Zhongli Cai

111In-Labeled Trastuzumab (Herceptin) Modified with Nuclear Localization Sequences (NLS): An Auger Electron-Emitting Radiotherapeutic Agent for HER2/neu-Amplified Breast Cancer

The cytotoxicity and tumor-targeting properties of the anti-HER2/neu monoclonal antibody trastuzumab modified with peptides (CGYGPKKKRKVGG) harboring the nuclear localization sequence ([NLS] italicized) of simian virus 40 large T-antigen and radiolabeled with 111In were evaluated. Methods: Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) for reaction with NLS-peptides and labeled with 111In using diethylenetriaminepentaacetic acid (DTPA). The immunoreactivity of 111In-NLS-trastuzumab was determined by its ability to displace the binding of trastuzumab to SK-BR-3 human breast cancer (BC) cells. Cellular uptake and nuclear localization were evaluated in SK-BR-3, MDA-MB-361, and MDA-MB-231 BC cells, expressing high, intermediate, or very low levels of HER2/neu, respectively, by cell fractionation and confocal microscopy. Biodistribution and nuclear uptake were compared in athymic mice bearing MDA-MB-361 xenografts. The cytotoxicity of 111In-trastuzumab and 111In-NLS-trastuzumab was studied by clonogenic assays, and DNA damage was assessed by probing for phosphorylated histone H2AX (γH2AX) foci. Results: The dissociation constant for binding of 111In-NLS-trastuzumab to SK-BR-3 cells was reduced <3-fold compared with that of 111In-trastuzumab, demonstrating relatively preserved receptor-binding affinity. The receptor-mediated internalization of 111In-trastuzumab in SK-BR-3, MDA-MB-361, and MDA-MB-231 cells increased significantly from 7.2% ± 0.9%, 1.3% ± 0.1%, and 0.2% ± 0.05% to 14.4% ± 1.8%, 6.3% ± 0.2%, and 0.9% ± 0.2% for 111In-NLS-trastuzumab harboring 6 NLS-peptides, respectively. NLS-trastuzumab localized in the nuclei of BC cells, whereas unmodified trastuzumab remained surface-bound. Conjugation of 111In-trastuzumab to NLS-peptides did not affect its tissue biodistribution but promoted specific nuclear uptake in MDA-MB-361 xenografts (2.4–2.9 %ID/g [percentage injected dose per gram] for 111In-NLS-trastuzumab and 1.1 %ID/g for 111In-trastuzumab). 111In-NLS-trastuzumab was 5- and 2-fold more potent at killing SK-BR-3 and MDA-MB-361 cells than 111In-trastuzumab, respectively, whereas toxicity toward MDA-MB-231 cells was minimal. 111In-NLS-trastuzumab was 6-fold more effective at killing SK-BR-3 cells than unlabeled trastuzumab. Formation of γH2AX foci occurred in a greater proportion of BC cells after incubation with 111In-NLS-trastuzumab compared with 111In-trastuzumab or unlabeled trastuzumab. Conclusion: NLS-peptides routed 111In-trastuzumab to the nucleus of HER2/neu-positive human BC cells, rendering the radiopharmaceutical lethal to the cells through the emission of nanometer−micrometer range Auger electrons. The greater cytotoxic potency of 111In-NLS-trastuzumab compared with unlabeled trastuzumab in vitro and its favorable tumor-targeting properties in vivo suggest that it could be an effective targeted radiotherapeutic agent for HER2/neu-amplified BC in humans.

Implications on clinical scenario of gold nanoparticle radiosensitization in regards to photon energy, nanoparticle size, concentration and location

Gold nanoparticle (AuNP) radiosensitization represents a novel approach to enhance the effectiveness of ionizing radiation. Its efficiency varies widely with photon source energy and AuNP size, concentration, and intracellular localization. In this Monte Carlo study we explored the effects of those parameters to define the optimal clinical use of AuNPs. Photon sources included (103)Pd and (125)I brachytherapy seeds; (169)Yb, (192)Ir high dose rate sources, and external beam sources 300 kVp and 6 MV. AuNP sizes were 1.9, 5, 30, and 100 nm. We observed a 10(3) increase in the rate of photoelectric absorption using (125)I compared to 6 MV. For a (125)I source, to double the dose requires concentrations of 5.33-6.26 mg g(-1) of Au or 7.10 × 10(4) 30 nm AuNPs per tumor cell. For 6 MV, concentrations of 1560-1760 mg g(-1) or 2.17 × 10(7) 30 nm AuNPs per cell are needed, which is not clinically achievable. Examining the proportion of energy transferred to escaping particles or internally absorbed in the nanoparticle suggests two clinical strategies: the first uses photon energies below the k-edge and takes advantage of the extremely localized Auger cascade. It requires small AuNPs conjugated to tumor targeted moieties and nuclear localizing sequences. The second, using photon sources above the k-edge, requires a higher gold concentration in the tumor region. In this approach, energy deposited by photoelectrons is the main contribution to radiosensitization; AuNP size and cellular localization are less relevant.

Design and characterization of HER-2-targeted gold nanoparticles for enhanced X-radiation treatment of locally advanced breast cancer.

Our purpose was to develop a human epidermal growth factor receptor-2 (HER-2) targeted nanotechnology-based radiosensitizer. HER-2 is overexpressed in 20-30% of all breast cancers and up to 2-fold higher in locally advanced disease (LABC). Trastuzumab was derivatized with a polyethylene glycol (OPSS-PEG-SVA) cross-linker to produce trastuzumab-PEG-OPSS. These immunoconjugates were analyzed by SDS-PAGE, and their immunoreactivity was assessed by flow cytometry using HER-2 overexpressing SK-BR-3 breast cancer cells. Reacting trastuzumab with increasing ratios of PEG resulted in an increase in molecular weight from approximately 148 kDa to 243 kDa, associated with increasing PEG substitution (0.6 to 18.9 PEG chains per trastuzumab). Attachment of approximately 7 PEG chains per trastuzumab resulted in 56% retention in immunoreactivity assessed by flow cytometry. The conjugates were then linked to 30 nm AuNPs. Using a novel (123)iodine-radiotracer based assay that overcomes the current limitations of spectrophotometric quantification of biological molecules on AuNPs we estimate 14.3 ± 2.7 antibodies were attached to each AuNP when 2 × 10(11) AuNPs were reacted with 20 μg of trastuzumab-PEG-OPSS. Specificity of trastuzumab-PEG-AuNPs for HER-2 and internalization in SK-BR-3 cells was demonstrated by comparing the uptake of trastuzumab-PEG-AuNPs or PEG-AuNPs by darkfield microscopy. The ability of trastuzumab-PEG-AuNPs in combination with 300 kVp X-rays to enhance DNA double strand breaks (DSBs) in SK-BR-3 cells was assessed by immunofluorescence using the γ-H2AX assay. γ-H2AX assay results revealed 5.1-fold higher DNA-DSBs with trastuzumab-PEG-AuNPs and X-radiation as compared to treatment with X-radiation alone. The trastuzumab-PEG-AuNPs are a promising targeted nanotechnology-based radiosensitizer for improving LABC therapy. The design and systematic approaches taken to surface modify and characterize trastuzumab-PEG-AuNPs described in this study would have application to other molecularly targeted AuNPs for cancer treatment.

Role of Antibody-Mediated Tumor Targeting and Route of Administration in Nanoparticle Tumor Accumulation in Vivo

In this study, we have looked at enhancing tumor uptake and intracellular delivery of gold nanoparticles (AuNPs) while reducing the systemic exposure by systematic evaluation of the impact of targeting and route of administration on organ distribution. High-resolution microSPECT/CT imaging was used to track the in vivo fate of (111)In-labeled nontargeted and human epidermal growth factor receptor-2 (HER-2) targeted AuNPs following intravenous (i.v.) or intratumoral (i.t.) injection. For i.v. injection, the effects of GdCl3 (for deactivation of macrophages) and nonspecific (anti-CD20) antibody rituximab (for blocking of Fc mediated liver and spleen uptake) were studied. It was found that HER-2 targeting via attachment of trastuzumab paradoxically decreased tumor uptake as a result of faster elimination of the targeted AuNPs from the blood while improving internalization in HER-2-positive tumor cells as compared to nontargeted AuNPs. I.T. injections with HER-2 targeted AuNPs resulted in high tumor retention with low systemic exposure and represents an attractive delivery strategy. Our results provide a strategy for optimizing tumor delivery and quantifying organ distribution of this widely studied class of nanomaterial.

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.

Micro-SPECT/CT with 111In-DTPA-Pertuzumab Sensitively Detects Trastuzumab-Mediated HER2 Downregulation and Tumor Response in Athymic Mice Bearing MDA-MB-361 Human Breast Cancer Xenografts

Pertuzumab is a HER2 dimerization inhibitor that binds to an epitope unique from that of trastuzumab. Our objective was to determine whether SPECT with 111In-diethylenetriaminepentaacetic acid–pertuzumab (111In-DTPA-pertuzumab) could sensitively detect an early molecular response to trastuzumab manifested by HER2 downregulation and a later tumor response revealed by a decreased number of HER2-positive viable tumor cells. Methods: Changes in HER2 density in SKBr-3 and MDA-MB-361 BC cells exposed to trastuzumab (14 μg/mL) in vitro were measured by saturation binding assays using 111In-DTPA-pertuzumab and by confocal immunofluorescence microscopy and flow cytometry with fluorescein isothiocyanate–labeled HER2/neu antibodies. Imaging of HER2 downregulation was studied in vivo in athymic mice with subcutaneous MDA-MB-361 tumors treated for 3 d with trastuzumab (4 mg/kg) or nonspecific human IgG (hIgG) or phosphate-buffered saline (PBS). Imaging of tumor response to trastuzumab was studied in mice bearing subcutaneous MDA-MB-361 xenografts treated with trastuzumab (4 mg/kg), followed by weekly doses of nonspecific hIgG or rituximab or PBS (2 mg/kg). Mice were imaged on a micro-SPECT/CT system at 72 h after injection of 111In-DTPA-pertuzumab. Tumor and normal-tissue biodistribution was determined. Results: 111In-DTPA-pertuzumab saturation binding to SKBr-3 and MDA-MB-361 cells was significantly decreased at 72 h after exposure in vitro to trastuzumab (14 μg/mL), compared with untreated controls (62% ± 2%, P < 0.0001; 32% ± 9%, P < 0.0002, respectively). After 3 d of trastuzumab, in vivo tumor uptake of 111In-DTPA-pertuzumab decreased 2-fold in trastuzumab- versus PBS-treated mice (13.5 ± 2.6 percentage injected dose per gram [%ID/g] vs. 28.5 ± 9.1 %ID/g, respectively; P < 0.05). There was also a 2-fold decreased tumor uptake in trastuzumab- versus PBS-treated mice by image volume-of-interest analysis (P = 0.05), suggesting trastuzumab-mediated HER2 downregulation. After 3 wk of trastuzumab, tumor uptake of 111In-DTPA-pertuzumab decreased 4.5-fold, compared with PBS-treated mice (7.6 ± 0.4 vs. 34.6 ± 9.9 %ID/g, respectively; P < 0.001); this decrease was associated with an almost-completed eradication of HER2-positive tumor cells determined immunohistochemically. Conclusion: 111In-DTPA-pertuzumab sensitively imaged HER2 downregulation after 3 d of treatment with trastuzumab and detected a reduction in viable HER2-positive tumor cells after 3 wk of therapy in MDA-MB-361 human breast cancer xenografts.

Relationship Between Induction of Phosphorylated H2AX and Survival in Breast Cancer Cells Exposed to 111In-DTPA-hEGF

The Auger electron–emitting radiopharmaceutical 111In-diethylenetriaminepentaacetic acid human epidermal growth factor (111In-DTPA-hEGF) binds the epidermal growth factor receptor (EGFR), is internalized, and translocates to the nucleus. The purpose of this study was to investigate the relationship between EGFR expression, DNA damage, and cytotoxicity in cells exposed to 111In-DTPA-hEGF. Methods: Breast cancer cell lines with a range of EGFR expression levels were exposed to 111In-DTPA-hEGF or γ-radiation. The cell lines (followed by number of EGFR per cell in parentheses) were MDA-MB-468 (1.3 × 106), MDA-MB-231 (1.3 × 105), and MCF-7 (1.5 × 104). The proportion of radioactivity partitioning into the nucleus was measured by cell fractionation. DNA double-strand breaks were evaluated using the γ-H2AX assay. Clonogenic survival assays were used to measure cytotoxicity. Results: All data are presented as mean ± SD. The amount of 111In-DTPA-hEGF that translocated to the nucleus (in mBq/nucleus) in MDA-MB-468, MDA-MB-231, and MCF-7 cells incubated with 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h was 131 ± 6, 8.1 ± 0.1, and 1.1 ± 0.9, respectively. The number of γ-H2AX foci per nucleus was 35 ± 15, 19 ± 10, and 1.7 ± 0.3, respectively. A reduction in the surviving fraction (SF) in MDA-MB-468 (0.013 ± 0.001) and MDA-MB-231 (0.5 ± 0.1) but not in MCF-7 cells after exposure to 111In-DTPA-hEGF (5.2 MBq/mL, 43 nM) for 20 h has been demonstrated. The SF of MDA-MB-468 cells after exposure to DTPA-EGF (43 nM) and 111In-acetate (5.2 MBq/mL) for 20 h was 0.5 ± 0.1 and 0.53 ± 0.05, respectively. MDA-MB-468 was the most sensitive of the cell lines to γ-irradiation, with an SF after 2 Gy of 0.45 ± 0.04, compared with 0.7 ± 0.1 and 0.8 ± 0.1 for MCF-7 and MDA-MB-231, respectively. The number of γ-H2AX foci per nucleus in MDA-MB-468 cells correlated with the concentration, specific activity, and incubation time of 111In-DTPA-hEGF. Conclusion: DNA damage caused by 111In-DTPA-hEGF correlates with the EGFR expression level of the exposed cells and with concentration, specific activity, and incubation time of 111In-DTPA-hEGF. The γ-H2AX assay may be a useful biomarker to predict and monitor the outcome of treatment with 111In-DTPA-hEGF.

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.

Cellular Dosimetry of 111In Using Monte Carlo N-Particle Computer Code: Comparison with Analytic Methods and Correlation with In Vitro Cytotoxicity

Our objective was to compare Monte Carlo N-particle (MCNP) self- and cross-doses from 111In to the nucleus of breast cancer cells with doses calculated by reported analytic methods (Goddu et al. and Farragi et al.). A further objective was to determine whether the MCNP-predicted surviving fraction (SF) of breast cancer cells exposed in vitro to 111In-labeled diethylenetriaminepentaacetic acid human epidermal growth factor (111In-DTPA-hEGF) could accurately predict the experimentally determined values. Methods: MCNP was used to simulate the transport of electrons emitted by 111In from the cell surface, cytoplasm, or nucleus. The doses to the nucleus per decay (S values) were calculated for single cells, closely packed monolayer cells, or cell clusters. The cell and nucleus dimensions of 6 breast cancer cell lines were measured, and cell line–specific S values were calculated. Results: For self-doses, MCNP S values of nucleus to nucleus agreed very well with those of Goddu et al. (ratio of S values using analytic methods vs. MCNP = 0.962–0.995) and Faraggi et al. (ratio = 1.011–1.024). MCNP S values of cytoplasm and cell surface to nucleus compared fairly well with the reported values (ratio = 0.662–1.534 for Goddu et al.; 0.944–1.129 for Faraggi et al.). For cross doses, the S values to the nucleus were independent of 111In subcellular distribution but increased with cluster size. S values for monolayer cells were significantly different from those of single cells and cell clusters. The MCNP-predicted SF for monolayer MDA-MB-468, MDA-MB-231, and MCF-7 cells agreed with the experimental data (relative error of 3.1%, −1.0%, and 1.7%). The single-cell and cell cluster models were less accurate in predicting the SF. For MDA-MB-468 cells, relative error was 8.1% using the single-cell model and −54% to −67% using the cell cluster model. Individual cell-line dimensions had large effects on S values and were needed to estimate doses and SF accurately. Conclusion: MCNP simulation compared well with the reported analytic methods in the calculation of subcellular S values for single cells and cell clusters. Application of a monolayer model was most accurate in predicting the SF of breast cancer cells exposed in vitro to 111In-DTPA-hEGF.

Epidermal Growth Factor Receptor Inhibition Modulates the Nuclear Localization and Cytotoxicity of the Auger Electron–Emitting Radiopharmaceutical 111In-DTPA–Human Epidermal Growth Factor

111In-DTPA–human epidermal growth factor (111In-DTPA-hEGF [DTPA is diethylenetriaminepentaacetic acid]) is an Auger electron–emitting radiopharmaceutical that targets EGF receptor (EGFR)–positive cancer. The purpose of this study was to determine the effect of EGFR inhibition by gefitinib on the internalization, nuclear translocation, and cytotoxicity of 111In-DTPA-hEGF in EGFR-overexpressing MDA-MB-468 human breast cancer cells. Methods: Western blot analysis was used to determine the optimum concentration of gefitinib to abolish EGFR activation. Internalization and nuclear translocation of fluorescein isothiocyanate–labeled hEGF were evaluated by confocal microscopy in MDA-MB-468 cells (1.3 × 106 EGFRs/cell) in the presence or absence of 1 μM gefitinib. The proportion of radioactivity partitioning into the cytoplasm and nucleus of MDA-MB-468 cells after incubation with 111In-DTPA-hEGF for 24 h at 37°C in the presence or absence of 1 μM gefitinib was measured by cell fractionation. DNA double-strand breaks caused by 111In were quantified using the γ-H2AX assay, and radiation-absorbed doses were estimated. Clonogenic survival assays were used to measure the cytotoxicity of 111In-DTPA-hEGF alone or in combination with gefitinib. Results: Gefitinib (1 μM) completely abolished EGFR phosphorylation in MDA-MB-468 cells. Internalization and nuclear translocation of fluorescein isothiocyanate–labeled EGF were not diminished in gefitinib-treated cells compared with controls. The proportion of internalized 111In that localized in the nucleus was statistically significantly greater when 111In-DTPA-hEGF was combined with gefitinib compared with 111In-DTPA-hEGF alone (mean ± SD: 26.0% ± 5.5% vs. 14.6% ± 4.0%, respectively; P < 0.05). Induction of γ-H2AX foci was greater in MDA-MB-468 cells that were treated with 111In-DTPA-hEGF (250 ng/mL, 1.5 MBq/mL) plus gefitinib (1 μM) compared with those treated with 111In-DTPA-hEGF alone (mean ± SD: 35 ± 4 vs. 24 ± 5 foci per nucleus, respectively). In clonogenic assays, a significant reduction in the surviving fraction was observed when 111In-DTPA-hEGF (5 ng/mL, 6 MBq/μg) was combined with gefitinib (1 μM) compared with 111In-DTPA-hEGF alone (42.9% ± 5.7% vs. 22.9% ± 3.6%, respectively; P < 0.01). Conclusion: The efficacy of 111In-DTPA-hEGF depends on internalization and nuclear uptake of the radionuclide. Nuclear uptake, DNA damage, and cytotoxicity are enhanced when 111In-DTPA-hEGF is combined with gefitinib. These results suggest a potential therapeutic role for peptide receptor radionuclide therapy in combination with tyrosine kinase inhibitors.