Journal: Nature Communications

Article Title: IGFBP5 is an ROR1 ligand promoting glioblastoma invasion via ROR1/HER2-CREB signaling axis

doi: 10.1038/s41467-023-37306-1

Figure Lengend Snippet: a Human phospho-RTK array of 83 GSCs treated with 100 ng/ml recombinant IGFBP5 (rIGFBP5) or vehicle control for 6 h. b Human phospho-kinase array of 83 GSCs treated with 100 ng/ml rIGFBP5 or vehicle control for 6 h. c–e Immunoblot (IB) analysis of pROR1, pHER2, pCREB, ROR1, HER2, and CREB in c non-invasive GSCs (83 and 131) treated with rIGFBP5 (100 ng/ml) or vehicle control for 6 h, d 83 and 131 cells infected with vector control or IGFBP5 lentivirus and e 448 and X01 GSCs infected with shIGFBP5 or shCtrl lentivirus. β-actin was used as a loading control. f, g Co-IP of X01 cells with antibodies targeting HER2, ROR1 or normal IgG. h Co-IP analysis for the interaction of IGFBP5 and ROR1 in 293 T cells transfected with Flag-tagged IGFBP5 and HA-tagged ROR1. Cell lysates were precipitated with anti-Flag antibody. i Co-IP analysis for the interaction of IGFBP5 and ROR1 in 293 T cells transfected with HA-tagged ROR1 and Flag-tagged IGFBP5. Cell lysates were precipitated with anti-HA antibody. j In vitro binding affinity between IGFBP5 and ROR1 tested by MST assay. The concentration of IGFBP5 proteins is kept constant at 50 nM, while the ROR1 concentration varies from 1.45 µM to 0.04 nM. The binding curve yields a Kd of 157.5 nM. Inset, thermophoretic movement of fluorescently labeled proteins. Fnorm = F 1 /F 0 (Fnorm: normalized fluorescence; F 1 : fluorescence after thermodiffusion; F 0 : initial fluorescence or fluorescence after T-jump). Kd, dissociation constant. k IB analysis of pROR1, pHER2, pCREB, ROR1, HER2, and CREB in X01 GSCs infected with shCtrl, shHER2-1, or shHER2-2 lentivirus. GAPDH was used as a loading control. l IB analysis of pROR1, pHER2, pCREB, ROR1, HER2, and CREB in X01 GSC infected with shCtrl, shROR1-1, or shROR1-2 lentivirus. GAPDH was used as a loading control. m IHC analysis of pHER2, pROR1, and pCREB in orthotopic xenografts of X01 GSCs infected with shCtrl, shIGFBP5-1, or shIGFBP5-2 lentivirus. Scale bar, 100 μm. n IHC analysis of pHER2, pROR1, and pCREB in orthotopic xenografts of 83 GSCs infected with vector control or IGFBP5 lentivirus. Scale bar, 100 μm. All the immunoblots were representative data from three independent experiments. Source data are provided as the Source Data file.

Article Snippet: The membranes were incubated with primary antibodies against IGFBP5 (1:200, Santa Cruz), HER2 (1:500, Cell Signaling Technology), pHER2 Y1248 (1:500, R&D System), ROR1 (1:500, Cell Signaling Technology), pROR1 Tyr786 (1:500, Thermo Fisher), CREB (48H2) (1:500, Cell Signaling Technology), pCREB Ser133 (1:500, Cell Signaling Technology), IGF1R (1:1000, Sangon Biotech), pIGF1R Tyr1165/1166 (1:1000, Sangon Biotech), GAPDH (1:1000, Cell Signaling Technology), and β-actin (1:1000, Santa Cruz) overnight at 4 °C.

Techniques: Recombinant, Western Blot, Infection, Plasmid Preparation, Co-Immunoprecipitation Assay, Transfection, In Vitro, Binding Assay, Concentration Assay, Labeling, Fluorescence

Journal: Nature Communications

Article Title: IGFBP5 is an ROR1 ligand promoting glioblastoma invasion via ROR1/HER2-CREB signaling axis

doi: 10.1038/s41467-023-37306-1

Figure Lengend Snippet: a Schematic illustration of Ang-SS-Cas9/sgRNA nanocapsule preparation. b Size distribution of Ang-SS-Cas9/sgRNA nanocapsules. c TEM images of Ang-SS-Cas9/sgRNA nanocapsules treated with or without 10 mM GSH at pH 7.4 for 12 h. Scale bar, 50 nm. d, e Indel detection by T7 endonuclease I (T7EI) of X01 and 448 GSCs treated by Ang-SS-Cas9/sgIGFBP5. T7EI cleavage assays were representative data from three independent experiments. f, g Invasion assays with X01 GSCs treated with Ang-SS-Cas9/sgIGFBP5 and Ang-SS-Cas9/sgScramble. f Images taken after 48 h of invasion are representative of three independent experiments (scale bar, 100 μm; n = 3), and g the graph shows the mean number of invasive cells ± SEM, two-tailed Student’s t -test ( *** P = 0.0001). h, i Invasion assays with 448 GSCs treated with Ang-SS-Cas9/sgIGFBP5 and Ang-SS-Cas9/sgScramble. h Images taken after 48 h of invasion are representative of three independent experiments (scale bar, 100 μm; n = 3), and i the graph shows the mean number of invasive cells ± SEM, two-tailed Student’s t -test ( *** P = 0.0004). j Luminescence images of orthotopic X01-Luc human glioblastoma tumor-bearing nude mice following treatment with Ang-SS-Cas9/sgIGFBP5, Ang-SS-Cas9/sgScramble or PBS. Mice were intravenously injected at a dose of 1.5 mg Cas9 equiv./kg on day 10, 12, 14, 16, and 18 post tumor implantations. k IB analysis of IGFBP5, pROR1, pHER2, pCREB, ROR1, HER2 and CREB in tumor tissues taken from mice treated with Ang-SS-Cas9/sgIGFBP5, Ang-SS-Cas9/sgScramble or PBS. β-actin was used as the loading control. The immunoblots were representative data from three independent experiments. l Body weight changes in mice following treatment with Ang-SS-Cas9/sgIGFBP5, Ang-SS-Cas9/sgScramble or PBS. Data are presented as mean ± SEM ( n = 6 in each group), one-way ANOVA (day 18, *** P = 0.0006) and two-tailed Student’s t -test (day 20 sgIGFBP5 vs sgScramble, **** P = 0.00008). m Kaplan–Meier survival curves of mice implanted with 1 × 10 5 X01-Luc GSCs and treated with Ang-SS-Cas9/sgIGFBP5, Ang-SS-Cas9/sgScramble or PBS ( n = 6 in each group). PBS vs sgIGFBP5, P = 0.0006, sgScramble vs sgIGFBP5, P = 0.001, log-rank test. Source data are provided as the Source Data file.

Article Snippet: The membranes were incubated with primary antibodies against IGFBP5 (1:200, Santa Cruz), HER2 (1:500, Cell Signaling Technology), pHER2 Y1248 (1:500, R&D System), ROR1 (1:500, Cell Signaling Technology), pROR1 Tyr786 (1:500, Thermo Fisher), CREB (48H2) (1:500, Cell Signaling Technology), pCREB Ser133 (1:500, Cell Signaling Technology), IGF1R (1:1000, Sangon Biotech), pIGF1R Tyr1165/1166 (1:1000, Sangon Biotech), GAPDH (1:1000, Cell Signaling Technology), and β-actin (1:1000, Santa Cruz) overnight at 4 °C.

Techniques: Two Tailed Test, Injection, Western Blot

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: 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: HER2 protein detection on EVs was performed by staining samples with HER2/ErbB2 (29D8) – PE-conjugated antibody or Isotype Control (Cell Signaling Technology) and CellMask plasma membrane stains (C10046, Life Technologies).

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: HER2 protein detection on EVs was performed by staining samples with HER2/ErbB2 (29D8) – PE-conjugated antibody or Isotype Control (Cell Signaling Technology) and CellMask plasma membrane stains (C10046, Life Technologies).

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: HER2 protein detection on EVs was performed by staining samples with HER2/ErbB2 (29D8) – PE-conjugated antibody or Isotype Control (Cell Signaling Technology) and CellMask plasma membrane stains (C10046, Life Technologies).

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: HER2 protein detection on EVs was performed by staining samples with HER2/ErbB2 (29D8) – PE-conjugated antibody or Isotype Control (Cell Signaling Technology) and CellMask plasma membrane stains (C10046, Life Technologies).

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

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: Primary antibodies used were rabbit anti -CD9 (#D801A; Cell signaling Technology; 1/500); rabbit anti – CD 9 (# MA5-33125; Invitrogen; 1/500); mouse anti – Apolipoprotein A1 (#5F4; Cell Signaling Technology; 1/250); rabbit anti– Albumin (#4929; Cell Signaling Technology; 1/1000), mouse anti-Tsg-101 (ab83, Abcam, 1/1000), rabbit anti-Flotillin 1 (#18634, Cell Signaling Technology; 1/1000), rabbit anti-Flotillin 2 (#3436, Cell Signaling Technology; 1/1000), rabbit anti-CD44 (ab157107, Abcam, 1/500), rabbit anti-EpCam (#D1B3, Cell Signaling Technology; 1/500), rabbit anti-HER2/ErbB2 (#29D8, Cell Signaling Technology; 1/1000).

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: Primary antibodies used were rabbit anti -CD9 (#D801A; Cell signaling Technology; 1/500); rabbit anti – CD 9 (# MA5-33125; Invitrogen; 1/500); mouse anti – Apolipoprotein A1 (#5F4; Cell Signaling Technology; 1/250); rabbit anti– Albumin (#4929; Cell Signaling Technology; 1/1000), mouse anti-Tsg-101 (ab83, Abcam, 1/1000), rabbit anti-Flotillin 1 (#18634, Cell Signaling Technology; 1/1000), rabbit anti-Flotillin 2 (#3436, Cell Signaling Technology; 1/1000), rabbit anti-CD44 (ab157107, Abcam, 1/500), rabbit anti-EpCam (#D1B3, Cell Signaling Technology; 1/500), rabbit anti-HER2/ErbB2 (#29D8, Cell Signaling Technology; 1/1000).

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: Primary antibodies used were rabbit anti -CD9 (#D801A; Cell signaling Technology; 1/500); rabbit anti – CD 9 (# MA5-33125; Invitrogen; 1/500); mouse anti – Apolipoprotein A1 (#5F4; Cell Signaling Technology; 1/250); rabbit anti– Albumin (#4929; Cell Signaling Technology; 1/1000), mouse anti-Tsg-101 (ab83, Abcam, 1/1000), rabbit anti-Flotillin 1 (#18634, Cell Signaling Technology; 1/1000), rabbit anti-Flotillin 2 (#3436, Cell Signaling Technology; 1/1000), rabbit anti-CD44 (ab157107, Abcam, 1/500), rabbit anti-EpCam (#D1B3, Cell Signaling Technology; 1/500), rabbit anti-HER2/ErbB2 (#29D8, Cell Signaling Technology; 1/1000).

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: Primary antibodies used were rabbit anti -CD9 (#D801A; Cell signaling Technology; 1/500); rabbit anti – CD 9 (# MA5-33125; Invitrogen; 1/500); mouse anti – Apolipoprotein A1 (#5F4; Cell Signaling Technology; 1/250); rabbit anti– Albumin (#4929; Cell Signaling Technology; 1/1000), mouse anti-Tsg-101 (ab83, Abcam, 1/1000), rabbit anti-Flotillin 1 (#18634, Cell Signaling Technology; 1/1000), rabbit anti-Flotillin 2 (#3436, Cell Signaling Technology; 1/1000), rabbit anti-CD44 (ab157107, Abcam, 1/500), rabbit anti-EpCam (#D1B3, Cell Signaling Technology; 1/500), rabbit anti-HER2/ErbB2 (#29D8, Cell Signaling Technology; 1/1000).

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

Journal: Nature Communications

Article Title: Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis

doi: 10.1038/s41467-023-36496-y

Figure Lengend Snippet: a Schematic representation of anti-HER2 biparatopic antibody BS4. The Trastuzumab (Tz) single-chain variable fragment (scFv) is attached to the N terminus of the heavy chain of 39 S IgG1 resulting in four antigen binding sites per molecule. Mutations in the Fc region (*) reduce binding to Fc gamma receptor. b , c Surface binding and endocytosis of dylight650-labelled monotopic antibodies Tz and 39 S and biparatopic BS4 (all at 3 µg/ml) after 1 h of uptake by confocal microscopy ( b ) and over a period of 6 h by flow cytometry ( c ), (means ± SD, n = 3 independent experiments). d , e Endocytosis of BS4 is independent of clathrin and dynamin. SkBr3 cells transfected with dominant-negative AP180ct (for clathrin-mediated endocytosis) and DynaminS45N N-terminally GFP-tagged expression constructs were incubated with dylight650-labelled BS4 and AlexaFluor546-transferrin for indicated times over a period of 4 h and analysed by flow cytometry. Endocytosis of transferrin and BS4 in cells from the same well expressing or not dominant-negative constructs (AP180ct + AP180ct - , DynS45N + , DynS45N − in ( d ) and ( e )), (means ± SD, n = 4 independent experiments). f BS4 uptake requires actin polymerisation. SkBr3 cells endocytosing pHrodo-labelled BS4 were treated with 10 µM CytoD, which was subsequently washed out as indicated, (means ± SEM, n = 6 from three independent experiments, two replicate wells each). Scale bars: 10 µm. Source data are provided as a Source Data file.

Article Snippet: Antibodies recognising the HER2 cytoplasmic domain (2242), pHER2 (2241), HER3 (12708), the HA-tag (2367) and EGFR (4267) were from Cell Signaling Technology.

Techniques: Binding Assay, Confocal Microscopy, Flow Cytometry, Transfection, Dominant Negative Mutation, Expressing, Construct, Incubation

Journal: Nature Communications

Article Title: Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis

doi: 10.1038/s41467-023-36496-y

Figure Lengend Snippet: a , b BS4 stimulates fluid-phase uptake. SkBr3 cells were co-incubated with dextran (70 kDa)-TMR and BS4-dylight650 for 10 min and after fixation analysed by confocal microscopy ( a ). The TMR channel is false-colour-coded in green, and individual cells are outlined. b Quantification of dextran (70 kDa)-TMR endocytosis in absence (mock) and presence of BS4 (dots represent measurements from individual cells, red lines indicate the median; n ≥ 50 cells from three independent experiments, **** P < 0.0001, two-tailed unpaired Student’s t test). c BS4-positive endocytic carriers exhibit dextran-filled lumina. d Concentration of surface-bound BS4 by inwards moving lamellipodium wave. Cells expressing GPI membrane-bound GFP (green) were imaged immediately after addition of BS4-dylight650 (magenta) (see also Supplementary Movies and 4). 3D views ( x , y , z dimensions) are shown on the right. The arrows indicate the position of the plasma membrane edge at the 40 s timepoint. e Rac1 re-localises to BS4-induced HER2 cell-surface aggregates. SkBr3 cells were incubated with BS4 for 2.5 min, fixed, stained for surface HER2 (red) and endogenous RAC (green) and analysed by confocal microscopy. BS4 endocytosis depends on Rac1 GTPase activity ( f , g ). SkBr3 cells transfected with Rac1 wt or dominant-negative T17N C-terminally GFP-tagged expression constructs were incubated with dylight650-labelled BS4 for 10 min. Samples were fixed, surface-bound BS4 was counterstained and cell sections analysed by confocal microscopy ( f ). Subtraction of surface from total BS4 signal shows endocytosed pool (BS4 uptake), transfected cells are outlined. Results are quantified in panel g , dots representing measurements from individual cells, red lines indicate the median; n ≥ 50 cells from three independent experiments, ns (non-significant) P > 0.05, **** P < 0.0001; one-way ANOVA with Dunnett’s multiple comparison test. BS4 is internalised via macroendocytic cups ( h ). SkBr3 cells expressing GFP-GPI (green) were imaged after addition of BS4-dylight650 (magenta) (see also Supplementary Movie ). Maximum intensity projections are shown and 3D views with surface rendering of the plasma membrane (green) below each panel (see also Supplementary Movie ). The arrows indicate the concentration of BS4 in endocytic vesicles after internalisation. Scale bars: 10 µm ( a , e , f ), 1 µm ( c ), 5 µm ( d , h ). Source data are provided as a Source Data file.

Article Snippet: Antibodies recognising the HER2 cytoplasmic domain (2242), pHER2 (2241), HER3 (12708), the HA-tag (2367) and EGFR (4267) were from Cell Signaling Technology.

Techniques: Incubation, Confocal Microscopy, Two Tailed Test, Concentration Assay, Expressing, Staining, Activity Assay, Transfection, Dominant Negative Mutation, Construct

Journal: Nature Communications

Article Title: Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis

doi: 10.1038/s41467-023-36496-y

Figure Lengend Snippet: a BS4 clusters HER2 in the plasma membrane prior to endocytosis. SkBr3 cells were incubated with HER2-specific, dylight650-labelled biparatopic antibody BS4 or monotopic antibody 39 S for indicated times. After fixation, total HER2 in cell sections was stained using an antibody against the cytoplasmic domain and samples were analysed by confocal microscopy. b , c Aggregation of HER2 receptors by BS4 precedes endocytosis. After surface biotinylation, SkBr3 cells were incubated with BS4 for indicated times, surface remaining biotin was removed and samples lysed in low detergent buffer. A fraction was spun to separate soluble (Sup) from insoluble/aggregated proteins (Pellet). Protein in the remaining sample was solubilised (see “Methods” for protocol) and endocytosed biotinylated proteins concentrated on Streptavidin beads (uptake). Samples were assayed by immunoblot for the HER2 protein ( b ). c Time dependence of BS4-triggered aggregation and endocytosis of HER2 receptor is quantified (means ± SD, n = 3 independent experiments). d BS4-triggered cross-linking and endocytosis of HER2 is abrogated for mutant HER2 lacking the Tz-binding site. CHO cells expressing full-length HER2 receptor, either wt or lacking the Tz-binding site, were surface biotinylated and incubated with BS4 for 10 min. Samples were analysed as described in ( b ). e , f Cross-linking of both monotopic antibodies phenocopies the effect of BS4 on HER2 aggregation and endocytosis. Cells were incubated with equal amounts of indicated antibodies, with or without a cross-linking anti-human Alexa488 antibody (2nd) for 1 h. After fixation surface-bound, dylight650-labelled antibody was quenched and cell sections analysed by confocal microscopy ( e ) or antibody uptake quantified by flow cytometry ( f ) (means ± SD, n = 3 independent experiments, ns (non-significant) P > 0.05, ** P = 0.0025, **** P < 0.0001, two-way ANOVA with Sidak’s multiple comparison test). Scale bars: 10 µm. Source data are provided as a Source Data file.

Article Snippet: Antibodies recognising the HER2 cytoplasmic domain (2242), pHER2 (2241), HER3 (12708), the HA-tag (2367) and EGFR (4267) were from Cell Signaling Technology.

Techniques: Incubation, Staining, Confocal Microscopy, Western Blot, Mutagenesis, Binding Assay, Expressing, Flow Cytometry

Journal: Nature Communications

Article Title: Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis

doi: 10.1038/s41467-023-36496-y

Figure Lengend Snippet: a WGA aggregates and induces endocytosis of glycosylated proteins in cells. After surface biotinylation, SkBr3 cells ± CytochalasinD (CytoD) were incubated with WGA for 10 min, and remaining surface biotin was removed. Samples were harvested after processing, as described in the “Methods” section, assayed by immunoblot for receptor tyrosine-protein kinase erbB-2 (HER2), epidermal growth factor receptor (EGFR), sodium/potassium-transporting ATPase Alpha1 (Na/K-ATPase) and transferrin-receptor 1 (TfR). Numbers indicate relative levels of uptake normalised to the control sample. b WGA uptake is mediated by both clathrin/dynamin-dependent and actin-dependent endocytic pathways. Cells transfected with dominant-negative DynaminS45N N-terminally GFP-tagged expression construct were incubated ± CytochalasinD (CytoD) with dylight650-labelled WGA and analysed by confocal microscopy. Scale bars: 10 µm. Source data are provided as a Source Data file.

Article Snippet: Antibodies recognising the HER2 cytoplasmic domain (2242), pHER2 (2241), HER3 (12708), the HA-tag (2367) and EGFR (4267) were from Cell Signaling Technology.

Techniques: Incubation, Western Blot, Transfection, Dominant Negative Mutation, Expressing, Construct, Confocal Microscopy

Journal: Nature Communications

Article Title: Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis

doi: 10.1038/s41467-023-36496-y

Figure Lengend Snippet: a , b Depletion of HER2 and EGFR surface receptor levels by treatment with BS4 and EGF, respectively. SkBr3 cells were incubated for 10 min with BS4 or EGF, and ± CytochalasinD (CytoD) followed by cell-surface biotinylation and concentration on Streptavidin beads (surface). Samples were analysed by immunoblot for HER2, EGFR and HER3, TfR as negative controls ( a ). A quantitation of the results is shown in ( b ) means ± SD, n = 4 independent experiments, ns (non-significant) P > 0.05, ** P = 0.0051, *** P = 0.0009, *** P < 0.0001; two-way ANOVA with Sidak’s multiple comparison test. c , d BS4 clusters HER2 but no other receptors in the plasma membrane resulting in HER2-specific endocytosis. SkBr3 cells were incubated with HER2-specific, dylight650-labelled biparatopic antibody BS4 for 2.5 min. After fixation, total receptor tyrosine-protein kinase ErbB-2 (HER2), receptor tyrosine-protein kinase ErbB-3 (HER3), epidermal growth factor receptor (EGFR), sodium/potassium-transporting ATPase Alpha1 (Na/K-ATPase) and Transferrin-receptor 1 (TfR) in cell sections were stained and samples were analysed by confocal microscopy. The spot overlap of fluorescent signal for BS4 and respective receptors is quantified in ( d ); means ± SD, n = 6 randomly chosen fields of view with a total of at least 50 cells per condition, *** P < 0.001, one-way ANOVA with Dunnett’s multiple comparison test. e Specific aggregation of HER2 receptor by BS4. SkBr3 cells were incubated with BS4 for indicated times and samples lysed in the low detergent buffer. Samples were spun to separate soluble (Sup) from insoluble/aggregated proteins (Pellet) and immunoblotted for indicated cell-surface receptors. f BS4 specifically endocytoses HER2 receptor. After surface biotinylation, cells were incubated with BS4 for indicated times. Protein was fully solubilised (see “Methods” for protocol) and endocytosed biotinylated proteins concentrated on Streptavidin beads (uptake). Samples were assayed by immunoblot for the HER2, HER3, EGFR, Na/K-ATPase and TfR. Scale bars: 10 µm. Source data are provided as a Source Data file.

Article Snippet: Antibodies recognising the HER2 cytoplasmic domain (2242), pHER2 (2241), HER3 (12708), the HA-tag (2367) and EGFR (4267) were from Cell Signaling Technology.

Techniques: Incubation, Concentration Assay, Western Blot, Quantitation Assay, Staining, Confocal Microscopy

Journal: Nature Communications

Article Title: Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis

doi: 10.1038/s41467-023-36496-y

Figure Lengend Snippet: BS4 induces HER2 autophosphorylation ( a, b ). SkBr3 cells ± dual EGFR/HER2 kinase inhibitor lapatinib (HER1/2i) were incubated with BS4 for 2.5 min as indicated. Samples were analysed by immunoblot for total HER2 and phospho-HER2 (pHER2) ( a ). SkBr3 cells were incubated with BS4-dylight650 (red) for 2.5 min, after fixation stained for phosphorylated tyrosine residues (p-Y, green) and analysed by confocal microscopy ( b ). Localisation of PIP3 to BS4-induced HER2 aggregates ( c ). SkBr3 cells were transfected with a vector expressing the PIP3 sensor PH-AKT-GFP (green) for 16 h treated or not with the PI3K inhibitor LY294002 (PI3Ki) followed by incubation with BS4-dylight650 (red) for 2.5 min. After fixation samples were analysed by confocal microscopy. BS4 uptake is reduced by HER2 kinase but not PI3-kinase inhibition ( d ). SkBr3 cells were treated with CytoD, lapatinib (HER1/2i) or LY294002 (PI3Ki) as indicated and incubated with BS4-dylight650 for 30 min. After fixation BS4 uptake was quantified by flow cytometry; means ± SD, n = 6 independent experiments, ns (non-significant) P = 0.252, **** P < 0.0001; one-way ANOVA with Dunnett’s multiple comparison test. BS4-induced ADE is independent of Ras ( e , f ). SkBr3 cells were transfected with a plasmid expressing a dominant-negative mutant (S17N) of Ras for 16 h followed by incubation with BS4-dylight650 for 30 min. After fixation surface-bound BS4 was counterstained and samples analysed by confocal microscopy. Subtraction of surface from total antibody signal yielded the endocytosed pool ( e ) with transfected cell outlined. Results are quantified in ( f ); dots represent measurements from individual cells, red lines indicate the median; n ≥ 50 cells from three independent experiments, ns (non-significant) P = 0.532; two-tailed unpaired Student’s t test. Localisation of VAV2 to BS4-induced HER2 aggregates ( g ). SkBr3 cells were incubated with BS4-dylight650 (red) for 2.5 min. After fixation, samples were stained for endogenous VAV2 and analysed by confocal microscopy. Scale bars: 10 µm ( b , e , g ), 3 µm ( c ). Source data are provided as a Source Data file.

Article Snippet: Antibodies recognising the HER2 cytoplasmic domain (2242), pHER2 (2241), HER3 (12708), the HA-tag (2367) and EGFR (4267) were from Cell Signaling Technology.

Techniques: Incubation, Western Blot, Staining, Confocal Microscopy, Transfection, Plasmid Preparation, Expressing, Inhibition, Flow Cytometry, Dominant Negative Mutation, Two Tailed Test

Journal: Nature Communications

Article Title: Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis

doi: 10.1038/s41467-023-36496-y

Figure Lengend Snippet: BS4-induced interaction of HER2 with VAV2 is phosphotyrosine dependent ( a ). SkBr3 cells ± lapatinib (HER1/2i) or LY294002 (PI3Ki) were incubated with BS4 for 2.5 min. BS4-HER2 aggregates were immunoprecipitated and samples analysed by western blot. VAV2 is recruited to BS4-HER2 aggregates via its SH2 domain ( b ). SkBr3 cells were transfected with VAV2 wt or lacking residues 665–772 (ΔSH2) C-terminally GFP-tagged expression vector (green) for 16 h followed by incubation with BS4-dylight650 (red) for 10 min. After fixation samples were analysed by confocal microscopy. BS4-induced ADE requires VAV2 GEF activity ( c , d ). SkBr3 cells were transfected with VAV2 wt, comprising amino acids 546–878, or lacking catalytic residues (Δ341–347) for 16 h followed by incubation with BS4-dylight650 (red) for 30 min. After fixation, surface-bound BS4 antibody was counterstained and samples were analysed by confocal microscopy. Subtraction of surface from total BS4 signal shows an endocytosed pool (BS4 uptake), transfected cells are outlined. Results are quantified in ( d ). VAV proteins are required for BS4 uptake ( e , f ). SkBr3 wt or VAV1-3 knockout (KO) cells were incubated with BS4-dylight650 (red) for 30 min. After fixation, the surface-bound BS4 antibody was counterstained (green) and samples were analysed by confocal microscopy ( e ). Subtraction of surface from total BS4 signal shows endocytosed pool (BS4 uptake). Results are quantified in ( f ). Rescue of BS4 uptake in SkBr3 VAV1-3 knockout cells by expression of VAV1-3 ( g ). SkBr3 wt or VAV1-3 knockout (KO) cells were transfected with C-terminally HA-tagged VAV1-3 expression vectors for 16 h followed by incubation with BS4-dylight650 (red) for 30 min. Samples were processed, analysed and BS4 uptake quantified as described in ( e ). Rescue of BS4 uptake in SkBr3 VAV1-3 knockout cells depends on the SH2 domain in VAV2 ( h ). SkBr3 wt or VAV1-3 knockout (KO) cells were transfected with VAV2 wt or a lacking residue 665–772 (ΔSH2) C-terminally GFP-tagged expression vectors for 16 h followed by incubation with BS4-dylight650 for 30 min. Samples were processed, analysed and BS4 uptake quantified as described in ( e ). Quantifications in ( d , f , g , h ): dots represent measurements from individual cells, red lines indicate the median; n ≥ 50 cells from three independent experiments, ns (non-significant) P > 0.05, **** P < 0.0001; one-way ANOVA with Dunnett’s multiple comparison test. Scale bars: 10 µm ( b , c , e ). Source data are provided as a Source Data file.

Article Snippet: Antibodies recognising the HER2 cytoplasmic domain (2242), pHER2 (2241), HER3 (12708), the HA-tag (2367) and EGFR (4267) were from Cell Signaling Technology.

Techniques: Incubation, Immunoprecipitation, Western Blot, Transfection, Expressing, Plasmid Preparation, Confocal Microscopy, Activity Assay, Knock-Out

Journal: Nature Communications

Article Title: Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis

doi: 10.1038/s41467-023-36496-y

Figure Lengend Snippet: a HER2 receptor aggregation and endocytosis upon chemical and physical stress. After surface biotinylation, SkBr3 cells ± CytochalasinD (CytoD) were washed in an acidic buffer (pH 3.5), heat shocked for 5 min (at 50 °C), or treated with BS4. After incubation of all samples at 37 °C for 30 min, surface remaining biotin was removed and samples lysed in low detergent buffer. A fraction was spun to precipitate insoluble/aggregated proteins (Pellet). Protein in the remaining sample was solubilised (see “Methods” for protocol) and endocytosed biotinylated proteins concentrated on Streptavidin beads (uptake). Samples were assayed by immunoblot for HER2. b Stress-induced aggregation-dependent endocytosis occurs independent of dynamin but requires actin polymerisation. SkBr3 cells transfected with RFP control, or dominant-negative DynaminS45N N-terminally RFP tagged expression vector (RFP channel displayed in white) were heat shocked for 5 min (at 50 °C) or washed in an acidic buffer (pH 3.5) ± CytochalasinD (CytoD) followed by 30 min incubation at 37 °C. After fixation surface HER2 was stained (false-colour-coded in red) before permeabilisation and staining for total HER2 (green channel). Cell sections were analysed by confocal microscopy and post-processed by subtraction of surface from total HER2 staining, which allowed for specific visualisation of intracellular HER2. c , d Heat stress induces fluid-phase uptake. HeLa cells ± CytochalasinD (CytoD) or ± 5‐(N‐ethyl‐N‐isopropyl)amiloride (EIPA) were incubated with dextran (70 kDa)-fluorescein incubated for 5 min at 50 °C followed by 30 min at 37 °C. After fixation samples were analysed by confocal microscopy ( c ). Quantification of heat stress-induced dextran uptake is shown in ( d ). Dots represent measurements from individual cells, red lines indicate the median; n ≥ 50 cells from three independent experiments, **** P < 0.0001; one-way ANOVA with Sidaks’s multiple comparison test. Scale bars: 10 µm ( b , c ), 1 µm insets panel c . Source data are provided as a Source Data file.

Article Snippet: Antibodies recognising the HER2 cytoplasmic domain (2242), pHER2 (2241), HER3 (12708), the HA-tag (2367) and EGFR (4267) were from Cell Signaling Technology.

Techniques: Incubation, Western Blot, Transfection, Dominant Negative Mutation, Expressing, Plasmid Preparation, Staining, Confocal Microscopy

Journal: Nature Communications

Article Title: Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis

doi: 10.1038/s41467-023-36496-y

Figure Lengend Snippet: a Time-dependent degradation of endocytosed antibody-receptor aggregates. Western blot analysis of steady-state HER2 protein and antibody levels after exposure to monotopic antibodies Tz and 39 S as well as biparatopic antibody BS4 for increasing length of time. b , c Stress-induced aggregates are degraded in the lysosome after endocytosis. SkBr3 cells ± CytochalasinD (CytoD) or BafilomycinA1 (BafA1) were surface biotinylated, heat shocked for 5 min (at 50 °C) and subsequently incubated for the indicated length of time at 37 °C. Samples were lysed in a low detergent buffer, insoluble/aggregated proteins precipitated by centrifugation, biotinylated receptors isolated as described in materials and methods and analysed by immunoblot for HER2, EGFR and TfR. Total cell lysates analysed for HER2 and actin are shown as controls. c The amount of intracellular HER2 aggregates at different times points is quantified. Means ± SD, n = 3 independent experiments, ** P ≤ 0.0067, *** P = 0.0004; one-way ANOVA with Sidaks’s multiple comparison test. d Presence of extracellular receptor aggregates negatively affect cell growth. SkBr3 cells were incubated with monotopic antibody Tz or biparatopic antibody BS4 ± CytochalasinD (CytoD) for 4 h at 37 °C. Cell confluence was determined hourly using an Incucyte live-cell imager. Data normalised to the 4-h timepoint, at which antibodies and CytoD were removed, are displayed. The representative result of three independent experiments is shown. means ± SD, n = 8 replicate wells, significance shown for CytoD vs CytoD+BS4 (red) and CytoD vs CytoD+Tz (turquoise) 5–12 h time points ns P > 0.05, **** P < 0.0001; two-way ANOVA with Tukey’s multiple comparison test. Source data are provided as a Source Data file.

Article Snippet: Antibodies recognising the HER2 cytoplasmic domain (2242), pHER2 (2241), HER3 (12708), the HA-tag (2367) and EGFR (4267) were from Cell Signaling Technology.

Techniques: Western Blot, Incubation, Centrifugation, Isolation

Journal: bioRxiv

Article Title: Anticancer effect of zanubrutinib in HER2-positive breast cancer cell lines

doi: 10.1101/2023.02.23.529815

Figure Lengend Snippet: Heregulin rescues HER2-positive cells from G1 arrest induced by BTK inhibitors. Cells were treated for 24 hours with increasing concentrations of the selected compounds, with or without activation by heregulin (HRG, 0.1 μg/ml).

Article Snippet: The following specific antibodies were used and purchased from Cell Signaling: anti-EGFR (D38B1), anti-HER2/ErbB2 (D8F12), anti-HER3/ErbB3 (D22C5), anti-HER4/ErbB4 (111B2), anti-phospho-EGFR Y1068 (D7A5), anti-phospho-HER2/ErbB2 Y1221/1222 (6B12), anti-phospho-HER3/ErbB3 Y1289 (D1B5), anti-phospho-HER4/ErbB4 Y1284/EGFR Y1173 (21A9), anti-Akt (pan) (C67E7), anti-phospho-Akt S473 (D9E), anti-p44/42 MAPK (Erk1/2), anti-phospho-p44/42 MAPK (phospho-Erk1/2) T202/Y204), and anti-PARP (46D11).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Anticancer effect of zanubrutinib in HER2-positive breast cancer cell lines

doi: 10.1101/2023.02.23.529815

Figure Lengend Snippet: (A) Ibrutinib and zanubrutinib inhibit colony formation in HER2+ SKBR3 cells, while the proliferation rate of HER2-MCF7 cells remains unaffected. Colonies were stained with crystal violet after 10 days of treatment. The numbers indicate the percentages of colonies formed (calculated from the absorbance values). (B) Immunoblot analysis of lysates of cells treated with 1 μM zanubrutinib and ibrutinib. β-Actin served as a control for equal loading.

Article Snippet: The following specific antibodies were used and purchased from Cell Signaling: anti-EGFR (D38B1), anti-HER2/ErbB2 (D8F12), anti-HER3/ErbB3 (D22C5), anti-HER4/ErbB4 (111B2), anti-phospho-EGFR Y1068 (D7A5), anti-phospho-HER2/ErbB2 Y1221/1222 (6B12), anti-phospho-HER3/ErbB3 Y1289 (D1B5), anti-phospho-HER4/ErbB4 Y1284/EGFR Y1173 (21A9), anti-Akt (pan) (C67E7), anti-phospho-Akt S473 (D9E), anti-p44/42 MAPK (Erk1/2), anti-phospho-p44/42 MAPK (phospho-Erk1/2) T202/Y204), and anti-PARP (46D11).

Techniques: Staining, Western Blot

Journal: bioRxiv

Article Title: Anticancer effect of zanubrutinib in HER2-positive breast cancer cell lines

doi: 10.1101/2023.02.23.529815

Figure Lengend Snippet: Dose-dependent effect of zanubrutinib in HER2-positive BT474 and SKBR3 cell lines. Cells were treated with increasing concentrations of zanubrutinib for 16 hours. Cells were stimulated by heregulin (HRG, 0.1 μg/mL) 30 min prior to harvesting. β-Actin served as a control for equal loading.

Article Snippet: The following specific antibodies were used and purchased from Cell Signaling: anti-EGFR (D38B1), anti-HER2/ErbB2 (D8F12), anti-HER3/ErbB3 (D22C5), anti-HER4/ErbB4 (111B2), anti-phospho-EGFR Y1068 (D7A5), anti-phospho-HER2/ErbB2 Y1221/1222 (6B12), anti-phospho-HER3/ErbB3 Y1289 (D1B5), anti-phospho-HER4/ErbB4 Y1284/EGFR Y1173 (21A9), anti-Akt (pan) (C67E7), anti-phospho-Akt S473 (D9E), anti-p44/42 MAPK (Erk1/2), anti-phospho-p44/42 MAPK (phospho-Erk1/2) T202/Y204), and anti-PARP (46D11).

Techniques: