquitinated HER2 was detected by anti-Myc antibody. (B) HER2-VIA stimulation leads to HER2 ubiquitination. SK-BR-3 cells were pretreated with MG132 for 2 hours before HER2-VIA stimulation. Cell lysates were immunoprecipitated with anti-HER2 29D8 antibody. The endogenous ubiquitinated HER2 was detected by the anti-ubiquitin antibody and the total HER2 was visualized by anti-HER2 3B5 antibody. (C), (D) HER2-VIA stimulation causes HER2 degradation: (C) SK-BR-3 and (D) HCC1569 cells were stimulated with HER2-VIA, LacZ-VIA, or trastuzumab for the indicated time, and an equal amount of cell lysates was subjected to western blot analysis. Expression levels of HER2 and β-actin were detected by corresponding antibodies. (E) HER2-VIA stimulation produces HER2 fragmentation. SK-BR-3 cells were treated with HER2-VIA for 6 hours in the absence or presence of prior MG132 treatment for 2 hours. Full-length and truncated HER2 are detected by anti-HER2 3B5 antibody. β-actin serves as loading control. (F) HER2-VIA stimulation produces tyrosine phosphorylation of the 130 kDa HER2 C-terminal fragment. SK-BR-3 cells were incubated with HER2-VIA for 6 hours after pretreatment with lapatinib, MG132, or lapatinib plus MG132 for 2 hours. Full-length and truncated HER2 are detected by anti-HER2 3B5 antibody, which recognizes the C-terminus (top panel). Phosphorylated full-length and truncated HER2 were recognized by tyrosine site-specific phospho-antibodies for phosphorylated tyrosine 877, 1,221/1,222 and 1,248 (middle three panels). IB, immunoblot.

Article Snippet: The cell lysate was then spun at 18,000 g for 10 minutes to remove the beads, and the supernatant was incubated overnight with 1 µl anti-HER2 antibody 29D8 (Cell Signaling).

Techniques: Expressing, Immunoprecipitation, Western Blot, Incubation

Journal: Breast Cancer Research : BCR

Article Title: Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells

doi: 10.1186/bcr3204

Figure Lengend Snippet: Reduced signaling by HER2 following prolonged vaccine-induced anti-HER2 antibody treatment . SK-BR-3 cells were stimulated for (A) 24 hours and (B) 72 hours with LacZ-VIA (A, lanes 2 to 4; B, lane 2), HER2-VIA (A, lanes 5 to 7; B, lane 3), and trastuzumab (A, lane 8 and 9; B, lane 4). Protein samples were immunoblotted with indicated antibodies. β-actin in the lower panel serves as a loading control. n = 3. ERK, extracellular signal-regulated protein kinase; IB, immunoblot; NS, non-stimulated.

Article Snippet: The cell lysate was then spun at 18,000 g for 10 minutes to remove the beads, and the supernatant was incubated overnight with 1 µl anti-HER2 antibody 29D8 (Cell Signaling).

Techniques: Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells

doi: 10.1186/bcr3204

Figure Lengend Snippet: Reduced signaling of HER2 following patient's serum treatment . (A) Imaging of endogenous HER2 internalization by the human HER2-specific antibodies and vaccine-induced anti-HER2 antibodies (HER2-VIA). All human serum samples were purified to deplete lapatinib prior to using in the internalization assay. SK-BR-3 cells were allowed to grow for 24 hours and were treated for 1 hour with different murine or human antibodies as indicated above each image. Following treatment, the cells were fixed and stained to visualize the cellular distribution of HER2 using HER2 antibody 29D8 as described in Materials and methods. Confocal images of cells that (a) were left untreated, or were treated with (b) control GFP-VIA, (c) HER2-VIA, (d, e) serum (week 0 and week 10) from Patient 8, (f, g) serum (week 0 and week 12) from Patient 2, and (h, i) serum (week 0 and week 12) from Patient 4. (B) Effect of human HER2-specific antibodies on Her2 tyrosine 877 phosphorylation. SK-BR-3 cells were stimulated for 9 hours with indicated sera from either patients or mice. Protein samples were immunoblotted with anti-Her2PY877 antibodies (upper panel), anti-Her2 antibodies (middle panel), or anti-β-actin antibodies as a loading control (lower panel). n = 3. IB, immunoblot; NS, non-stimulated.

Article Snippet: The cell lysate was then spun at 18,000 g for 10 minutes to remove the beads, and the supernatant was incubated overnight with 1 µl anti-HER2 antibody 29D8 (Cell Signaling).

Techniques: Imaging, Purification, Staining, Western Blot

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Genetic characteristics of a case of colorectal cancer harboring HER2 G776S mutation detected by clinical NGS. ( A ) Summary of the patient's clinical profile. ( B ) Comprehensive genomic analysis report of the cancer of the patient. Positive biomarkers are shown. ( C ) Sanger sequencing of a part of HER2 gene of the cancer genome DNA. The amino acid (a.a.) sequence from 774 to 778 is shown. The sequence of the corresponding wild-type (WT) site is also presented. The red arrow indicates the mutation position of G776S. ( D ) The mutation position of G776 in HER2 protein domains based on the COSMIC database. The nucleotide sequences of the WT, G776S and G776 > VC are described. The underlined part indicates a substitution mutation and the yellow background site indicates a short insertion. Recep_L, receptor L domain; Furin-like, furin-like cysteine-rich region; GF_recep_IV, growth factor receptor domain IV. In the lower part of the figure, the number of reports in the COSMIC database and annotations from OnkoKB ( https://www.oncokb.org/ ) are shown. The data are current as of April 2021.

Article Snippet: Recombinant HER2 WT and mutant proteins in the lysate were purified by immunoprecipitation using Protein A Dynabeads (Thermo Fisher Scientific) and added rabbit monoclonal anti-HER2 (29D8) antibody (Cell Signaling Technology, Danvers, MA) following the manufacturer’s instructions.

Techniques: Mutagenesis, Sequencing

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Effects of HER2 mutations on kinase activity and phosphorylation, and their functional evaluation in classical cell-based transfection assays. ( A ) Western blot results showing recombinant proteins of HER2 WT and mutants (G776S and G776 > VC). HeLa cells were transiently transfected with the HER2 expression vectors, and 48 h after the transfection, the cells were lysed and purified by immunoprecipitation using HER2 antibody. ( B ) Measurement of the kinase activity of 100 ng of recombinant HER2 protein using the kinase assay. Kinase activity was measured in triplicate, and the data were standardized to the mean HER2 WT activity. One-way ANOVA: P < 0.01, * P < 0.05, ** P < 0.01. ( C ) Western blot results for the expression and phosphorylation of HER2 and EGFR in HeLa cells transfected with the HER2 expression vectors. β-Actin served as an internal control. The bar graph indicates the normalized ratios of densitometric values of phosphorylated to total HER2 protein. ( D ) Focus formation assays using NIH/3T3 cells stably transfected with the indicated plasmids. ( E ) Ba/F3 transformation assays using Ba/F3 (interleukin 3-dependent cells) stably transfected with the indicated plasmids. The bar graph shows the fold changes in number of viable cells. ns, not significant. One-way ANOVA: P < 0.01, ** P < 0.01 ( n = 3).

Article Snippet: Recombinant HER2 WT and mutant proteins in the lysate were purified by immunoprecipitation using Protein A Dynabeads (Thermo Fisher Scientific) and added rabbit monoclonal anti-HER2 (29D8) antibody (Cell Signaling Technology, Danvers, MA) following the manufacturer’s instructions.

Techniques: Activity Assay, Functional Assay, Transfection, Western Blot, Recombinant, Expressing, Purification, Immunoprecipitation, Kinase Assay, Stable Transfection, Transformation Assay

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Effects of HER2 G776S mutation on the HER2 signaling pathway and anchorage-independent growth in several cell lines. ( A ) Detection of APC proteins in each cell by Western blotting. Full-length APC was detected with APC antibody (#2504, Cell Signaling Technology), and truncated APC was detected with APC antibody (sc-9998, Santa Cruz Biotechnology) raised against the N-terminus of APC. ( B ) The activity of the Wnt/β-catenin signaling pathway measured using the TCF/LEF luciferase reporter assay. The assay was performed in triplicate and the results were standardized to the mean of the activity of HeLa cells. ( C ) Phosphorylation and expression of HER2 and the downstream signaling in WT HER2 - or HER2 G776S-transfected HeLa and colon cells (FHC, CACO-2 and COLO-320). ( D ) Soft agar colony-forming assays showing the effects of stable transfection with HER2 WT or HER2 G776S in FHC (WT APC ) and COLO-320 (mutant APC ) cells. The cells were seeded into six-well plates in triplicate, and the number of colonies per well was counted. ns, not significant. ** P < 0.01 HER2 WT vs HER2 G776S.

Article Snippet: Recombinant HER2 WT and mutant proteins in the lysate were purified by immunoprecipitation using Protein A Dynabeads (Thermo Fisher Scientific) and added rabbit monoclonal anti-HER2 (29D8) antibody (Cell Signaling Technology, Danvers, MA) following the manufacturer’s instructions.

Techniques: Mutagenesis, Western Blot, Activity Assay, Luciferase, Reporter Assay, Expressing, Transfection, Stable Transfection

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Effects of APC KO on HER signaling in HeLa cells (with WT APC ). ( A ) Confirmation of APC KO and its effect on the HER2–ERK pathway in APC -KO HeLa cells using Western blotting. β-Actin served as a loading control. ( B ) Activity of the Wnt/β-catenin signaling pathway measured using the TCF/LEF luciferase reporter assay. Nontargeting control cells (NTC) incubated with Wnt3a (100 ng/ml) for 24 h were used as a positive control (rightmost bar). The data were standardized to the mean of the activity of NTC cells. One-way ANOVA: P < 0.01, * P < 0.05, ** P < 0.01 vs NTC cells ( n = 3). ( C ) Measurement of activated RAS (RAS–GTP) using the G-LISA assay. NTC plus Wnt3a (100 ng/ml) was used as a control (rightmost bar). The data were standardized to the mean of the activity of NTC cells. One-way ANOVA: P < 0.05, * P < 0.05 vs NTC cells ( n = 3). ( D ) Western blot results showing the effects of HER2 WT or HER2 G776S transfection on the HER2 signaling pathway in APC -KO cells. APC -KO cells or NTC cells were transfected with mock or HER2 expression vectors, and the experiments were performed 48 h after transfection. β-Actin served as a loading control. ( E ) Measurement of RAS-GTP using the G-LISA assay performed 48 h after HER2 expression vector transfection. The data were standardized to the mean of the activity of NTC cells transfected with mock vectors. Two-way ANOVA: interaction P < 0.01, * P < 0.05, ** P < 0.01 ( n = 3). ( F ) Colony-forming assay results showing the effects of stable transfection with HER2 WT or HER2 G776S in APC -KO cells. The number of colonies was counted in six random low-power fields. Scale bar, 200 μm. Two-way ANOVA: interaction P < 0.01, ** P < 0.01.

Article Snippet: Recombinant HER2 WT and mutant proteins in the lysate were purified by immunoprecipitation using Protein A Dynabeads (Thermo Fisher Scientific) and added rabbit monoclonal anti-HER2 (29D8) antibody (Cell Signaling Technology, Danvers, MA) following the manufacturer’s instructions.

Techniques: Western Blot, Activity Assay, Luciferase, Reporter Assay, Incubation, Positive Control, Transfection, Expressing, Plasmid Preparation, Stable Transfection

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Effects of WT APC overexpression on the HER2 signaling pathway in COLO-320 cells (with mutant APC ). ( A ) Confirmation of APC overexpression in cells transfected with pCMV_APC vector using Western blotting. ( B ) Activity of the Wnt/β-catenin pathway in COLO-320 cells transfected with pCMV_APC measured using the TCF/LEF luciferase reporter assay. The reporter vectors were cotransfected into COLO-320 cells along with the pCMV empty vector (mock) or pCMV-APC. The data were standardized to the mean of the activity of mock cells. ** P < 0.01 vs mock ( n = 3). ( C ) Measurement of activated RAS using the G-LISA assay performed 48 h after the transfection with pCMV. The data were standardized to the mean of the activity of cells transfected with mock vectors. * P < 0 .05 vs mock ( n = 3) ( D ) Western blot results showing the effects of APC overexpression on the HER2 signaling pathway in COLO-320 cells. The cells were cotransfected with pcDNA_HER2-WT/G776S vectors and/or pCMV_APC vectors. Western blotting was performed 48 h after transfection. β-Actin served as a loading control. ( E ) Measurement of activated RAS using the G-LISA assay performed 48 h after transfection. The data were standardized to the mean of the activity of cells transfected with mock vectors. Two-way ANOVA: interaction P < 0.01, ** P < 0.01 ( n = 3). ( F ) Activity of the Wnt/β-catenin pathway in COLO-320 cells measured 24 h after addition of ICG-001 at different concentrations. The assay was performed in triplicate. ( G ) Measurement of activated RAS using the G-LISA assay in COLO-320 cells 24 h after addition of ICG-001 at different concentrations. The assay was performed in triplicate.

Article Snippet: Recombinant HER2 WT and mutant proteins in the lysate were purified by immunoprecipitation using Protein A Dynabeads (Thermo Fisher Scientific) and added rabbit monoclonal anti-HER2 (29D8) antibody (Cell Signaling Technology, Danvers, MA) following the manufacturer’s instructions.

Techniques: Over Expression, Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Activity Assay, Luciferase, Reporter Assay

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Efficacy of afatinib treatment in COLO-320 cells stably transfected with HER2 G776S. ( A ) Western blot results show the effects of gefitinib (1 μM) or afatinib (0.1 μM) treatment in COLO-320 cells transfected with WT HER2 or HER2 G776S. One day after seeding, the cells were treated for 24 h. ( B ) Cell proliferation assay (WST-1 assay) results show the effects of afatinib treatment in COLO-320 cells transfected with HER2 expression vectors. The cells were exposed to afatinib at the indicated concentrations for 24 h. ( C ) Number of colonies per well in the colony-forming assay treated with DMSO, gefitinib (1 μM) or afatinib (0.1 μM). Cells were seeded into six-well plates in triplicate and treated for 10 days. The number of colonies was counted in six random low-power fields. ns, not significant. ** P < 0.01 vs DMSO treatment. ( D ) The efficacy of afatinib treatment in HER2 WT or HER2 G776S stably expressing COLO-320 xenograft tumors. Xenograft tumors were treated with DMSO or afatinib (25 mg/kg/day p.o.). Two-way ANOVA: interaction P < 0.01, ns, not significant. * P < 0.05 ( n = 6). ( E ) Images of hematoxylin and eosin staining of xenograft tumor tissues in each treatment group and images of immunohistochemical staining for Ki-67 on day 18. Scale bar, 100 μm. ( F ) Rates of Ki-67-positively stained cells observed in six random fields. ns, not significant. Two-way ANOVA: interaction P < 0.01, * P < 0.05, ** P < 0.01.

Article Snippet: Recombinant HER2 WT and mutant proteins in the lysate were purified by immunoprecipitation using Protein A Dynabeads (Thermo Fisher Scientific) and added rabbit monoclonal anti-HER2 (29D8) antibody (Cell Signaling Technology, Danvers, MA) following the manufacturer’s instructions.

Techniques: Stable Transfection, Transfection, Western Blot, Proliferation Assay, WST-1 Assay, Expressing, Staining, Immunohistochemical staining

Journal: Scientific Reports

Article Title: HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

doi: 10.1038/s41598-022-13189-y

Figure Lengend Snippet: Schema of the relationship between HER2 mutations and APC mutations in colorectal cancer cells. HER2 G776S is a mutation that increases HER2 kinase activity, although the activity is weak. APC loss-of-function increases Wnt/β-catenin pathway activation but also increases the amount of RAS-GTP, which helps the HER2 G776S mutation to activate the HER2-ERK pathway.

Article Snippet: Recombinant HER2 WT and mutant proteins in the lysate were purified by immunoprecipitation using Protein A Dynabeads (Thermo Fisher Scientific) and added rabbit monoclonal anti-HER2 (29D8) antibody (Cell Signaling Technology, Danvers, MA) following the manufacturer’s instructions.

Techniques: Mutagenesis, Activity Assay, Activation Assay

Journal: Journal of the American Chemical Society

Article Title: Hairpin-like siRNA-Based Spherical Nucleic Acids

doi: 10.1021/jacs.1c12750

Figure Lengend Snippet: Hairpin-like RNAs self-hybridize to function as active siRNA duplexes. (A) Chemical structure of hairpin-like siRNA with passenger strand (light blue) and guide strand (dark blue). The sequence shown is used for targeting HER2 mRNA. (B) Scheme showing hairpin-like RNA conformations and predicted native PAGE band locations. (C) Native PAGE gel of hairpin-like siRNA, noncomplementary RNA, and cocomplementary RNA. (D) Gene silencing activity of hybridized and hairpin-like siRNA. SK-OV-3 cells were transfected with 100 nM siRNA. Relative (rel.) gene expression (exp.) was measured 48 h after siRNA administration by RT-qPCR, normalized to transfection agent-only treatment. Error bars are standard deviation (SD) of 3 experimental replicates (***P ≤ 0.001).

Article Snippet: The cells were then incubated with HER2 antibody (29D8) diluted 1:200 in Intercept blocking buffer (Cell Signaling) for 2 h. The wells were washed with 0.1% Tween-20 in 1× DPBS 3 times and incubated with 2 μ g/mL IRDye 800CW goat anti-rabbit secondary antibody (LI-COR) and 500 nM CellTag 700 (LI-COR) diluted 1:500 in Intercept blocking buffer for 1 h protected from light, with shaking.

Techniques: Sequencing, Clear Native PAGE, Activity Assay, Transfection, Expressing, Quantitative RT-PCR, Standard Deviation

Journal: Journal of the American Chemical Society

Article Title: Hairpin-like siRNA-Based Spherical Nucleic Acids

doi: 10.1021/jacs.1c12750

Figure Lengend Snippet: Hairpin-like design increases gene silencing durability of siRNA-SNAs. (A) Full gene silencing activity of HER2-targeting siRNA-SNAs. SK-OV-3 cells were transfected with 100 nM siRNA equivalent SNAs. Relative gene expression was measured 48 h after SNA administration by RT-qPCR, normalized to transfection agent-only treatment. Error bars are SD of 3 experimental replicates. (B) Knockdown potency of HER2-targeting siRNA-SNAs 2 days after SNA administration. SK-OV-3 cells were transfected with a range of SNA concentrations. Protein expression was measured using an in-cell Western assay, normalized to transfection agent-only treatment. Dotted lines are 95% CI. Error bars are SD of 3 biological replicates. (C) Knockdown potency of HER2-targeting siRNA-SNAs 4 weeks after SNA administration. Dotted lines are 95% CI. Error bars are SD of 3 biological replicates. (D) IC50 of HER2-targeting siRNA-SNAs at 2 days and 4 weeks after SNA administration, based on protein expression curves in (B) and (C). Error bars are SD of 3 biological replicates (ns, P > 0.05, *P ≤ 0.05).

Article Snippet: The cells were then incubated with HER2 antibody (29D8) diluted 1:200 in Intercept blocking buffer (Cell Signaling) for 2 h. The wells were washed with 0.1% Tween-20 in 1× DPBS 3 times and incubated with 2 μ g/mL IRDye 800CW goat anti-rabbit secondary antibody (LI-COR) and 500 nM CellTag 700 (LI-COR) diluted 1:500 in Intercept blocking buffer for 1 h protected from light, with shaking.

Techniques: Activity Assay, Transfection, Expressing, Quantitative RT-PCR, In-Cell ELISA