Journal: Bioengineering & Translational Medicine

Article Title: Near‐infrared‐induced drug release from antibody–drug double conjugates exerts a cytotoxic photo‐bystander effect

doi: 10.1002/btm2.10388

Figure Lengend Snippet: in vitro evaluation of the NIR‐PIT and cytotoxic photo‐bystander effects via photo‐triggered drug release from T‐DM1–IR700. (a) Flow cytometric analysis of the binding of Tra–IR700 and T‐DM1–IR700 to HER2+ (3T3/HER2) and HER2−(MDAMB‐468) cells. Preincubation with excess Tra or T‐DM1 inhibited the binding of Tra–IR700 or T‐DM1–IR700 to 3T3/HER2 cells, respectively, indicating that Tra–IR700 and T‐DM1–IR700 specifically bound to the HER2 antigen. Neither Tra–IR700 nor T‐DM1–IR700 showed IR700 fluorescence signals in the presence of HER2−MDAMB‐468 cells. (b) Microscopic observations before and immediately after HER2‐targeted NIR‐PIT. Mixed cultures of HER2+ 3T3/HER2 and HER2− MDAMB‐468‐luc‐GFP cells were incubated with Tra–IR700 or T‐DM1–IR700 overnight and observed under a microscope before and immediately after irradiation with NIR light (4 J/cm 2 ). Necrotic cell death (revealed using PI staining) was observed only for HER2+ 3T3/HER2 cells after NIR light exposure, whereas HER2− MDAMB‐468‐luc‐GFP cells remained intact. Confirmation of the selective cytotoxicity induced by NIR‐PIT with both Tra–IR700 and T‐DM1–IR700. Scale bars: 20 μm. (c) In vitro NIR‐PIT (4 J/cm 2 ) with Tra–IR700 (1 μg/ml) or T‐DM1–IR700 (1 μg/ml) on HER2+ (3T3/HER2‐luc‐GFP) and HER2− (MDAMB‐468‐luc‐GFP) cells. Luciferase activities were measured as RLU values ( n = 4, * p < 0.001). (d) Co‐culture of 3T3/HER2 and MDAMB‐468‐luc‐GFP cells. NIR‐PIT was performed following treatment with T‐DM1–IR700 (1, 5, or 10 μg/ml) or Tra–IR700 (10 μg/ml), after which the mixed cultures were incubated for 4 days (left panel). Luciferase activities were measured as RLUs, and the viability of nontargeted MDAMB‐468‐luc‐GFP cells was measured 4 days after NIR‐light irradiation. Upon NIR‐PIT following Tra–IR700 treatment of the mixed culture, HER2+ 3T3/HER2 cells were eradicated, resulting in more space available for the nontargeted MDAMB‐468‐luc‐GFP cells to grow ( n = 4). NIR‐PIT showed no immediate effect on MDAMB‐468‐luc‐GFP cells in the mixed culture (Figure ). Only MDAMB‐468‐luc‐GFP cells treated with T‐DM1–IR700 did not show any significant decrease in luciferase activity 4 days after irradiation (Figure ). Other cell‐line combinations (HER2+ and HER2− or ‐low cells) were also examined and showed cytotoxic photo‐bystander effects (Figure ). In panels (e) and (f), the data are presented as the mean ± standard error of the mean (SEM). In panel (g), the data are presented as the mean ± standard deviation. * p < 0.0001, ** p < 0.01 (Student's t test). (e) Schematic depicting the MS‐analysis procedure for investigating the supernatants of mixed 3T3/HER2 and MDAMB‐468‐luc‐GFP cells treated with T‐DM1–IR700‐mediated NIR‐PIT (left) and LC–MS/MS data showing a specific peak only in an NIR‐light‐irradiated (16 J/cm 2 ) tube (right).

Article Snippet: Humanized Tra (Herceptin) and T‐DM1 (Kadcyla) IgG 1 mAbs against HER2 were purchased from Chugai Pharmaceutical Co. (Tokyo, Japan).

Techniques: In Vitro, Binding Assay, Fluorescence, Incubation, Microscopy, Irradiation, Staining, Luciferase, Co-Culture Assay, Activity Assay, Standard Deviation, Liquid Chromatography with Mass Spectroscopy

Journal: Bioengineering & Translational Medicine

Article Title: Near‐infrared‐induced drug release from antibody–drug double conjugates exerts a cytotoxic photo‐bystander effect

doi: 10.1002/btm2.10388

Figure Lengend Snippet: Evaluation of the in vivo cytotoxic photo‐bystander effect. (a) Tumors excised 6 days after the inoculation of nude mice with HER2+ 3T3/HER2 cells and HER2− MDAMB‐468‐luc‐GFP cells. Scale bar: 300 μm. The tumor sample was imuunostained with HER2 or GFP antibody. (b) Representative IR700 fluorescence images of Tra–IR700 and T‐DM1–IR700‐injected mice. We used the mice tumor model with over 1 cm tumors. (c) Fluorescence intensity measurements of the tumor and liver. The target‐to‐background ratios of the tumor and liver are indicated ( n = 3 mice). (d) In vivo therapeutic regimen involving tumor cell inoculation, Tra–IR700 or T‐DM1–IR700 injection, and NIR‐light exposure. BLI was performed at the indicated points (arrowheads). BLI indicated HER2− nontargeted MDAMB‐468‐luc‐GFP tumor activity in the mixed tumor. (e) Mixed tumors inoculated on both dorsa of mice, with only the right‐sided tumor irradiated with NIR light. Representative BLI of right‐sided NIR‐PIT with Tra–IR700 or T‐DM1–IR700 is shown. (f) Quantitative RLUs, indicating nontargeted HER2− MDAMB‐468‐luc‐GFP cells inside the mixed tumors ( n = 6 mice/group). (g) Mixed tumor volume (mm 3 ) of the ratio (defined as day 0 = 100; n = 8 mice/group). (h) Survival of HER2‐targeted NIR‐PIT with T‐DM1–IR700 or Tra‐IT700 on mixed tumors ( n = 8 mice/group). In panel (c), the data represent the mean ± SEM. In panel (f), * p = 0.033 < 0.05 (Kruskal–Wallis test with Dunn's post hoc test). In panel (g), ** p = 0.0005 < 0.001 (Kruskal–Wallis test with Dunn's post hoc test). In panel (h), # p = 0.035 < 0.05 (log‐rank test)

Article Snippet: Humanized Tra (Herceptin) and T‐DM1 (Kadcyla) IgG 1 mAbs against HER2 were purchased from Chugai Pharmaceutical Co. (Tokyo, Japan).

Techniques: In Vivo, Fluorescence, Injection, Activity Assay, Irradiation

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Upregulation of ER signaling as an adaptive mechanism of cell survival in HER2-positive breast tumors treated with anti-HER2 therapy

doi: 10.1158/1078-0432.CCR-14-2728

Figure Lengend Snippet: Correlation among biomarkers at baseline.

Article Snippet: Tissue sections were incubated with primary antibody against ER (Vector Labs, Burlingame, CA, USA), PR, Bcl2, and Ki67 (Dako Cytomation, Carpinteria, CA, USA), t-HER2 (Thermo Scientific/ Neomarkers Waltham, MA, USA), or p-Tyr1221/1222 HER2 (Cell Signaling Technology, Beverly, MA, USA).

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Upregulation of ER signaling as an adaptive mechanism of cell survival in HER2-positive breast tumors treated with anti-HER2 therapy

doi: 10.1158/1078-0432.CCR-14-2728

Figure Lengend Snippet: A. Mice bearing MCF7 HER2-18 tumor xenografts were subjected to estrogen deprivation (ED) until development of resistance, which was associated with complete loss of ER expression (18). At that time, the combination of trastuzumab, pertuzumab, and gefitinib (TPG) was added to ED. Anti-HER therapy induced tumor regression in 53% of mice (responders), whereas the remaining tumors continued to grow despite the inhibition of both ER and HER pathways. Restoration of ER expression was significantly associated with tumor response.

Article Snippet: Tissue sections were incubated with primary antibody against ER (Vector Labs, Burlingame, CA, USA), PR, Bcl2, and Ki67 (Dako Cytomation, Carpinteria, CA, USA), t-HER2 (Thermo Scientific/ Neomarkers Waltham, MA, USA), or p-Tyr1221/1222 HER2 (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Inhibition

Journal: Experimental & Molecular Medicine

Article Title: A functional comparison between the HER2 high /HER3 and the HER2 low /HER3 dimers on heregulin-β1-induced MMP-1 and MMP-9 expression in breast cancer cells

doi: 10.3858/emm.2012.44.8.054

Figure Lengend Snippet: HRG-induced MMP-1 and MMP-9 expression was regulated by MEK/ERK dependent pathway but not by PI-3K/Akt pathway in MCF7 cells. After serum-starvation for 24 h, (A) cells were treated with or without 50 ng/ml HRG and further incubated at 37℃ for the indicated times. (B-D) Cells were pretreated with U0126 or LY294002 at the indicated concentrations for 60 min, then treated with or without 50 ng/ml HRG for 30 min (B) and 24 h (C, D), respectively. MMP-1 and MMP-9 protein and mRNA expression was determined by (C) Western blotting (upper band) and Zymography (lower band) and (D) RT-PCR, respectively. (E) After transfection of CA-MEK for 24 h, cells were further incubated for 24h in serum-free culture media. p-HER3, p-HER2, p-ERK, p-Akt, and β-actin were measured in whole cell lysates by Western blotting. Con, control; HRG, heregulin-β1; U, U0126; LY, LY294002.

Article Snippet: Rabbit monoclonal anti-p-HER2, t-HER2, p-ERK, p-SAPK/JNK, and p-Akt antibodies were purchased from Epitomics (Burlingame, CA).

Techniques: Expressing, Incubation, Western Blot, Zymography, Reverse Transcription Polymerase Chain Reaction, Transfection, Control

Journal: Experimental & Molecular Medicine

Article Title: A functional comparison between the HER2 high /HER3 and the HER2 low /HER3 dimers on heregulin-β1-induced MMP-1 and MMP-9 expression in breast cancer cells

doi: 10.3858/emm.2012.44.8.054

Figure Lengend Snippet: HRG-induced MMP-1 and MMP-9 expression was significantly increased in HER2-overexpressed cells. (A) The expression of t-HER2 and t-HER3 was measured by Western blotting in whole cell lysates. (B) The analysis of cell cycle by FACS was detected as described in Materials and Methods . (C, D) Cells were treated with or without 50 ng/ml HRG for 24 h. MMP-1 and MMP-9 mRNA and protein expression was determined by (C) Western blotting (upper band) and Zymography (lower band) and (D) RT-PCR, respectively. The results shown are representative of three independent experiments. Con, control; HRG, heregulin-β1.

Article Snippet: Rabbit monoclonal anti-p-HER2, t-HER2, p-ERK, p-SAPK/JNK, and p-Akt antibodies were purchased from Epitomics (Burlingame, CA).

Techniques: Expressing, Western Blot, Zymography, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Experimental & Molecular Medicine

Article Title: A functional comparison between the HER2 high /HER3 and the HER2 low /HER3 dimers on heregulin-β1-induced MMP-1 and MMP-9 expression in breast cancer cells

doi: 10.3858/emm.2012.44.8.054

Figure Lengend Snippet: Activation of downstream signaling molecules of receptor by HRG were significantly increased in HER2-overexpressed MCF7 cells. After serum-starvation for 24 h, cells were treated with or without 50 ng/ml HRG for 30 min. The phosphorylation of HER3, HER2, ERK, JNK, Akt, c-Jun, and β-actin was determined in whole cell lysates by Western blotting. The results shown are representative of three independent experiments.

Article Snippet: Rabbit monoclonal anti-p-HER2, t-HER2, p-ERK, p-SAPK/JNK, and p-Akt antibodies were purchased from Epitomics (Burlingame, CA).

Techniques: Activation Assay, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: A functional comparison between the HER2 high /HER3 and the HER2 low /HER3 dimers on heregulin-β1-induced MMP-1 and MMP-9 expression in breast cancer cells

doi: 10.3858/emm.2012.44.8.054

Figure Lengend Snippet: HRG-induced MMP-1 and MMP-9 expression of HER2-overexpressed MCF7 cells was also regulated by MEK/ERK dependent pathway. After serum-starvation for 24 h, (A) HER2-overexpressed cells were treated with or without 50 ng/ml HRG for the indicated times. Co-immnunoprecipitation was detected as described in Materials and Methods . (B, C) Cells were pretreated with UO126 or LY294002 at the indicated concentrations for 60 min, then treated with or without 50 ng/ml HRG for 24 h. MMP-1 and MMP-9 protein and mRNA expression was determined by (B) Western blotting (upper band) and Zymography (lower band) and (C) RT-PCR, respectively. The level of t-HER3 and t-HER2 expression was determined in whole cell lysates by Western blotting. The results shown are representative of three independent experiments. Con, control; HRG, heregulin-β1.

Article Snippet: Rabbit monoclonal anti-p-HER2, t-HER2, p-ERK, p-SAPK/JNK, and p-Akt antibodies were purchased from Epitomics (Burlingame, CA).

Techniques: Expressing, Western Blot, Zymography, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Experimental & Molecular Medicine

Article Title: A functional comparison between the HER2 high /HER3 and the HER2 low /HER3 dimers on heregulin-β1-induced MMP-1 and MMP-9 expression in breast cancer cells

doi: 10.3858/emm.2012.44.8.054

Figure Lengend Snippet: HRG-induced MMP-1 and MMP-9 expression was completely eliminated by lapatinib in both vector alone and HER2-overexpressed cells. After serum-starvation for 24 h, (A) cells were pretreated with 2 µM lapatinib for 60 min, then treated with or without 50 ng/ml HRG for 24 h. MMP-1 and MMP-9 protein expression was determined in culture media by Western blotting (upper band) and Zymography (lower band), respectively. (B) Cells were pretreated with 2 µM lapatinib for 60 min and then treated with or without 50 ng/ml HRG for 30 min. The phosphorylation of HER3, HER2, ERK, Akt, and β-actin was determined in whole cell lysates by Western blotting. (C) After transfection of scrambled and 25 and 50 nM HER3 siRNA for 48 h, respectively, cells were further incubated for 24 h in serum-free culture media, then treated with or without 50 ng/ml HRG at 37℃ for 24 h. MMP-1 and MMP-9 protein expression was determined by Western blotting (upper band) and Zymography (lower band) in culture media. The results shown are representative of three independent experiments. Con, control; HRG, heregulin-β1.

Article Snippet: Rabbit monoclonal anti-p-HER2, t-HER2, p-ERK, p-SAPK/JNK, and p-Akt antibodies were purchased from Epitomics (Burlingame, CA).

Techniques: Expressing, Plasmid Preparation, Western Blot, Zymography, Transfection, Incubation, Control

Journal: Experimental & Molecular Medicine

Article Title: A functional comparison between the HER2 high /HER3 and the HER2 low /HER3 dimers on heregulin-β1-induced MMP-1 and MMP-9 expression in breast cancer cells

doi: 10.3858/emm.2012.44.8.054

Figure Lengend Snippet: Schematic models. Heregulin-β1 triggers the phosphorylation of HER3 and HER4 and then activates several signaling molecules such as ERK and Akt. These activations are additively augmented by HER2 overexpression. In particular, the phosphorylation of ERK and Akt is higher in HER2 high /HER3 MCF7 cells than in HER2 low /HER3 MCF7 cells. MMP-1 and -9 expressions also significantly increase in HER2 high /HER3 MCF7 cells, compared with in HER2 low /HER3 MCF7 cells.

Article Snippet: Rabbit monoclonal anti-p-HER2, t-HER2, p-ERK, p-SAPK/JNK, and p-Akt antibodies were purchased from Epitomics (Burlingame, CA).

Techniques: Over Expression