Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: Expression levels of ErbB2 and ErbB3 on cell lines

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: Affinity and binding kinetics of the anti-ErbB3 A5 scFv. k on and k off rates were determined by surface plasmon resonance and used to determine the binding affinity ( K D ) of the A5 scFv. ( A ) Sensorgram fit to 1 : 1 Langmuir binding model. ( B ) Analysis of data.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Binding Assay, SPR Assay

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: The anti-ErbB2/ErbB3 bs-scFv ALM. ( A ) Cartoon of ALM depicting scFv orientation, linker sequence and kinetic constants of ALM for each target antigen. ( B ) UV adsorption spectrum chromatograph of ALM over Superdex 75 size-exclusion column.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Sequencing, Adsorption

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: ALM selectively targets ErbB2/ErbB3 positive cells in vitro

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: In Vitro

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: The A5-linker-ML3.9 bs-scFv selectively binds BT-474 tumour cells in vitro . Non-labelled BT-474 (ErbB2‘+’/ErbB3‘+’) breast tumour cells were mixed with either an equal ( A and B ) or 18-fold excess ( C ) of fluorescently labelled MCF10a (ErbB2‘±’/ErbB3‘±’) normal breast epithelial cells. Cell mixtures were then incubated with buffer ( A ) or 100 n M ALM ( B and C ) and binding of ALM to each cell population was determined by flow cytometry with an anti-6XHis tag secondary antibody. MCF10a cells were sorted to the upper quadrants and the non-labelled BT-474 cells were sorted to the lower quadrants. Cells bound by the secondary antibody sorted to the respective right hand quadrants. Images on the left depict the raw flow cytometry data. Values on the right represent the absolute number and overall percentage of each cell type in the respective quadrants.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: In Vitro, Incubation, Binding Assay, Flow Cytometry

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: Bispecific binding is required for optimal tumour targeting of the ALM bs-scFv in vivo . The biodistributions of radioiodinated ALM, ALD and DLM bs-scFv were analysed 24 h post-injection into xenograft-bearing SCID mice ( n =5 per cohort). ( A ) Co-expression of ErbB2 and ErbB3 by the targeted tumour is required for optimal targeting of ALM in vivo . 125 I-ALM targeted ErbB2+/ErbB3+ tumour xenografts to ⩾3-fold higher levels than xenografts that express only one of the target antigens. ( B ) Radioiodinated ALM ( 125 I-ALM), which is capable of bivalent association with the surface of Sk-OV-3 tumour cells, exhibited increased targeting as compared with ALD and DLM that targeted the tumours monovalently. Error bars represent the standard error of the mean (s.e.m.).

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Binding Assay, In Vivo, Injection, Expressing

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: The A5-linker-ML3.9 bs-scFv has intrinsic anti-tumour cell activity. ( A ) Treatment of BT-474 and MDA-361/DYT2 cells with ALM inhibits colony formation in clonogenicity assays. Treatment of ( B ) BT-474 or ( C ) MDA-361/DYT2 cells with A5 scFv, ML3.9 scFv or the combination of both indicates that the majority of the intrinsic anti-tumour cell activity of ALM is due to the anti-ErbB3 A5 scFv arm. Colonies larger than 0.35 m M were counted using an automatic colony counter. Error bars represent the standard deviation.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Activity Assay, Standard Deviation

Journal: Science Advances

Article Title: Tracking extracellular vesicle phenotypic changes enables treatment monitoring in melanoma

doi: 10.1126/sciadv.aax3223

Figure Lengend Snippet: ( A ) A melanoma cell with a BRAF V600E mutation secretes EVs into circulation or cell culture medium. ( B ) The sample is directly injected into EPAC, where the applied nanomixing fluid flow increases EV collisions with the capture antibody and SERS nanotags and shears off nontarget molecules (e.g., protein aggregates and apoptotic bodies) and free SERS nanotags. ( C ) The characterization of EV phenotypes is performed by SERS mapping. The false-color SERS spectral images are established on the basis of the characteristic peak intensities of SERS nanotags (MCSP-MBA, red; MCAM-TFMBA, blue; ErbB3-DTNB, green; LNGFR-MPY, yellow). ( D ) EV phenotypes defined by the relative expression levels of four biomarkers are extracted from the average signal spectra of false-color SERS spectral images. EV phenotypes are unique to each EV subpopulation. By analyzing EV samples before, during, and after BRAF inhibitor treatment, the phenotypic evolution can be tracked to provide information on treatment responses and early signs of drug resistance.

Article Snippet: The collected cells were first labeled with either mouse anti-human MCSP (R&D Systems, MAB2585), MCAM (R&D Systems, MAB932), ErbB3 (R&D Systems, MAB3481), LNGFR (R&D Systems, MAB367) monoclonal antibodies, or isotype-matched control [normal mouse immunoglobulin G (IgG), sc-2025, Santa Cruz Biotechnology], followed by Alexa Fluor 488–labeled goat anti-mouse IgG (H+L) secondary antibodies.

Techniques: Mutagenesis, Cell Culture, Injection, Expressing

Journal: Science Advances

Article Title: Tracking extracellular vesicle phenotypic changes enables treatment monitoring in melanoma

doi: 10.1126/sciadv.aax3223

Figure Lengend Snippet: The specificity was studied using EV samples released from SK-MEL-28 and MCF7 cell lines, as well as control experiments including (++) EV-free cell culture medium, (−+) without the CD63 capture antibody, and (+−) with nontarget CD45 detection antibodies on SERS nanotags. ( A ) The expressions of MCSP, MCAM, ErbB3, and LNGFR in SK-MEL-28 and MCF7 cells were detected by flow cytometry. ( B ) Representative false-color SERS spectral images, ( C ) average SERS spectra obtained from corresponding SERS mapping datasets, and ( D ) average SERS intensities at 1075 cm −1 (red, MCSP), 1375 cm −1 (blue, MCAM), 1335 cm −1 (green, ErbB3), and 1000 cm −1 (yellow, LNGFR). Data in (D) are represented as means ± standard deviation, where error bars represent standard deviation of three separate experiments. Scale bars, 10 μm.

Article Snippet: The collected cells were first labeled with either mouse anti-human MCSP (R&D Systems, MAB2585), MCAM (R&D Systems, MAB932), ErbB3 (R&D Systems, MAB3481), LNGFR (R&D Systems, MAB367) monoclonal antibodies, or isotype-matched control [normal mouse immunoglobulin G (IgG), sc-2025, Santa Cruz Biotechnology], followed by Alexa Fluor 488–labeled goat anti-mouse IgG (H+L) secondary antibodies.

Techniques: Control, Cell Culture, Flow Cytometry, Standard Deviation

Journal: Science Advances

Article Title: Tracking extracellular vesicle phenotypic changes enables treatment monitoring in melanoma

doi: 10.1126/sciadv.aax3223

Figure Lengend Snippet: EVs released from ( A ) LM-MEL-64 cells without treatment were used as a control and followed for 30 days. EVs derived from ( B ) LM-MEL-33, ( C ) SK-MEL-28, ( D ) LM-MEL-64, and ( E ) LM-MEL-35 cell lines were collected before (day 0), during (days 3 to 30), and after treatment (days 33 and 39). (A to E) Average biomarker signals are represented by red (MCSP), blue (MCAM), green (ErbB3), and yellow (LNGFR). LM-MEL-35 cell line is BRAF wild type but NRAS mutant, and the other three cell lines are BRAF mutant. Data in (A) to (E) are represented as means ± standard deviation, where error bars represent standard deviation of three separate experiments. ( F to J ) Clustering of EV populations before, during, and after treatment via LDA of SERS signals.

Article Snippet: The collected cells were first labeled with either mouse anti-human MCSP (R&D Systems, MAB2585), MCAM (R&D Systems, MAB932), ErbB3 (R&D Systems, MAB3481), LNGFR (R&D Systems, MAB367) monoclonal antibodies, or isotype-matched control [normal mouse immunoglobulin G (IgG), sc-2025, Santa Cruz Biotechnology], followed by Alexa Fluor 488–labeled goat anti-mouse IgG (H+L) secondary antibodies.

Techniques: Control, Derivative Assay, Biomarker Discovery, Mutagenesis, Standard Deviation

Journal: Science Advances

Article Title: Tracking extracellular vesicle phenotypic changes enables treatment monitoring in melanoma

doi: 10.1126/sciadv.aax3223

Figure Lengend Snippet: ( A ) EV phenotypes of 15 melanoma samples (P1 to P15) and 12 healthy controls (H1 to H12). P1, P4, P7, and P9 are from the same patient but different time points, as are P5 and P10. ( B ) Representative false-color SERS spectral images and ( C ) corresponding average SERS spectra from patient and normal samples (P1, P8, and H1). For (A) and (B), the biomarker signals are represented by red (MCSP), blue (MCAM), green (ErbB3), and yellow (LNGFR). Data in (A) are represented as means ± standard deviation, where error bars represent standard deviation of three separate experiments. Scale bars, 10 μm.

Article Snippet: The collected cells were first labeled with either mouse anti-human MCSP (R&D Systems, MAB2585), MCAM (R&D Systems, MAB932), ErbB3 (R&D Systems, MAB3481), LNGFR (R&D Systems, MAB367) monoclonal antibodies, or isotype-matched control [normal mouse immunoglobulin G (IgG), sc-2025, Santa Cruz Biotechnology], followed by Alexa Fluor 488–labeled goat anti-mouse IgG (H+L) secondary antibodies.

Techniques: Biomarker Discovery, Standard Deviation