Raymond Reilly

Computational analysis of the number, area and density of γ-H2AX foci in breast cancer cells exposed to 111In-DTPA-hEGF or γ-rays using Image-J software

Purpose: To develop a simple method for the quantification of γ-H2AX focus number, density and size. Methods: MDA-MB-468 human breast cancer cells were treated overnight with 111In-diethylenetriaminepentaacetic acid human epidermal growth factor (111In-DTPA-hEGF, 0–142 kBq/pmol) or exposed to γ-radiation to induce DNA double strand breaks (DSB). DNA DSB formation was evaluated by detection of phosphorylated histone H2AX on serine 139 (γ-H2AX) using immunofluorescence. Confocal microscopy was used to capture images of γ-H2AX foci and cell nuclei. Image-J software with customized macros was used to quantify γ-H2AX foci. Results: The number of γ-H2AX foci per nucleus scored using Image-J correlated strongly with the number scored using direct visual confirmation (coefficient of determination, R2 = 0.950; 60 nuclei scored). The mean density (grayscale values per pixel), area and integrated density (IntDen) of individual foci increased linearly as the specific radioactivity (SR) increased up to 67 kBq/pmol (R2 values of 0.826, 0.964, 0.978, respectively). The mean number of foci per nucleus, the combined area of γ-H2AX foci per nucleus and the IntDen per nucleus also increased linearly with SR, giving R2 values of 0.926, 0.974 and 0.983, respectively. Similar linear relationships were observed with the γ-ray absorbed dose up to 3.0 Gy. Conclusions: The density, area and IntDen of individual foci, as well as the number of γ-H2AX foci, total focus area and IntDen per nucleus were successfully quantified using Image-J with customized macros.

Optimized digital counting colonies of clonogenic assays using ImageJ software and customized macros: Comparison with manual counting

Abstract Purpose: To develop a digital method for counting colonies that highly replicates manual counting. Materials and methods: Breast cancer cells were treated with trastuzumab-conjugated gold nanoparticles in combination with X-ray irradiation, 111In labeled trastuzumab, or γ-radiation, followed by clonogenic assays. Colonies were counted manually or digitally using ImageJ software with customized macros. Key parameters, intensity threshold and minimum colony size, were optimized based on three preliminary manual counts or blindly chosen. The correlation of digital and manual counting and inter- and intra-experimenter variability were examined by linear regression. Survival curves derived from digital and manual counts were compared by F-test (P < 0.05). Results: Using optimized parameters, digital counts corresponded linearly to manual counts with slope (S) and R2 value close to 1 and a small y-intercept (y0): SK-BR-3 (S = 0.96 ± 0.02, R2 = 0.969, y0 = 5.9 ± 2.2), MCF-7/HER2-18 (S = 0.98 ± 0.03, R2 = 0.952, y0 = 0.74 ± 0.47), and MDA-MB-231 cells (S = 1.00 ± 0.02, R2 = 0.995, y0 = 3.3 ± 4.5). Both reproducibility and repeatability of digital counts were better than the manual method. Survival curves generated from digital and manual counts were not significantly different; P-values were 0.3646 for SK-BR-3 cells and 0.1818 for MCF-7/HER2-18 cells. Using blind parameters, survival curves generated by both methods showed some differences: P-values were 0.0897 for SK-BR-3 cells and 0.0024 for MCF-7/HER2-18 cells. Conclusions: The colony counting using ImageJ and customized macros with optimized parameters was a reliable method for quantifying the number of colonies.

Development and preclinical studies of 64Cu-NOTA-pertuzumab F(ab′)2 for imaging changes in tumor HER2 expression associated with response to trastuzumab by PET/CT

ABSTRACT We previously reported that microSPECT/CT imaging with 111In-labeled pertuzumab detected decreased HER2 expression in human breast cancer (BC) xenografts in athymic mice associated with response to treatment with trastuzumab (Herceptin). Our aim was to extend these results to PET/CT by constructing F(ab′)2 of pertuzumab modified with NOTA chelators for complexing 64Cu. The effect of the administered mass (5–200 µg) of 64Cu-NOTA-pertuzumab F(ab′)2 was studied in NOD/SCID mice engrafted with HER2-positive SK-OV-3 human ovarian cancer xenografts. Biodistribution studies were performed in non-tumor bearing Balb/c mice to predict radiation doses to normal organs in humans. Serial PET/CT imaging was conducted on mice engrafted with HER2-positive and trastuzumab-sensitive BT-474 or trastuzumab-insensitive SK-OV-3 xenografted mice treated with weekly doses of trastuzumab. There were no significant effects of the administered mass of 64Cu-NOTA-pertuzumab F(ab′)2 on tumor or normal tissue uptake. The predicted total body dose in humans was 0.015 mSv/MBq, a 3.3-fold reduction compared to 111In-labeled pertuzumab. MicroPET/CT images revealed specific tumor uptake of 64Cu-NOTA-pertuzumab F(ab′)2 at 24 or 48 h post-injection in mice with SK-OV-3 tumors. Image analysis of mice treated with trastuzumab showed 2-fold reduced uptake of 64Cu-NOTA-pertuzumab F(ab′)2 in BT-474 tumors after 1 week of trastuzumab normalized to baseline, and 1.9-fold increased uptake in SK-OV-3 tumors after 3 weeks of trastuzumab, consistent with tumor response and resistance, respectively. We conclude that PET/CT imaging with 64Cu-NOTA-pertuzumab F(ab′)2 detected changes in HER2 expression in response to trastuzumab while delivering a lower total body radiation dose compared to 111In-labeled pertuzumab.