Journal: Journal of Cancer Research and Clinical Oncology

Article Title: CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate

doi: 10.1007/s00432-023-05031-3

Figure Lengend Snippet: Live-cell imaging to assess internalization and lysosomal routing of the αCSPG4(scFv)-SNAP fusion protein conjugated to Alexa 488. a Antigen-positive Hs578T and b antigen-negative MCF-7 tumor cell lines were incubated with 5 µM αCSPG4(scFv)-SNAP-Alexa488 (green signal) for 30 min at 37 °C. The lysosomal compartments were stained with 50 ng of LysoTracker (red signal) for 30 min at 37 °C. The nuclei were counterstained with Hoechst (blue signal) diluted 1:5000 in media. Images were captured with the Zeiss confocal-scanner microscope (LSM880) with Airyscan at ×63 magnification

Article Snippet: This also allowed the generation of antibody titration curves (using GraphPad Prism v5) for each cell line and αCSPG4(scFv)–SNAP–Alexa488 combinations, depicting the change in frequency and median fluorescence intensity (MFI) of the Alexa488-positive population.

Techniques: Live Cell Imaging, Incubation, Staining, Microscopy

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate

doi: 10.1007/s00432-023-05031-3

Figure Lengend Snippet: Generation of αCSPG4(scFv)-SNAP. a ORF coding for αCSPG4(scFv)-SNAP. Here, unique Sfi I and Not I restriction sites were used in the cloning of the scFv genes into the pCB-SNAP mammalian expression vector. Important components of the ORFs include: Ig K (Ig- Kappa ) leader sequence for secretion of the fusion protein expressed by host cells; His-tag (×6), 6 histidine tags for protein purification by IMAC and detection in western blot analysis; EKS (enterokinase cleavage site) for the enzymatic removal of the N-terminal elements and STOP, a stop codon for halting protein synthesis); b microscopic visualization of eGFP in HEK293T cells transfected with pCB-αCSPG4(scFv)-SNAP DNA. Enrichment was performed using 100 µg/mL of Zeocin. The green channel (right panel) was used to assess eGFP expression, while the brightfield (or phase contrast) channel (left panel) showed the number of cells in a specific region. Images were taken using a ZOE™ Fluorescent Cell Imager at 100 µm magnification; c chromatogram of αCSPG4(scFv)-SNAP after purification using IMAC. The y -axis is a measure of the elution buffer percentage, while the x -axis represents the ÄKTA flow-through volume with respect to increasing time. The blue line shows the elution profile of fusion protein, and the green line demonstrates the concentration gradient of imidazole. Fractions were eluted in the form of two distinct peaks on the chromatogram

Article Snippet: This also allowed the generation of antibody titration curves (using GraphPad Prism v5) for each cell line and αCSPG4(scFv)–SNAP–Alexa488 combinations, depicting the change in frequency and median fluorescence intensity (MFI) of the Alexa488-positive population.

Techniques: Clone Assay, Expressing, Plasmid Preparation, Sequencing, Protein Purification, Western Blot, Transfection, Purification, Concentration Assay

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate

doi: 10.1007/s00432-023-05031-3

Figure Lengend Snippet: Characterization of IMAC-purified αCSPG4(scFv)-SNAP. a SDS-PAGE and western blot analysis of enriched αCSPG4(scFv)-SNAP (51.1 kDa) protein fractions. Left panel: comparison of protein profiles on a 10% SDS-PAGE gel stained with Aqua staining solution. Right panel: immunoblot of proteins transferred to a nitrocellulose membrane from a duplicate SDS-PAGE gel. A chemiluminescent ladder (SuperSignal™ Molecular Weight Protein Ladder) was used to assess the size of the protein bands. An anti-his rabbit antibody (1:1000 primary antibody) and a goat anti-rabbit HRP-conjugate antibody (1:5000 secondary antibody) were used. Red arrow indicates presence of full-length recombinant scFv-SNAP fusion protein. The membrane was visualized using a Gel Documentation System; b assessing the binding activity of αCSPG4(scFv)-SNAP to BG-Alexa Fluor 488. A ratio of 1:2 of protein to BG-Alexa Fluor 488 was used in the conjugation reaction. Left panel: Alexa488-conjugated protein ran on a 10% SDS-PAGE gel stained with Aqua staining solution. Right panel: the same SDS-PAGE gel visualized under blue light for potential fluorescence. A Dark Reader Transilluminator was used for visualization of the fluorescent signal

Article Snippet: This also allowed the generation of antibody titration curves (using GraphPad Prism v5) for each cell line and αCSPG4(scFv)–SNAP–Alexa488 combinations, depicting the change in frequency and median fluorescence intensity (MFI) of the Alexa488-positive population.

Techniques: Purification, SDS Page, Western Blot, Comparison, Staining, Membrane, Molecular Weight, Recombinant, Binding Assay, Activity Assay, Conjugation Assay, Fluorescence

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate

doi: 10.1007/s00432-023-05031-3

Figure Lengend Snippet: Assessing the binding activity of αCSPG4(scFv)-SNAP-Alexa488 by screening target cells for potential CSPG4 expression. a HEK293T, b Hs578T, c MDA-MB-231, d MDA-MB-468, and e SK-Mel-28. Cell lines were incubated with 15 µM of conjugated protein (green signal) for 15–20 min at 37 °C. Hoechst (1:5000 dilution in media) was used as a stain for the nuclei (blue signal). Washes were performed three times with 1 × PBS, before fixing with 4% PFA and mounting the coverslips on a microscope slide. Images were captured using a Zeiss confocal-scanner microscope (LSM880) with Airyscan at 20 µm magnification

Article Snippet: This also allowed the generation of antibody titration curves (using GraphPad Prism v5) for each cell line and αCSPG4(scFv)–SNAP–Alexa488 combinations, depicting the change in frequency and median fluorescence intensity (MFI) of the Alexa488-positive population.

Techniques: Binding Assay, Activity Assay, Expressing, Incubation, Staining, Microscopy

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate

doi: 10.1007/s00432-023-05031-3

Figure Lengend Snippet: Binding of αCSPG4(scFv)-SNAP on South African breast cancer patient tissue sections and the pooled mean fluorescence intensities of CSPG4 expression. Using the LSM confocal 510 microscope, FFPE tissue sections were imaged. The mean of each patient’s fluorescence intensity data was extracted and tabulated for comparison. a αCSPG4(scFv)-SNAP conjugated to BG-Alexa647 which labels the cell membrane (in red) of a tumorigenic tissue section; b DAPI panel showing nuclear staining of cells (in blue) on a non-tumor sample and c auto-fluorescence control. The αCSPG4(scFv)-SNAP pooled label data for patient means were compared using one-way ANOVA (**** p < 0.0001). The mean intensity data indicated significant differences between all tumor (T) and non-tumor (NT) tissues in the selected patient samples as shown in j . Qualitative differences are indicated as a comparison of the fluorescence image panels (as previously indicated) of patient 12 ( d – f ) and patient 3 ( g – i ). These samples were normalized against an autofluorescent control for each patient

Article Snippet: This also allowed the generation of antibody titration curves (using GraphPad Prism v5) for each cell line and αCSPG4(scFv)–SNAP–Alexa488 combinations, depicting the change in frequency and median fluorescence intensity (MFI) of the Alexa488-positive population.

Techniques: Binding Assay, Fluorescence, Expressing, Microscopy, Comparison, Membrane, Staining

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate

doi: 10.1007/s00432-023-05031-3

Figure Lengend Snippet: Comparison of the CSPG4 expression status across target cell lines. Cells were incubated with the optimal Alexa488-conjugated αCSPG4(scFv)-SNAP concentration and acquired on a BD™ LSR II flow cytometer. Data shown are representative of two biological repeat experiments. a Representative pseudocolor plots indicating the gating strategy employed in the determination of the receptor expression status; b representative pseudocolor plots with gates showing the position of the Alexa488-positive/negative cell populations at the optimal antibody titer. Frequencies of the Alexa488-positive populations (expressed as a percentage of the total population) are indicated at the top right-hand corner of the plots; c antibody titration curves showing the frequencies of the Alexa488-positive population at the optimal antibody concentration (indicated by red box); d histograms depicting the relative fluorescence of the Alexa488-positive/negative populations (gray curve: untreated cells, blue curve: Alexa488-negative cells at the optimal antibody concentration, green curve: Alexa488-positive cells at the optimal antibody concentration); bar graphs demonstrating the e frequencies of the Alexa488-positive population and f median fluorescence intensity (MFI). Statistical comparisons (relative to the CSPG4-negative HEK293T cell line) were calculated using Student’s t tests [* p < 0.05, ** p < 0.01, *** p < 0.001, ns (not significant)]

Article Snippet: This also allowed the generation of antibody titration curves (using GraphPad Prism v5) for each cell line and αCSPG4(scFv)–SNAP–Alexa488 combinations, depicting the change in frequency and median fluorescence intensity (MFI) of the Alexa488-positive population.

Techniques: Comparison, Expressing, Incubation, Concentration Assay, Flow Cytometry, Titration, Fluorescence

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate

doi: 10.1007/s00432-023-05031-3

Figure Lengend Snippet: Overview of binding activities for Alexa Fluor 488-conjugated αCSPG4(scFv)-SNAP fusion protein on target cell lines

Article Snippet: This also allowed the generation of antibody titration curves (using GraphPad Prism v5) for each cell line and αCSPG4(scFv)–SNAP–Alexa488 combinations, depicting the change in frequency and median fluorescence intensity (MFI) of the Alexa488-positive population.

Techniques: Binding Assay, Confocal Microscopy, Flow Cytometry

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate

doi: 10.1007/s00432-023-05031-3

Figure Lengend Snippet: Summary of IC 50 values based on the cytocidal activity of αCSPG4(scFv)-SNAP-AURIF, unmodified MMAF, and unconjugated BG–linker–AURIF on target cell lines

Article Snippet: This also allowed the generation of antibody titration curves (using GraphPad Prism v5) for each cell line and αCSPG4(scFv)–SNAP–Alexa488 combinations, depicting the change in frequency and median fluorescence intensity (MFI) of the Alexa488-positive population.

Techniques: Activity Assay

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate

doi: 10.1007/s00432-023-05031-3

Figure Lengend Snippet: Evaluation of the cytotoxicity of AURIF-conjugated αCSPG4(scFv)-SNAP fusion proteins. a Unconjugated αCSPG4(scFv)-SNAP fusion protein displays negligible toxicity on target cell lines; b confirming the saturation of scFv-SNAP fusion proteins with BG–linker–AURIF through a double conjugation with BG-Alexa Fluor 488. After conjugation with BG–linker–AURIF for 4 h at room temperature, 5 µM of the conjugation reaction was incubated with 10 µM of BG-Alexa Fluor 488 for 60 min at 37 °C, before being loaded on a 10% SDS-PAGE gel, which was visualized under blue light using a Dark Reader Transilluminator (right panel) and stained using Aqua staining solution (left panel). Unbound BG-Alexa Fluor 488 is indicated by red arrow; c dose–response curves demonstrating the cytotoxic activity of αCSPG4(scFv)-SNAP-AURIF in vitro. The cytotoxic activity was assessed using an XTT-based viability assay after incubation with the drug for 72 h. Cells were treated with (threefold serially diluted) increasing concentrations of the drug and the IC 50 values (relative to the untreated and zeocin-treated cells) were calculated using GraphPad Prism v5. Data are mean ± standard deviation (SD) of each measurement (presented as a percentage of cell viability), and the measurements were performed in triplicate at least three times

Article Snippet: This also allowed the generation of antibody titration curves (using GraphPad Prism v5) for each cell line and αCSPG4(scFv)–SNAP–Alexa488 combinations, depicting the change in frequency and median fluorescence intensity (MFI) of the Alexa488-positive population.

Techniques: Conjugation Assay, Incubation, SDS Page, Staining, Activity Assay, In Vitro, Viability Assay, Standard Deviation