Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: ERBB2 knockout inhibits autophagy in ERBB2-positive SKBR3 breast cancer cells. ( A ) Knockout of ERBB2 was performed by a double-nickase Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) system and demonstrated by Western blotting. Autophagy was measured by Western blotting LC3-II and p62. Chloroquine (CQ, 20 µM) was used to block lysosomal degradation for autophagic flux measurement (the same hereafter). No AA, amino acid starvation. Anti-actin beta (ACTB) was used as a loading control (the same hereafter). Uncropped Western blot images and quantification of the protein band intensities were shown in . ( B ) Autophagy was measured by demonstrating the mRFP-LC3 puncta in cells and counting the numbers of mRFP-LC3 puncta per cell. Data represent or demonstrate the results from at least three independent experiments (the same hereafter). More example images of mRFP-LC3 puncta are shown in the . *, p < 0.5; **, p < 0.01; ***, p < 0.001 (the same hereafter). NIC- Con , control cells; NIC- ERBB2 , ERBB2 knockout cells.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Knock-Out, CRISPR, Western Blot, Blocking Assay, Control

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: Overexpression of ERBB2 promotes autophagy in ERBB2-negative and -ERBB2-positive breast cancer cells. Western blotting demonstration of ERBB2 overexpression in the ERBB2-negative MDA-MB-231 cells and the ERBB2-positive MCF7 cells ( A ). ERBB2 overexpression increased the basal levels of autophagy ( B ) and amino acid starvation (No AA)-induced levels of autophagy ( C ). Autophagy was measured by Western blotting LC3-II in the absence and presence of CQ. Uncropped Western blot images and quantification of the protein band intensities were shown in .

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Over Expression, Western Blot

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: Effects of lapatinib on autophagy in NIH3T3 cells expressing EGFR or ERBB2. ( A ) Western blotting showed the expression of EGFR and ERBB2. ( B ) Effects of EGFR and ERBB2 on basal autophagy. ( C ) Effects of lapatinib (1 µM) on autophagy in cells expressing vector alone, EGFR or ERBB2. Autophagy was measured by Western blotting LC3-II and SQSTM1/p62 in the absence and presence of CQ. Uncropped Western blot images and quantification of the protein band intensities were shown in . *, p < 0.5; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Plasmid Preparation

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: ERBB2 regulates the expression of autophagy genes. ( A ) Bioinformatics analysis of the mRNA levels of different autophagy genes (ATG genes) in ERBB2-positive and -negative human breast tumor tissues. The data were extracted from the Cancer Genome Atlas (TCGA) database. RE, relative mRNA expression of an autophagy gene in ERBB2-positive breast tumors compared to that in ERBB2-negative breast tumors. The mRNA expression was quantified by RNA-seq by Expectation–Maximization (RSEM) followed by log2 transformation to estimate the gene-level transcription. ( B ) Western blotting demonstration of the effects of ERBB2 overexpression and knockout on protein expression of autophagy proteins ULK1, FIP200 (RB1CC1), BECN1, ATG7, ATG5, and ATG12. Overexpression of ERBB2 in MDA-MB-231 and MCF7 cells and ERBB2 knockout in SKBR3 cells are demonstrated in A and A, respectively. Expression of ATG5 or ATG12 was demonstrated by the level of ATG12–ATG5 complex since it is well known that ATG5 and ATG12 form a conjugate in the form of ATG12–ATG5 during autophagy. Uncropped Western blot images and quantification of the protein band intensities are shown in . NIC- Con , control cells; NIC- ERBB2 , ERBB2 knockout cells.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, RNA Sequencing, Transformation Assay, Western Blot, Over Expression, Knock-Out, Control

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: The Kaplan–Meier plots of the relapse-free survival (RFS)-based survival probability of patients with ERBB2-positive and -negative breast tumors. The plots were downloaded from the Kaplan–Meier Plotter website ( http://kmplot.com/analysis/index.php?p=service&cancer=breast (accessed on 19 November 2019)). ( A ) Data of patients with ATG12 expression ( ATG12 mRNA) in breast tumors. ( B ) Data of patients with MAP1LC3B (LC3B) expression ( MAP1LC3B mRNA) in breast tumors. ( C ) Data of patients with BECN1 expression ( BECN1 mRNA) in breast tumors.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: Autophagy is ATG12-dependent in ERBB2-positive and -negative breast cancer cells. Autophagy was measured by Western blotting LC3-II in the absence and presence of CQ. ( A ) Western blotting demonstration of siRNA knockdown of ATG12 in the ERBB2-positive SKBR3 breast cancer cells and ATG12 overexpression in the ERBB2-negative MDA-MB-231 breast cancer cells. ( B ) ATG12 knockdown decreased the basal level and amino acid starvation (No AA)-induced level of autophagy in SKBR3 cells. ( C ) ATG12 overexpression increased the basal level and amino acid starvation (No AA)-induced level of autophagy in MDA-MB-231 cells. Uncropped Western blot images and quantification of the protein band intensities are shown in .

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Western Blot, Knockdown, Over Expression

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: ERBB2 promotion of autophagy inhibits cell death induced by chemotherapy drug Taxol in breast cancer cells. ( A ) ERBB2 knockout decreased autophagy induced by Taxol (paclitaxel, 10 µM) in SKBR3 cells. Autophagy was measured by Western blotting LC3-II in the absence and presence of NH 4 Cl (30 mM). ERBB2 knockout is demonstrated in A. ( B ) Cell death induced by Taxol was increased by ERBB2 knockout or ATG12 knockdown in SKBR3 cells. Knockdown of ATG12 and the related effect on autophagy are demonstrated in A,B. ( C ) ATG12 overexpression increased Taxol-induced autophagy in MDA-MB-231 cells. Overexpression of ERBB2 is demonstrated in A. Autophagy was measured by Western blotting LC3-II in the absence and presence of NH 4 Cl. ( D ) Cell death induced by Taxol was decreased by overexpression of ERBB2 or ATG12 in MDA-MB-231 cells. ATG12 overexpression and the related effect on autophagy are demonstrated in A,C. NH 4 Cl, instead of CQ, was chosen to measure Taxol-induced autophagy because CQ can induce higher cell death than NH 4 Cl ( and unpublished data). Uncropped Western blot images and quantification of the protein band intensities were shown in . NIC- Con , control cells; NIC- ERBB2 , ERBB2 knockout cells. *, p < 0.5; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Knock-Out, Western Blot, Knockdown, Over Expression, Control

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: Effects of ERBB2 antibody on ATG12 expression, autophagy, and cell death. An anti-human ERBB2 antibody (Ab) (research-grade trastuzumab biosimilar) was used to treat SKBR3 cells. ( A ) ERBB2 Ab treatment reduced the protein levels of ERBB2 and ATG12. ( B ) Real-time PCR experiments showed that ERBB2 Ab treatment decreased ATG12 mRNA level. ( C ) Immunoprecipitation (IP) demonstration of the interaction between ERBB2 and ATG12. ( D ) ERBB2 Ab treatment inhibited autophagy at the basal and amino acid starvation (No AA)-induced levels. Autophagy was measured by Western blotting LC3-II in the absence and presence of CQ. ( E ) ERBB2 Ab treatment increased cell death without and with Taxol treatment. Uncropped Western blot images and quantification of the protein band intensities are shown in . *, p < 0.5; **, p < 0.01; ***, p < 0.001.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot

Journal: Cancers

Article Title: Erb-b2 Receptor Tyrosine Kinase 2 (ERBB2) Promotes ATG12-Dependent Autophagy Contributing to Treatment Resistance of Breast Cancer Cells

doi: 10.3390/cancers13051038

Figure Lengend Snippet: A graphic model for ERBB2 promotion of autophagy and its effect on cancer treatment and outcome to patients. ERBB2 expression upregulates the protein levels autophagy-related (ATG) proteins ULK1, FIP200, ATG5, ATG7, and ATG12 increasing ATG12-dependent autophagy. This leads to treatment resistance of breast cancer and a worse outcome for patients with ERBB2-positive breast cancer. Treatment with ERBB2 antibody (Herceptin or others) causes degradation of ERBB2 and downregulation of ATG12 resulting in autophagy inhibition.

Article Snippet: Anti-human ERBB2 antibody (research-grade trastuzumab biosimilar) (MAB9589) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Inhibition

Journal: bioRxiv

Article Title: Personalized models of breast cancer desmoplasia reveal biomechanical determinants of drug penetration

doi: 10.1101/2021.12.12.472296

Figure Lengend Snippet: (a) Confocal microscopy images of perfused tumoroid MVNs. The arrows indicate focal leaks. The scale bar is 400 μm. (b) Permeability to fluorescent dextran, Trastuzumab, and Cetuximab of tumoroid MVNs compared to control MVNs and (c) effective permeability of those MVNs as a function of applied intravascular pressure; n = 3. (d) Schematic diagram of computation model of the MVNs (left), and model results of interstitial fluid pressure, IFP , as a function of changes in vascular hydraulic conductivity, L p , and matrix permeability, k . (e) Confocal microscopy image of vascular HA in control and SKBR3 MVNs. (f) Gene expression of HA-associated proteins for ECs; n = 3. (g) Quantification of vascular HA concentration in 1 mm biopsies centered 2 mm from the tumoroids in the MVN devices. (h) Effective permeability of MVNs treated with HA-ase, resulting in increased filtration and hydraulic conductivity; n = 3. Significance assessed by one-way ANOVA after confirming a normal distribution of the data; p < 0.05 *, p < 0.01 **, p < 0.001 ***.

Article Snippet: Permeability of fluorescent molecules in the MVNs was measured by confocal microscopy and image analysis with the software ImageJ as previously described , using fluorescein isothiocyanate (FITC, #F4274, Sigma Aldrich), dextran (FITC-conjugated, 70 kDa, 0.1 mg mL −1 , #FD70, Sigma Aldrich), Trastuzumab (Alexa Fluor 488-conjugated, #FAB9589G, 0.1 mg mL −1 , R&D Systems), and Cetuximab (Alex Fluor 647-conjugated, #FAB9577R, 0.1 mg mL −1 , R&D Systems).

Techniques: Confocal Microscopy, Permeability, Control, Gene Expression, Concentration Assay, Filtration

Journal: bioRxiv

Article Title: Personalized models of breast cancer desmoplasia reveal biomechanical determinants of drug penetration

doi: 10.1101/2021.12.12.472296

Figure Lengend Snippet: (a) Effective permeability of MDA-MB-468 MVNs with intravascular pressure as a result of different treatments targeting stromal HA; n = 3. (b) Normalized MVN concentration of dextran near the tumoroids to that at a distance > 5mm, as measured by proxy of fluorescence intensity, as a result of treatment with HA-ase, a CD44 blocking antibody (“CD44 block”), and a TGFβ blocking antibody (“TGFβ block”), and (c) representative confocal images of treated tumoroid (circles) MVNs perfused with dextran. (d) Quantification of CD44 isoforms and TGFβ in 1 mm biopsies centered at tumoroids in the MVN devices, and (e) quantification of stromal HA concentrations in the tumoroids as a result of the stromal HA-targeting treatments. (f) Confocal microscopy images of SKBR3 tumoroid cryosections showing increased Trastuzumab penetration after HA degradation. (g) Cell death in the tumoroids as a function of combined treatment with Trastuzumab + Cetuximab (“mAB”) with different targeting strategies for stromal HA, as measured by proxy of fluorescent SYTOX intensity. Significance assessed by one-way ANOVA after confirming a normal distribution of the data; p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****.

Article Snippet: Permeability of fluorescent molecules in the MVNs was measured by confocal microscopy and image analysis with the software ImageJ as previously described , using fluorescein isothiocyanate (FITC, #F4274, Sigma Aldrich), dextran (FITC-conjugated, 70 kDa, 0.1 mg mL −1 , #FD70, Sigma Aldrich), Trastuzumab (Alexa Fluor 488-conjugated, #FAB9589G, 0.1 mg mL −1 , R&D Systems), and Cetuximab (Alex Fluor 647-conjugated, #FAB9577R, 0.1 mg mL −1 , R&D Systems).

Techniques: Permeability, Concentration Assay, Fluorescence, Blocking Assay, Confocal Microscopy

Journal: bioRxiv

Article Title: Personalized models of breast cancer desmoplasia reveal biomechanical determinants of drug penetration

doi: 10.1101/2021.12.12.472296

Figure Lengend Snippet: (a) Confocal microscopy image of cell death signal in an MCF7 tumoroid in the MVN device after treatment with Trastuzumab and Cetuximab (“mAB”). (b) Change in size of TC line tumoroids after combination mAB treatment with stromal HA-targeting therapeutic strategies. (c) Normalized cell death signal in TC line tumoroids in well plates treated with Trastuzumab (“T”), Cetuximab (“C”), or both (“T+C”). The concentration indicted is in μg mL −1 . Significance assessed by one-way ANOVA; p < 0.5 *, p < 0.001 ***, p < 0.0001 ****.

Article Snippet: Permeability of fluorescent molecules in the MVNs was measured by confocal microscopy and image analysis with the software ImageJ as previously described , using fluorescein isothiocyanate (FITC, #F4274, Sigma Aldrich), dextran (FITC-conjugated, 70 kDa, 0.1 mg mL −1 , #FD70, Sigma Aldrich), Trastuzumab (Alexa Fluor 488-conjugated, #FAB9589G, 0.1 mg mL −1 , R&D Systems), and Cetuximab (Alex Fluor 647-conjugated, #FAB9577R, 0.1 mg mL −1 , R&D Systems).

Techniques: Confocal Microscopy, Concentration Assay

Journal: bioRxiv

Article Title: Personalized models of breast cancer desmoplasia reveal biomechanical determinants of drug penetration

doi: 10.1101/2021.12.12.472296

Figure Lengend Snippet: (a) Projected confocal microscopy images of patient-derived tumoroids show high levels of vascularization and different levels of cancer vascular colonization. (b) Effective permeability of patient-derived tumoroid MVNs with intravascular pressure; n = 3. (c) Quantification of stromal HA concentrations in the tumoroids as a result of stromal and vascular HA-targeting treatments, and (d) resulting cell death in the tumoroids when the treatments are administered in combination with Trastuzumab and Cetuximab, as measured by proxy of fluorescent SYTOX intensity. Significance assessed by one-way ANOVA after confirming a normal distribution of the data; p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****.

Article Snippet: Permeability of fluorescent molecules in the MVNs was measured by confocal microscopy and image analysis with the software ImageJ as previously described , using fluorescein isothiocyanate (FITC, #F4274, Sigma Aldrich), dextran (FITC-conjugated, 70 kDa, 0.1 mg mL −1 , #FD70, Sigma Aldrich), Trastuzumab (Alexa Fluor 488-conjugated, #FAB9589G, 0.1 mg mL −1 , R&D Systems), and Cetuximab (Alex Fluor 647-conjugated, #FAB9577R, 0.1 mg mL −1 , R&D Systems).

Techniques: Confocal Microscopy, Derivative Assay, Permeability

Journal: Nature Communications

Article Title: Unlocking the potential of allogeneic Vδ2 T cells for ovarian cancer therapy through CD16 biomarker selection and CAR/IL-15 engineering

doi: 10.1038/s41467-023-42619-2

Figure Lengend Snippet: a – g In vitro ADCC assay. Three human ovarian cancer cell lines (OVCAR3-FG, OVCAR8-FG, and SKOV3-FG) and four effector cells (Vδ2T, MCAR-Vδ2T, MCAR15-Vδ2T, and MCAR-T as a benchmark control) were included in the study. The same CD16 Hi donor PBMCs were used to generate all 4 types of effector cells. a Experimental design. Data were collected at 24 h after co-culture. Anti-HER2 Ab: monoclonal antibody trastuzumab. b FACS detection of HER2 expression on the indicated tumor cell lines. c Killing of OVCAR3-FG tumor cells by Vδ2T cells in the presence of titrated amounts of either isotype control or anti-HER2 Ab ( n = 3). Tumor cell killing data of ( d ) OVCAR3-FG, ( e ) OVCAR8-FG, and ( f ) SKOV3-FG in the presence or absence of anti-HER2 Ab (anti-HER2 Ab concentration = 0.1 μg/mL; E:T ratio = 0.5:1; n = 3). g ELISA measurements of IFN-γ production at 24 h in the presence or absence of anti-HER2 Ab (anti-HER2 Ab concentration = 0.1 μg/mL; E:T ratio = 1:1; n = 3). h , i In vitro repeated tumor cell challenge assay studying ADCC. h Experimental design. Effector cells were mixed with tumor cells and rechallenged every 3 days. Tumor cell killing data was measured at 24 h post co-culture with or without the addition of anti-HER2 antibody (anti-HER2 Ab concentration = 0.1 μg/mL; E:T ratio = 2:1; n = 3). i KO OVCAR3-FG tumor cell killing data collected over time. Representative of 3 experiments. Data are presented as the mean ± SEM. ns, not significant; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by Student’s t test ( c ) or by one-way ANOVA ( d – g ). Sour c e data and exact p values are provided as a Source Data file.

Article Snippet: InVivo SIM anti-human HER2 (Trastuzumab Biosimilar, Cat. SIM0005) was purchased from BioXCell.

Techniques: In Vitro, ADCC Assay, Control, Co-Culture Assay, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: Sensitization of breast cancer to Herceptin by redox active nanoparticles

doi:

Figure Lengend Snippet: CONPs enhance the cell round-up effect of Herceptin particularly under the hypoxic conditions. (A) The Herceptin-sensitive (BT474) and Herceptin-resistant cells (R20) were grown under the normoxic (normoxia) or hypoxic (hypoxia) conditions. At sub-confluent density, the cells were treated with saline (Mock), Herceptin (5 μg/ml), cerium oxide nanoparticles (CONPs) (75 μM) or Herceptin in combination with CONPs (H + C) for 24 hours prior to phase contrast microscopy. (B) Cells were analyzed for viability by separately collecting and staining the adherent and round up cells with trypan blue as described in Materials and Methods. The number inside each image in (A) indicates percentage of cells that became rounded up. *P<0.05, **P<0.01 and ***P<0.001 compared to respective mock-treated group.

Article Snippet: Herceptin was purchased from Bio-Rad (MCA6092. a.k.a.

Techniques: Saline, Microscopy, Staining

Journal: American Journal of Cancer Research

Article Title: Sensitization of breast cancer to Herceptin by redox active nanoparticles

doi:

Figure Lengend Snippet: CONPs sensitizes both Herceptin-sensitive and Herceptin-resistant cells to the inhibitory effect of Herceptin on cell survival and proliferative capacities more potently under hypoxia than under normoxia. The cells were grown and treated as described in Figure 1. The cells were then prepared for analysis of colony-forming capability by clonogenic assay (A) and proliferative capacity by WST-1 assay (B) as detailed in Materials and Methods. *P<0.05, **P<0.01 and ***P<0.001 compared to respective mock-treated group.

Article Snippet: Herceptin was purchased from Bio-Rad (MCA6092. a.k.a.

Techniques: Clonogenic Assay, WST-1 Assay

Journal: American Journal of Cancer Research

Article Title: Sensitization of breast cancer to Herceptin by redox active nanoparticles

doi:

Figure Lengend Snippet: CONPs potently and rapidly inhibit HIF-1α induction by hypoxia particularly in the Herceptin-resistant cells. A. Time-course change in HIF-1α expression under the hypoxic conditions. After incubated for indicated periods, the cells were processed for western blotting. HIF-1α levels relative to β-actin were quantified and normalized to 4-h-treated Herceptin-sensitive cells. *P<0.05 compared to columns 7 or 8. ***P<0.001 compared to column 1 as well as columns 7-10. B. CONPs markedly inhibit HIF-1α induction by hypoxia in the cells. Indicated cells were incubated either under that normoxic conditions and mock-treated only (basal) or under the hypoxic conditions and treated with vehicle (Mock), Herceptin, CONPs or their combination (H + C) for 4 hours. The cells were then processed for western blotting and quantitative analysis. *P<0.05 compared to column 7 or 8. **P<0.01 compared to column 7 or 8. ***P<0.001 compared to columns 2-4.

Article Snippet: Herceptin was purchased from Bio-Rad (MCA6092. a.k.a.

Techniques: Expressing, Incubation, Western Blot

Journal: American Journal of Cancer Research

Article Title: Sensitization of breast cancer to Herceptin by redox active nanoparticles

doi:

Figure Lengend Snippet: Treatment with CONPs causes slow reduction in intracellular levels of VEGF under the hypoxic conditions. A. Levels of VEGF and CAIX proteins were not affected within 8 hour treatment with Herceptin and/or CONPs. Indicated cells were incubated either under the normoxic conditions and mock-treated only (basal) or under the hypoxic conditions and treated with vehicle (Mock), Herceptin, CONPs or their combination (H + C) for 4 or 8 hours. The whole cell extracts were then processed for western blotting. B. Treatment with CONPs alone or in combination with Herceptin leads to severe decrease in levels of VEGF protein particularly in the Herceptin-resistant cells after 24 hour treatment. The cells were treated similarly as described above in A for 24 hours prior to western analysis. *1P<0.01 compared to column 1; *2P<0.01 compared to column 2; *3P<0.001 compared to column 1; *4P<0.05 compared to column 6; *5P<0.05 or 0.01 compared to column 6 or 7; *6P<0.01 or 0.001 compared to column 6 or 7. C. Levels of VEGF mRNA were not affected within 8 hours treatment with Herceptin and/or CONPs. VEGF mRNA was analyzed by semi-quantitative PCR shown here and quantitative real-time PCR (not shown).

Article Snippet: Herceptin was purchased from Bio-Rad (MCA6092. a.k.a.

Techniques: Incubation, Western Blot, Real-time Polymerase Chain Reaction

Journal: American Journal of Cancer Research

Article Title: Sensitization of breast cancer to Herceptin by redox active nanoparticles

doi:

Figure Lengend Snippet: Treatment with CONPs causes rapid and significant decrease in extracellular VEGF levels under hypoxic conditions. (A-D) Dot blotting analysis of VEGF protein in the culture media of the cells. Cells were incubated either under the normoxic conditions and mock-treated (basal) or under the hypoxic conditions and treated with vehicle (Mock), Herceptin, CONPs or their combination (H + C) for 4 hours. The medium of the cells was then collected and processed for dot blotting (A, C) and quantitative (B, D) analysis of extracellular VEGF levels. *P<0.05 and **P<0.01 compared to column 2. (E) ELISA analysis of VEGF protein in the media of the cells. The cells were incubated under either the normoxic or hypoxic conditions for 24 hours. The media of these cells were then collected and processed for ELISA analysis. *1P<0.01 compared to column 1; *2P<0.01 compared to column 1’; *3P<0.05 and *4P<0.01 compared to column 5’.

Article Snippet: Herceptin was purchased from Bio-Rad (MCA6092. a.k.a.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: Sensitization of breast cancer to Herceptin by redox active nanoparticles

doi:

Figure Lengend Snippet: CONPs sensitize Herceptin-resistant cells to the inhibitory effect of Herceptin on primary tumor growth. The HR20-nGL breast cancer cells were injected into the mammary fat pad of 4-6-week-old female NSG mice. The next day mice were randomly grouped into four groups and received intraperitoneal injection of 100 μl saline solution of Herceptin (2 mg/kg) in combination with CONPs (0.1 mg/kg), Herceptin, CONPs or saline alone, twice a week for two consecutive weeks. Primary tumor progression was monitored and examined by weekly BLI analysis for 8 weeks (A, B). After sectioned, the tumors were measured for weight and volume (C, D), and the tissues were further processed for IHC staining for expression of the proliferative marker Ki67 (E, F). P<0.05 compared between *H + C and saline alone, #H + C and CONPs alone, and $H + C and Herceptin alone.

Article Snippet: Herceptin was purchased from Bio-Rad (MCA6092. a.k.a.

Techniques: Injection, Saline, Immunohistochemistry, Expressing, Marker

Journal: American Journal of Cancer Research

Article Title: Sensitization of breast cancer to Herceptin by redox active nanoparticles

doi:

Figure Lengend Snippet: CONPs sensitize Herceptin-resistant cells to the inhibitory effect of Herceptin on tumor angiogenesis and survival. The tumor tissues collected from the mice described in Figure 6 were processed for IHC staining for expression of the angiogenic factor protein VEGF (A), the vascular endothelial marker protein CD31 (B), and the apoptotic marker protein cleaved caspase-3 (C). Optical density (OD) for expression quantification was obtained by ImageJ Fiji color deconvolution analysis and normalized to the Saline group value. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: Herceptin was purchased from Bio-Rad (MCA6092. a.k.a.

Techniques: Immunohistochemistry, Expressing, Marker, Saline

Journal: American Journal of Cancer Research

Article Title: Sensitization of breast cancer to Herceptin by redox active nanoparticles

doi:

Figure Lengend Snippet: CONPs sensitize the Herceptin-resistant breast cancer cells to the inhibitory effect of Herceptin on the lung metastasis. During tissue collection from the mice described in Figure 6, the lung specific BLI was performed (A) and bioluminescent intensity for the lung metastasis quantified (B). *P<0.05.

Article Snippet: Herceptin was purchased from Bio-Rad (MCA6092. a.k.a.

Techniques:

Journal: Nature Biomedical Engineering

Article Title: Antibody-displaying extracellular vesicles for targeted cancer therapy

doi: 10.1038/s41551-024-01214-6

Figure Lengend Snippet: a , Flow cytometry measurement of uptake by MFI of mNG + EVs (Fc-EVs versus ctrl-EVs versus no EVs) in HER2 positive breast cancer cells (SKBR-3 cells) when incubated with HER2-Ab (trastuzumab), control-Ab (IgG ctrl) or no Ab, showing significant increase in uptake of Fc-EVs by trastuzumab. b , Flow cytometry measurement of uptake (by MFI) of mNG + Fc-EVs decorated with the PD-L1-Ab atezolizumab in PD-L1 expression stimulated (IFNγ) or unstimulated malignant melanoma (B16F10) cells. c , Fluorescence microscopy images of B16F10 cells stained with DAPI (blue), untreated (UT) or treated with mNG + (green) Fc-EVs alone or with the PD-L1-Ab atezolizumab, showing increased uptake of the Fc-EVs when decorated with PD-L1-Ab. Panels a and b are shown as mean ± s.d. n = 3 biological replicates. Statistical significance was calculated using one-way ( b ) or two-way ( a ) ANOVA with Tukey’s post-test compared with each value; P values are indicated above the plots throughout.

Article Snippet: The tissues were processed as described in ref. , with lysed tissue and nLuc + EV input being analysed for nanoluc luminescence or for single-cell suspension for flow cytometry, as described above, but with the HER2 Ab (R&D Systems, FAB9896V-100UG) used for flow cytometry applications.

Techniques: Flow Cytometry, Incubation, Control, Expressing, Fluorescence, Microscopy, Staining

Journal: Nature Biomedical Engineering

Article Title: Antibody-displaying extracellular vesicles for targeted cancer therapy

doi: 10.1038/s41551-024-01214-6

Figure Lengend Snippet: IV injection of Fc-EVs with HER2-Ab (trastuzumab, Fc-EV + HER2-Ab) compared to control-Ab (Fc-EV + IgG-ctrl) in HER2 + breast cancer (SKBR-3) tumour-bearing Swiss nude mice. a , The experimental set-up with inoculation of SKBR-3 cells followed by tumour formation for 2 months before IV injection of nLuc + Fc-EVs with Abs, followed by tissue collection 30 min post injection. b , Fold change in detected EVs (based on luminescence) per gram tumour tissue compared to Fc-EV + IgG-ctrl. c , d , Accumulation of Fc-EV + HER2-Ab (based on luminescence) compared to Fc-EV + IgG-ctrl per gram spleen ( c ) and liver ( d ). All data are shown as mean ± s.d. n = 10 mice. Statistical significance was calculated using two-tailed unpaired t -test analysis compared with each value; P values are indicated above the plots throughout.

Article Snippet: The tissues were processed as described in ref. , with lysed tissue and nLuc + EV input being analysed for nanoluc luminescence or for single-cell suspension for flow cytometry, as described above, but with the HER2 Ab (R&D Systems, FAB9896V-100UG) used for flow cytometry applications.

Techniques: IV Injection, Control, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: A Supramolecular Self-assembly Approach to Site-Specific Antibody Conjugates via a Coiled-coil Peptides Platform

doi: 10.1101/2025.07.21.665979

Figure Lengend Snippet: A. AlphaFold illustration of coiled-coil heterodimer formed from the receiving and docking peptides B. Schematic diagram illustrating drug loading process to an antibody through formation of coiled-coil structure between docking peptide conjugated to the drug and receiving peptide fused to the heavy chain of an antibody C. SEC-UV traces at 280 nm of native trastuzumab (αHER2) and trastuzumab fused to different receiving peptides (αHER2-P1, αHER2-P2, αHER2-P3, αHER2-P4) D. Coomassie-stained SDS-PAGE gel analysis under non-reducing (left) and reducing (right) conditions of commercial trastuzumab (commercial αHER2), in house produced native trastuzumab (αHER2) and trastuzumab fused to P3 peptide (αHER2-P3) E. ELISA assay demonstrating antigen-binding ability of native αHER2 and αHER2 fused to P3 peptide (αHER2-P3) F. Binding affinity of αHER2-P3 to biotinylated P4 peptide measured by biolayer interferometry (BLI) . G. ELISA measurements of binding of native αHER2 and peptide modified αHER2-P3 antibodies to human FcRn receptor. Data are presented as mean ± s.d., n = 3.

Article Snippet: Serial dilutions of native αHER2 (Human ErbB2/Her2 Antibody, Cat. # MAB9589-100, R&D Systems Inc) or antibodies fused to receiving peptide αHER2-P3 were prepared in blocking buffer (starting concentration 3 ug/mL).

Techniques: Staining, SDS Page, Produced, Enzyme-linked Immunosorbent Assay, Binding Assay, Modification

Journal: bioRxiv

Article Title: A Supramolecular Self-assembly Approach to Site-Specific Antibody Conjugates via a Coiled-coil Peptides Platform

doi: 10.1101/2025.07.21.665979

Figure Lengend Snippet: A. Schematic illustration of an antibody reacting with payload-modified coil peptide resulting into antibody-payload conjugate B. Direct ELISA assay assessing the binding and enzyme activity of αHER2-HRP conjugate C. UV-Vis analysis of antibody conjugated to polyA 15 GGG oligo annealed with a fluorophore labeled polyT 15 CCC-TAMRA oligo D. Cytotoxicity of the αHER2 ADC containing MMAE drug against SKBR-3 cells compared to equivalent unloaded αHER2-P3 antibody concentrations over 72 hours E. Schematic illustration of an antibody reacting with two orthogonal payload-modified coil peptides resulting into antibody bearing two different payloads F-H. UV-Vis analyses of antibodies assembled with single F. Fluor488 payload or G. Flour647 payload and H. dual-loaded conjugates.

Article Snippet: Serial dilutions of native αHER2 (Human ErbB2/Her2 Antibody, Cat. # MAB9589-100, R&D Systems Inc) or antibodies fused to receiving peptide αHER2-P3 were prepared in blocking buffer (starting concentration 3 ug/mL).

Techniques: Modification, Direct ELISA, Binding Assay, Activity Assay, Labeling

Journal: bioRxiv

Article Title: A Supramolecular Self-assembly Approach to Site-Specific Antibody Conjugates via a Coiled-coil Peptides Platform

doi: 10.1101/2025.07.21.665979

Figure Lengend Snippet: A. Stability of conventionally prepared AFCs via hinge cysteines conjugation, B . via short 28 amino acid long coiled-coil peptides, C. via long 35 amino acid long coiled-coil peptides and D. via long 35 amino acid long coiled-coil peptides with cysteine residue substitution in human plasma incubated at 37 °C. The ratio of intact AFC was determined by ELISA normalizing the detected payload amount to the total amount of IgG compared to the standard curve prepared from intact AFC (no incubation in serum, flash frozen at zero time point) E. Schematic illustration of ADC conjugation strategy and molecular structure of the cleavable linker-payload F. PK of unmodified trastuzumab (αHER2), unloaded ADC (αHER2-P3 5mer -cys), and fully assembled coiled-coil ADC (ADC) in female Balb/c (N = 3, n=6 for trastuzumab and ADC; N = 2, n=4 for unloaded ADC group). Blood was collected at indicated time points and the total amount of human IgG was quantified by indirect ELISA using standard curve G. Ex vivo organ biodistribution of AFC injected i.v. at 10 mg/kg dose into SKOV-3 tumor bearing BALB/c nude mice at different time points (n=3 per time point). The total radiant efficiency was calculated using IVIS software keeping area of ROI the same across all the samples. H . Representative fluorescent picture of ex vivo organs post AFC injection on day 5. I. Orthotopic xenograft model of ErbB2/Her2-positive cancer study design and timeline. Female NSG mice were engrafted with 2 million SKOV-3 tumor cells by intraperitoneal injection, were randomized on day 14 and treated with single dose of PBS or trastuzumab antibody (10 mg/kg) or coiled-coil ADC (10 mg/kg) or high dose coiled-coil ADC (2xADC, 20 mg/kg) or commercial ADC disitamab vedotin (10 mg/kg), n= 5 mice per group. All mice were euthanized on day 48 post-treatment, when PBS-treated mice exhibited severe hunching posture and signs of morbidity, to record endpoint tumor volume and mass J . Average mouse body weight measured twice a week (±SD) for each treatment group demonstrating no significant difference between the groups over the observation period. K. Images of harvested tumors at study termination at day 48 post treatment. L. Recorded tumor size and M. tumor mass. Statistical significance is indicated as follows: ns, non-significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005 (unpaired t-test).

Article Snippet: Serial dilutions of native αHER2 (Human ErbB2/Her2 Antibody, Cat. # MAB9589-100, R&D Systems Inc) or antibodies fused to receiving peptide αHER2-P3 were prepared in blocking buffer (starting concentration 3 ug/mL).

Techniques: Conjugation Assay, Residue, Clinical Proteomics, Incubation, Enzyme-linked Immunosorbent Assay, Indirect ELISA, Ex Vivo, Injection, Software

Journal: bioRxiv

Article Title: An electro-optical bead-nanochip technology for the ultrasensitive and multi-dimensional detection of small extracellular vesicles and their markers

doi: 10.1101/2022.04.11.487936

Figure Lengend Snippet: (A) Biotinylated (detection) antibodies recognise and bind to target surface proteins (detection markers) on sEVs. (B) Magnetic Beads (MBs) pre-coated with (capture) antibodies, capture (detection) antibody-labelled target sEVs and non-labelled sEVs. (C) O-NEXOS detection of target sEV epitopes is performed by the stepwise addition of poly-HRP streptavidin, fluorescence substrate and by the measurement of the resultant fluorescence product. (D) Alternatively, 1 – E-NEXOS is performed by adding streptavidin-GNPs (s-GNPs) to the mixture of MB-captured sEVs. sEV-bound GNPs are recovered by washing the MBs-sEVs-s-GNPs complexes in a magnetic field. 2 – s-GNPs are eluted by dissolving the complexes in pH 2.6, then restoring it back to pH 7. 3 – Buffer containing s-GNPs is exchanged by centrifugation to ultra-pure water or 1 % PBS and, finally, the s-GNPs are attracted and detected in an E-NEXOS nanochip.

Article Snippet: HER2 was detected with a recombinant human biotinylated antibody (FAB9589B, RnDSystems).

Techniques: Magnetic Beads, Fluorescence, Centrifugation