Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: ( A ) HER2 (diamonds), TNBC (circles), and luminal (squares and triangles) breast cancer lines were seeded into 96-well plates and treated with increasing doses of MAL3-101 for 72 hr. Viability is expressed as the average of three or more independent experiments, ± SEM. ( B ) MAL3-101-sensitive (MCF7 and MDA MB 231, denoted in blue) and resistant (MDA MB 453 and MDA MB 361, denoted in black) cells were treated with 12 µM MAL3-101 for the indicated times, and lysates were prepared and immunoblotted for cleaved caspase-3, caspase-7, and caspase-8. β-actin serves as a loading control. ( C ) The corresponding fold-increase of the indicated apoptotic markers relative to the DMSO control are plotted, ± SEM (n≥3 for cleaved caspase-3, n=3 for cleaved caspase-7, and n≥4 for cleaved caspase-8). Black asterisks correspond to statistical significance between MDA MB 231 cells (closed circle) and MDA MB 453 and MDA MB 361 (open circle and triangle, respectively), and the red asterisk represents statistical significance between MCF7 (closed triangle) and MDA MB 453 and MDA MB 361 (open circle and triangle) cells; * denotes p<0.05, ** denotes p<0.005. Figure 1—source data 1. Source data for cell viability assay and apoptotic marker accumulation in .

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay, Marker

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101, a specific Hsp70 inhibitor. The indicated breast cancer lines were seeded into 96-well plates and treated with increasing doses of the indicated compounds for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for an undetermined value. MAL3-101 sensitivities of Hsp70 inhibitor resistant cells are in bold.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: The cell numbers and autophagy or proteasome inhibitor concentrations used for the cell viability assay in combination with increasing doses of MAL3-101 are shown. The concentrations of bortezomib, CQ, and bafilomycin to induce no greater than 30% of cell death in each line after 72 hr treatment are shown. ND stands for undetermined value.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques: Viability Assay

Journal: eLife

Article Title: Unique integrated stress response sensors regulate cancer cell susceptibility when Hsp70 activity is compromised

doi: 10.7554/eLife.64977

Figure Lengend Snippet: Breast cancer cells exhibit a range of sensitivities to MAL3-101 in the presence of either autophagy or proteasome inhibitors. Cells were seeded into 96-well plates and treated with increasing doses of MAL3-101 in the presence or absence of subcritical doses of bortezomib (proteasome inhibitor), or CQ or bafilomycin (autophagy inhibitors) for 72 hr. Viability was measured using the CellTiter-Glo assay. IC 50 values were generate using a sigmoidal nonlinear regression with SigmaPlot 11.0. ND stands for undetermined value. MAL3-101 resistant cells are highlighted in yellow.

Article Snippet: For the remaining proteins aliquots from the same lysates were instead heated to 37°C for 30 min prior to SDS-PAGE using 4–20% Tris-Glycine gradient gels (XP04202Box, Thermo Fisher Scientific), and after transfer on nitrocellulose membranes (#84–874, Prometheus Laboratories Inc, California), the blots were incubated with anti-cleaved Caspase-8 (18C8, #9496; at 1:1000), anti-BiP (C50B12, #3177S; at 1:1000), anti-Hsp70 (smc-113, StressMarq Biosciences, Victoria, British Columbia; at 1:1000), anti-Hsc/Hsp70 (#4872S; at 1:1000), anti-HER2 (29D8, #2165; at 1:2000), anti-eIF2α (#9722S; at 1:2000), anti-phospho-eIF2α (#9721L; at 1:500), anti-PERK (D11A8, #5683S; at 1:2000), anti-PKR (D7F7, #12297S; at 1:2000), anti-Ire1α (14C10, #3294S; at 1:1000), anti-HRI (MBS2538114, MyBioSource, San Diego, CA; at 1:500) and anti-GCN2 and anti-GCN2 pT899 antibody (ab-134053 and ab-75836, Abcam, Cambtidge, UK; at 1:2000).

Techniques:

Journal: Journal of Nuclear Medicine

Article Title: Advancing Targeted Radionuclide Therapy Through the National Cancer Institute’s Small Business Innovation Research Pathway

doi: 10.2967/jnumed.118.214684

Figure Lengend Snippet: NCI SBIR–Funded TRT Contracts for 2015

Article Snippet: 2016 , Rockland Immunochemicals, Inc. , Phase I , HER2-positive cancers , 68 Ga and 177 Lu , HER2 and digoxigenin , Phase I: Develop bispecific recombinant single-domain antibodies with one arm targeting cancer biomarker HER2 and another arm against digoxigenin; investigate in vivo clearance, tumor accumulation, stability, and ability to capture digoxigenylated fluorescence dyes and radionuclides.

Techniques: In Vitro, In Vivo, Marker, Fluorescence, Drug discovery, Imaging

Journal: Journal of Nuclear Medicine

Article Title: Advancing Targeted Radionuclide Therapy Through the National Cancer Institute’s Small Business Innovation Research Pathway

doi: 10.2967/jnumed.118.214684

Figure Lengend Snippet: NCI SBIR–Funded TRT Contracts from 2016 to 2017

Article Snippet: 2016 , Rockland Immunochemicals, Inc. , Phase I , HER2-positive cancers , 68 Ga and 177 Lu , HER2 and digoxigenin , Phase I: Develop bispecific recombinant single-domain antibodies with one arm targeting cancer biomarker HER2 and another arm against digoxigenin; investigate in vivo clearance, tumor accumulation, stability, and ability to capture digoxigenylated fluorescence dyes and radionuclides.

Techniques: Radioactivity, Drug discovery, Recombinant, Biomarker Discovery, In Vivo, Fluorescence, Binding Assay, Labeling, In Vitro, Produced, Software

Journal: ACS Measurement Science Au

Article Title: Prototype Smartphone-Based Device for Flow Cytometry with Immunolabeling via Supra-nanoparticle Assemblies of Quantum Dots

doi: 10.1021/acsmeasuresciau.1c00033

Figure Lengend Snippet: Counting of SK-BR3 cells, stained with DAPI (cell nuclei) and immunolabeled with SiO 2 @(QD635-Dex)-(anti-HER2 TAC). (A) SK-BR3 cells only: (i–v) frames from smartphone FC videos of different concentrations of suspended cells. For the most dilute suspension, the arrow indicates a single cell. An intensity profile across selected cells can be found in Figure S13 . The image in (i) is a zoomed section of (v) with one (of several) SK-BR3 cells circled. Correlation plots (vi) of HER2-positive SK-BR3 cell counts derived from the smartphone FC versus a commercial cell counter. The diagonal line has a slope of unity. (B) Target SK-BR3 cells and background MDA-MB-231 cells: (i–v) frames from smartphone FC videos of mixed suspensions of SK-BR3 cells (variable amount) and MDA-MB-231 cells (approximately constant amount; DAPI stained). An intensity profile across selected cells of each type can be found in Figure S13 . Video S3 is a 10 s clip from one of the smartphone FC videos. The image in (i) is a zoomed section of (v) with one example each of a SK-BR3 cell and a MDA-MB-231 cell circled. Correlation plots of cell counts (vi) measured with a smartphone FC versus a commercial cell counter. Frame image brightness has been digitally increased for display purposes.

Article Snippet: Anti-HER2 antibody was from Novus Biologicals (Centennial, CO).

Techniques: Staining, Immunolabeling, Suspension, Derivative Assay

Journal: ACS Measurement Science Au

Article Title: Prototype Smartphone-Based Device for Flow Cytometry with Immunolabeling via Supra-nanoparticle Assemblies of Quantum Dots

doi: 10.1021/acsmeasuresciau.1c00033

Figure Lengend Snippet: Assessment of multiple colors of QDs. (A) PL emission spectra of QD490, QD540, QD585, QD605, QD635, and QD650 (solid lines). The approximate RGB color filter transmission spectra for the smartphone are overlaid (dashed lines; data courtesy of Olivier Burggraaff ). (B) Frames from smartphone FC videos showing SK-BR3 cells immunolabeled with SiO 2 @(QDλ-CM-Dex)-(anti-HER2) for QD540, QD585, QD605, or QD650. Frame image brightness has been digitally increased for display purposes. Intensity profiles across selected cells of each color of QD can be found in Figure S16 . (C) Plots of the G/B and R/B intensity ratios for individual SK-BR3 cells labeled with these four colors of SiO 2 @(QDλ-CM-Dex)-(anti-HER2). (D) Plots of the R, G, and B channel intensity values for individual SK-BR3 cells nonspecifically labeled with GSH-QD490, GSH-QD540, or GSH-QD635 (none as a SiO 2 @QD assembly).

Article Snippet: Anti-HER2 antibody was from Novus Biologicals (Centennial, CO).

Techniques: Transmission Assay, Immunolabeling, Labeling

Journal: ACS Measurement Science Au

Article Title: Prototype Smartphone-Based Device for Flow Cytometry with Immunolabeling via Supra-nanoparticle Assemblies of Quantum Dots

doi: 10.1021/acsmeasuresciau.1c00033

Figure Lengend Snippet: Counting of SK-BR3 and MDA-MB-231 cells in parallel using SiO 2 @(QD540-CM-Dex)-(anti-HER2) and SiO 2 @(QD650-CM-Dex)-(anti-MUC1), respectively. (A) Cartoon schematic of the experiment (not drawn to scale). (B) Example of a frame from a smartphone FC video. Frame image brightness has been digitally increased for display purposes. An intensity profile across selected cells of each type can be found in Figure S17 . Video S4 is a 20 s clip from one of the smartphone FC videos. (C) Examples of normalized G/B and R/B intensity ratios and cell classifications for five samples with an increasing number of MDA-MB-231 cells and an approximately constant number of SK-BR3 cells (see Figure S18 for additional data sets). Normalization of intensity ratios assisted with application of the SVM training set to the experimental data (see the SI for details). (D) Correlation plots for the measured and expected numbers of SK-BR3 and MDA-MB-231 cells, and for the measured and expected ratios of MDA-MB-231 and SK-BR3 cells. This plot includes the samples in panel (C) and in Figure S18 .

Article Snippet: Anti-HER2 antibody was from Novus Biologicals (Centennial, CO).

Techniques:

Journal: ACS Measurement Science Au

Article Title: Prototype Smartphone-Based Device for Flow Cytometry with Immunolabeling via Supra-nanoparticle Assemblies of Quantum Dots

doi: 10.1021/acsmeasuresciau.1c00033

Figure Lengend Snippet: Identification of selected breast cancer cell types (SK-BR3, MDA-MB-231, and MCF-7) using SiO 2 @(QD540-CM-Dex)-(anti-HER2), SiO 2 @(QD585-CM-Dex)-(anti-MUC1), and SiO 2 @(QD650-CM-Dex)-(anti-ER). (A) Cartoon schematic of the experiment (not drawn to scale). (B) Examples of G/B and R/B intensity ratios for a control sample without cells and suspensions of each cell type. The shaded region in the plots corresponds to the mean of the control sample data points plus four standard deviations along each intensity ratio axis. The circled regions highlight the dominant vertical, diagonal, and horizontal spread of cell counts for SK-BR3, MDA-MB-231, and MCF-7 cells, respectively.

Article Snippet: Anti-HER2 antibody was from Novus Biologicals (Centennial, CO).

Techniques: Control

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Consolidated Standards of Reporting Trials (CONSORT) diagram of the study population. In this study, 87 patients with human epidermal growth factor receptor 2-positive ( HER2 pos ) breast cancer were enrolled; all tumors were histologically confirmed as invasive breast cancer ( IBC ) with HER2 overexpression (3+ or 2+/fluorescence in situ hybridization (FISH)-positive). Cohorts are labeled ( a – g ) for ease of comparison (of immune responses), and are referred to in Results . Time points at which blood was drawn are indicated ( red callout boxes ). Median follow up in the cohort treated with trastuzumab and chemotherapy ( T + C ) was 26 (IQR 16.5–31.0) months. pCR pathologic complete response, DCI type 1-polarized dendritic cell, Adj adjuvant, mo months

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: Over Expression, Fluorescence, In Situ Hybridization, Labeling, Comparison, Adjuvant

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Interferon (IFN)-γ + anti-human epidermal growth factor receptor 2 ( HER2 ) CD4 + T cell response variations in HER2 pos patients with invasive breast cancer (IBC). IFN-γ enzyme-linked immunosorbent spot analysis of HER2 peptide-pulsed peripheral blood mononuclear cells examined anti-HER2 CD4 + T cell responses in patients with HER2 pos IBC, stratified by anti-HER2 responsivity , response repertoire ( mean # of reactive peptides ), and cumulative response ( mean total SFC/10 6 cells ). Differences between ( a ) Treatment-naïve patients with HER2 pos IBC (n = 22) and those treated with trastuzumab and chemotherapy ( T + C ) (n = 65); and ( b ) patients treated with Neoadjuvant T + C (n = 40) and those treated with Adjuvant T + C (n = 25). SFC spot-forming cells

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: ELISpot Assay, Adjuvant

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Significant disparity in anti-human epidermal growth factor receptor 2 ( anti-HER 2) interferon (IFN)-γ + T cell immune responses between patients with pathologic complete response ( pCR ) and patients with non-pCR . a Significantly elevated anti-HER2 CD4 + T cell responses by IFN-γ enzyme-linked immunosorbent spot analysis (ELISPOT) are observed in patients with HER2 pos invasive breast cancer achieving pCR (n = 16) following neoadjuvant trastuzumab and chemotherapy (T + C), compared with patients with non-pCR (n = 24). Peripheral blood mononuclear cells (PBMC) from patients with pCR and non-pCR were stimulated ex vivo with six HER2-derived class II peptides and IFN-γ production via ELISPOT was compared. Responses are stratified by anti-HER2 responsivity, repertoire, and cumulative response. b Evaluable HLA-A2.1 pos PBMC from patients with pCR (n = 6; black bars ) and non-pCR (n = 4; white bars ) were stimulated ex vivo with two HER2-derived class I peptides, HER2 369 – 377 and HER2 689–697 , and IFN-γ production via ELISPOT was compared. Phorbol-12-myristate 13-acetate ( PMA ) and ionomycin served as positive control. Results are expressed as mean spot-forming cells ( SFC )/2 × 10 5 cells ± standard error of the mean

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: ELISpot Assay, Enzyme-linked Immunospot, Ex Vivo, Derivative Assay, Positive Control

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Anti-human epidermal growth factor receptor 2 ( anti-HER2 ) CD4 + T-helper type-1 (Th1) is the dominant phenotype contributing to interferon-γ + ( IFN-γ + ) CD4 + T cell deficit in patients without pathologic complete response ( non-pCR ). a Relative contributions of Th1 ( T-bet + IFN-γ + ) versus Th2 ( GATA-3 + IFN-γ + ) phenotypes to HER2 peptide-specific IFN-γ + cells in peripheral blood mononuclear cells ( PBMC ) from pCR and non-pCR patients. Representative stains within groups are shown after gating on CD4 + cells; results in adjoining histograms are expressed as mean proportions (%) ± standard error of the mean (SEM) as indicated. b Circulating HER2-specific IL-4 production does not vary between pCR and non-pCR patients, when assessed by responsivity , repertoire , and cumulative response . Results expressed as proportion or mean ± SEM ( top panel ). Donor-matched cumulative IFN-γ and IL-4 production (spot-forming cells ( SFC )/10 6 cells) across six HER2 class II peptides compared in pCR and non-pCR patients. Relative HER2-specific IFN-γ:IL-4 proportions (% depicted in graph) was significantly higher in pCR (IFN-γ/(IFN-γ + IL-4) = 73.9 %:IL-4/(IFN-γ + IL-4) = 26.1 %) compared with non-pCR patients (34.6 %:65.4 %). Absolute IFN-γ:IL-4 production ratio changed from 2.8:1 (pCR) to 0.5:1 (non-pCR) ( bottom left panel ). No relative shifts in IFN-γ:IL-4 production were observed to positive controls (anti-CD3/CD28 or anti-CD3, respectively) ( bottom right panel ). c HER2-specific IL-10 production is similar between pCR and non-pCR patients across all Th1 metrics. Results are expressed as proportion or mean ± SEM ( top panel ). Relative HER2-specific IFN-γ:IL-10 production was significantly higher in pCR (62.0:38.0 %) compared with non-pCR (23.8:76.2 %) patients. Absolute IFN-γ:IL-10 production ratio changed from 1.6:1 (pCR) to 0.3:1 (non-pCR) ( bottom left panel ). No relative shifts in IFN-γ:IL-4 production were observed to positive controls (anti-CD3/CD28 or anti-CD3, respectively) ( bottom right panel ); * p <0.05, ** p <0.01, *** p <0.001

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques:

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Anti-human epidermal growth factor receptor 2 (anti-HER2) T-helper type-1 (Th1) deficit in patients without pathologic complete response ( non-pC R) is not attributable to lack of immune competence, host-level T cell anergy, or increase in immunosuppressive phenotypes. Peripheral blood mononuclear cells ( PBMC ) from pCR and non-pCR patients did not differ significantly in ( a ) immune competence – measured by IFN-γ production to anti-CD3/anti-CD28 stimulus or recall stimuli tetanus toxoid and Candida albicans – by enzyme-linked immunosorbent spot. Results presented as median ± IQR interferon-γ (IFN-γ) spot-forming cells (SFC)/2 × 10 5 cells. b , c Relative proportions of CD3 + CD4 + ( b , top ) or CD3 + CD8 + T-cells ( b , bottom ), T reg (CD4 + CD25 + FoxP3 + ) ( c , top ) or myeloid-derived suppressor cells (MDSC) (CD11b + CD33 + HLA-DR − CD83 − ) ( c , bottom ) by flow cytometry. Representative stains within groups are shown; results in histograms are expressed as mean proportions (%) ± standard error of the mean as indicated

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: ELISpot Assay, Derivative Assay, Flow Cytometry

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Demographic and clinicopathologic characteristics of patients recruited to the ongoing phase I trial of HER2-pulsed DC1 vaccination in HER2 pos IBC patients with residual disease following neoadjuvant trastuzumab and chemotherapy (NCT02061423)

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: Fluorescence In Situ Hybridization

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Depressed anti-human epidermal growth factor receptor 2 (anti-HER2) T-helper type-1 (Th1) immunity is restored following HER2-pulsed type 1-polarized dendritic cell (DC1) immunization. a Anti-HER2 CD4 + T cell immune reactivity profiles in four patients with non-pCR ( UPCC # 26113–01 to −04 ) undergoing HER2-pulsed DC1 vaccination in the ongoing phase I trial NCT02061423 are demonstrated at three time points: pre-vaccination ( black ), 3 months post vaccination ( gray ), and 6 months post vaccination ( white ). For these time points, responses to individual HER2-derived class II peptides ( x-axis ; p42, p98, p328, p776, p927, p1166) are graphed, with anti-CD3/CD28 serving as positive control. Anti-HER2 Th1 repertoire and cumulative responses at these time points are listed in adjoining tables. b Anti-HER2 Th1 repertoire ( mean # of reactive peptides ) and cumulative response ( mean total SFC/10 6 cells ) increase progressively in patients with non-pCR at the 3-month and 6-month time points. SFC spot-forming cells

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: Derivative Assay, Positive Control

Journal: Breast Cancer Research : BCR

Article Title: Anti-HER2 CD4 + T-helper type 1 response is a novel immune correlate to pathologic response following neoadjuvant therapy in HER2-positive breast cancer

doi: 10.1186/s13058-015-0584-1

Figure Lengend Snippet: Demographic and tumor-related characteristics of the study population: age, race, AJCC pathologic stage, hormone receptor status, and time from completion of trastuzumab (when applicable)

Article Snippet: After plates were blocked, cryopreserved PBMCs, isolated using density gradient centrifugation, were plated in triplicate (2 × 10 5 cells/well) and incubated at 37 °C for 24–36 h with either HER2 peptides (4 μg; Genscript, Piscataway, NJ, USA); media alone (unstimulated control); or positive control (anti-human CD3/CD28 antibodies (0.5 μg/mL; BD Pharmingen, San Jose, CA, USA)).

Techniques: Adjuvant, Biomarker Discovery

Journal: Cancers

Article Title: HER-2-Targeted Nanoparticles for Breast Cancer Diagnosis and Treatment

doi: 10.3390/cancers14102424

Figure Lengend Snippet: Summary of all significant examples of NPs developed for HER-2 + BC diagnosis.

Article Snippet: , Anti-HER-2 antibody-conjugated silver nanoparticles (Anti-HER-2 antibody, Fuzhou Maxim Biotech, Inc., Fuzhou, China) , - High sensitivity for targeting HER-2 - Easy fabrication, high SERS sensitivity and biocompatibility , in vitro , [ ] .

Techniques: Biomarker Discovery, Magnetic Resonance Imaging, Expressing, In Vitro, Recombinant, In Vivo, Dispersion, Fluorescence, Imaging, Ex Vivo, Binding Assay, Injection, Modification, Raman Spectroscopy, Staining, Inhibition