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

Article Title: HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-based PET Imaging

doi: 10.1158/1078-0432.CCR-17-2754

Figure Lengend Snippet: (A) Scheme illustrating the radiolabeling reaction of DFO-conjugated ZHER3:8698 affibody molecule with the positron emitter 89Zr. (B) Saturation binding of 89Zr-DFO-ZHER3:8698 to MCF-7 cells. The data are expressed as the mean values ± SEM (n = 3 independent experiments). (C) HER3 expression in a panel of breast cancer cell lines. Representative Western blot from whole cell lysates, with GAPDH used as the loading control. (D) In vitro binding specificity of 89Zr-DFO-ZHER3:8698 in breast cancer cells and specific blocking using 100-fold molar excess of either unlabeled ZHER3:8698 or the natural HER3 ligand HRG. The data are expressed as the mean values ± SEM (n = 3 independent experiments). *P = 0.0357; *P = 0.0446; for MDA-MB468, **P = 0.0087; *P = 0.015 for MCF-7, and **P = 0.009; **P = 0.0097 for BT-474 cells.

Article Snippet: Tissue lysates (300 μg) and whole cell lysates (200 μg) were incubated overnight in a thermomixer at 4°C and 650 rpm, with 10 μg of anti-HER3 mouse mAb (Merck Millipore, UK).

Techniques: Radioactivity, Binding Assay, Expressing, Western Blot, Control, In Vitro, Blocking Assay

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

Article Title: HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-based PET Imaging

doi: 10.1158/1078-0432.CCR-17-2754

Figure Lengend Snippet: (A) Representative 15 min coronal fused PET/CT images of mice bearing MCF-7, MDA-MB-468, or MDA-MB-231 xenografts. The mice received ~ 8 MBq of either 89Zr-DFO-ZHER3:8698 or 89Zr-DFO-ZTAQ via tail vein injection, with image acquisition taking place 3 h after injection. The arrowheads indicate the tumors and the kidneys. (B) Ex vivo biodistribution at 3 h after injection of the radioconjugates. Data are expressed as the mean values ± SD (n = 3 animals). *P = 0.0136; ***P = 0.0002; ****P < 0.0001. (C) Representative Western blot of whole tumor tissue lysates evaluating HER3, HER2 and EGFR expression in the indicated xenograft models. (D) Histopathological analysis of HER3 expression in MCF-7, MDA-MB-468, and MDA-MB-231 xenografts displaying the highest HER3 expression in MCF-7 xenografts and the lowest in MDA-MB-231. (E) Representative ex vivo autoradiography sections taken 3 h after injection of 89Zr-DFO-ZHER3:8698. (F) Autoradiography quantification from panel E as the intensity per region of interest area (A.U./cm2). Data are expressed as the mean values ± SD (n = 10 sections). **P = 0.0028; ****P < 0.0001. (G) Representative segmented xenografts following PET/CT image acquisition 3 h after 89Zr-DFO-ZHER3:8698 injection. These images highlight the greater radioactivity accumulation observed in MCF-7 xenografts, and the heterogeneity of uptake across the tumor burden. Color map defined within the tumor volume only.

Article Snippet: Tissue lysates (300 μg) and whole cell lysates (200 μg) were incubated overnight in a thermomixer at 4°C and 650 rpm, with 10 μg of anti-HER3 mouse mAb (Merck Millipore, UK).

Techniques: Positron Emission Tomography-Computed Tomography, Injection, Ex Vivo, Western Blot, Expressing, Autoradiography, Radioactivity

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

Article Title: HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-based PET Imaging

doi: 10.1158/1078-0432.CCR-17-2754

Figure Lengend Snippet: MCF-7 xenografts were randomized into two groups: control and treatment. The treatment group received 40 mg/kg of AUY922 i.p. every second day for a period of two weeks. (A) Representative axial fused PET/CT images of mice bearing MCF-7 tumors. Each mouse received 7.2-8.1 MBq of 89Zr-DFO-ZHER3:8698 via tail vein injection, with image acquisition taking place at 3 h after injection. The mice were imaged before initiating AUY922 treatment (day 0), and following administration of the last treatment dose (day 14). The %ID/g ratios were determined by dividing the %ID/g on day 14 by that obtained on day 0. (B) Scatter plot of the %ID/g ratios for both control (n = 6) and AUY922-treated mice (n = 7). The horizontal lines indicate the mean for each group. *P = 0.0131. (C) Scatter plot of the ex vivo tumor biodistribution at 3 h after injection of the radioconjugate on day 14, for both control (n = 4) and AUY922-treated mice (n = 6). The horizontal lines indicate the mean per group. **P = 0.0036. (D) Histopathological analysis of control and AUY922-treated MCF-7 xenografts. Tumor sections were stained with hematoxylin and eosin (H&E), HER3, or CD31.

Article Snippet: Tissue lysates (300 μg) and whole cell lysates (200 μg) were incubated overnight in a thermomixer at 4°C and 650 rpm, with 10 μg of anti-HER3 mouse mAb (Merck Millipore, UK).

Techniques: Control, Positron Emission Tomography-Computed Tomography, Injection, Ex Vivo, Staining

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

Article Title: HER3-Mediated Resistance to Hsp90 Inhibition Detected in Breast Cancer Xenografts by Affibody-based PET Imaging

doi: 10.1158/1078-0432.CCR-17-2754

Figure Lengend Snippet: (A) HER receptors, IGF-1Rβ, and PI3K/AKT pathway activation were monitored by Western blot using whole tissue lysates from all control and AUY922-treated mice. Hsp70/72 expression was used as a surrogate for AUY922 treatment efficacy, and GAPDH as a loading control. (B) Minimum to maximum box & whiskers plot of the quantified protein expression represented in A. Data are expressed as the normalized protein expression per antibody for control and AUY922 groups. The black lines represent the median value. *P = 0.0306; **P = 0.0022; ****P < 0.0001. (C, D) Correlation between HER3/IGF-1Rβ and Hsp70/72 protein expression per mouse. The dashed lines represent the ninety-five percent confidence levels. (E) Correlation between %ID/g ratios obtained from 89Zr-DFO-ZHER3:8698 PET images and Hsp70/72 protein expression per mouse. The dashed lines represent the ninety-five percent confidence levels. (F) BT-474 and MCF-7 cells were treated with 32 nM of AUY922 for 48 h. Equal amounts of whole cell lysates were immunoprecipitated with a mouse anti-HER3 antibody followed by Western blot analysis of HER3 and IGF-1Rβ. Whole tissue lysates from control mouse C5 and AUY922-treated mouse A6 were also immunoprecipitated against HER3 and analyzed by Western blot. Ten percent of the input lysates were used as loading controls.

Article Snippet: Tissue lysates (300 μg) and whole cell lysates (200 μg) were incubated overnight in a thermomixer at 4°C and 650 rpm, with 10 μg of anti-HER3 mouse mAb (Merck Millipore, UK).

Techniques: Activation Assay, Western Blot, Control, Expressing, Immunoprecipitation