Amplified delivery of indium-111 to EGFR-positive human breast cancer cells

A method is described to amplify the delivery of ¹¹¹In to human breast cancer cells utilizing a novel human serum albumin-human EGF (HSA-hEGF) bioconjugate substituted preferentially in the HSA domain with multiple DTPA metal chelators for ¹¹¹In. ¹¹¹In-DTPA-HSA-hEGF exhibited a lower receptor-binding affinity than ¹¹¹In-DTPA-hEGF but was rapidly and specifically bound, internalized and translocated to the nucleus in EGFR-positive MDA-MB-468 breast cancer cells. ¹¹¹In-DTPA-HSA-hEGF was cytotoxic in vitro mainly through the emission of short-range Auger electrons and partially through the effects of the hEGF moiety to MDA-MB-468 cells overexpressing EGFR (1–2 × 10⁶ receptors/cell) but not towards MCF-7 breast cancer cells with a 100-fold lower level of EGFR on their surface. The cytotoxicity in vitro against MDA-MB-468 cells of ¹¹¹In-DTPA-HSA-hEGF substituted with nine DTPA chelators was enhanced 4-fold compared to ¹¹¹In-DTPA-hEGF monosubstituted with DTPA. Studies are planned to further evaluate ¹¹¹In-DTPA-HSA-hEGF in vivo as a new imaging and targeted radiotherapeutic agent for breast cancer.     

A comparison of EGF and MAb 528 labeled with 111In for imaging human breast cancer.

Our objective was to compare 111In-labeled human epidermal growth factor (hEGF), a 53-amino acid peptide with anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb) 528 (IgG2a) for imaging EGFR-positive breast cancer. METHODS: hEGF and MAb 528 were derivatized with diethylenetriamine pentaacetic acid (DTPA) and labeled with 111In acetate. Receptor binding assays were conducted in vitro against MDA-MB-468 human breast cancer cells. Biodistribution and tumor imaging studies were conducted after intravenous injection of the radiopharmaceuticals in athymic mice bearing subcutaneous MCF-7, MDA-MB-231, or MDA-MB-468 human breast cancer xenografts or in severe combined immunodeficiency mice implanted with a breast cancer metastasis (JW-97 cells). MCF-7, MDA-MB-231, JW-97, and MDA-MB-468 cells expressed 1.5 x 10(4), 1.3 x 10(5), 2.7 x 10(5), and 1.3 x 106 EGFR/cell, respectively in vitro. RESULTS: 111In-DTPA-hEGF and 111In-DTPA-MAb 528 bound with high affinity to MDA-MB-468 cells (Ka of 7.5 x 10(8) and 1.2 x 10(8) L/mol, respectively). 111In-DTPA-hEGF was eliminated rapidly from the blood with < 0.2% injected dose/g (%ID/g) circulating at 72 h after injection, whereas 111In-DTPA-MAb 528 was cleared more slowly (3%ID/g in the blood at 72 h). Maximum localization of 111In-DTPA-hEGF in MDA-MB-468 tumors (2.2 %ID/g) was 10-fold lower than with 111In-DTPA-MAb 528 (21.6 %ID/g). There was high uptake in the liver and kidneys for both radiopharmaceuticals. Tumor-to-blood ratios were greater for 111In-labeled hEGF than for MAb 528 (12:1 versus 6:1), but all other tumor-to-normal tissue ratios were higher for MAb 528. MDA-MB-468 and JW-97 tumors were imaged successfully with both radiopharmaceuticals, but tumors were more easily visualized using 111In-labeled MAb 528. There was no direct quantitative relationship between EGFR expression on breast cancer cell lines in vitro, and tumor uptake of the radiopharmaceuticals in vivo, but control studies showed that tumor uptake was receptor mediated. CONCLUSION: Our results suggest that the tumor uptake in vivo of receptor-binding radiopharmaceuticals is controlled to a greater extent by their elimination rate from the blood than by the level of receptor expression on the cancer cells. Radiolabeled anti-EGFR MAbs would be more effective for tumor imaging in cancer patients than peptide-based radiopharmaceuticals such as hEGF, because they exhibit higher tumor uptake at only moderately lower tumor-to-blood ratios.     

Meta-[123I]iodobenzylguanidine is selectively radiotoxic to neuroblastoma cells at concentrations that spare cells of haematopoietic lineage

BACKGROUND: The Auger electron-emitting agents meta-[125I]iodobenzylguanidine (125I-MIBG) and 123I-MIBG have been proposed as alternatives to 131I-MIBG for the treatment of neuroblastoma, due to the absence of a cross-fire effect which may minimize bone marrow toxicity. However, the differential toxicity of 123I-MIBG towards neuroblastoma cells and cells of haematopoietic lineage has not been studied. OBJECTIVE: To compare the toxic effects of 123I-MIBG on SK-N-SH and SK-N-BE(2) neuroblastoma cells and on cells of haematopoietic lineage, specifically HL-60 human myeloid leukemia cells and bone marrow stem cells (BMSCs) from human adult donors. METHODS: The antiproliferative effects of exchange-labelled or no carrier added (n.c.a.) 123I-MIBG, unlabelled MIBG or the trimethylsilylbenzylguanidine (MTBG) precursor used to prepare n.c.a. 123I-MIBG against SK-N-SH or SK-N-BE(2) cells or HL-60 cells were evaluated using a cell proliferation assay. The toxicity of 123I-MIBG towards SK-N-SH cells or BMSCs from healthy adult human donors was studied using a clonogenic assay. RESULTS: 123I-MIBG was strongly growth inhibitory to SK-N-SH or SK-N-BE(2) cells at concentrations (IC50 185-370 mBq.ml(-1); IC90 740 mBq.ml(-1)) that were sparing to HL-60 cells. Treatment of SK-N-SH cells with 74 mBq of 123I-MIBG decreased colony formation by >90%, whereas colonies from all three populations of stem cells were formed at amounts up to 370 mBq. It was discovered that the MTBG precursor was non-specifically toxic towards both SK-N-SH cells and HL-60 cells, suggesting the need to purify n.c.a. 123I-MIBG for clinical use. CONCLUSION: Our results suggest that 123I-MIBG is a promising novel radiotherapeutic agent for neuroblastoma. For the first time, we report that the MTBG precursor used to prepare n.c.a. 123I-MIBG was toxic towards neuroblastoma cells as well as to HL-60 cells, representing cells of the haematopoietic lineage, suggesting the need for purification.     

Molecular therapy of human neuroblastoma cells using Auger electrons of 111In-labeled N-myc antisense oligonucleotides.

Auger electrons can create breaks in nucleic acids, giving them possible therapeutic utility. We investigated the therapeutic effect of Auger electrons emitted by 111In-labeled phosphorothioate antisense oligonucleotides on human neuroblastoma cells in which N-myc was overexpressed. METHODS: Human SK-N-DZ neuroblastoma cells (5 x 10(6) cells) were treated with cationic reverse-phase evaporation vesicles (REVs) encapsulating 111In-labeled antisense (40 MBq/2 nmol of oligonucleotides/mumol of total phospholipids) that had an average diameter of 250 nm. Hybridization of the radiolabeled oligonucleotides with N-myc messenger RNA (mRNA), N-myc expression, and cell proliferation were investigated. The tumorigenicity of treated cells was analyzed in nude mice. Nonradiolabeled antisense, 111In-labeled sense, or empty cationic REVs were used as controls. RESULTS: 111In-Labeled antisense, which hybridized with N-myc mRNA, was detected in cells at 12 and 24 h after the initiation of treatment. Reduced N-myc expression and inhibited cell proliferation were shown in the same cells at 48 h after the completion of treatment. N-myc expression-suppressed cells produced intraperitoneal tumors in nude mice, but the average weight of the tumors was lower than that of tumors in control mice. CONCLUSION: Auger electrons emitted from 111In in close proximity to their target N-myc mRNA may prolong the time to cell proliferation in human neuroblastoma cells due to inhibition of the translation of N-myc. Auger electron therapy therefore has potential as an internally delivered molecular radiotherapy targeting the mRNA of a tumor cell.     

131I-recombinant human EGF has antitumor effects against MCF-7 human breast cancer xenografts with low levels of EGFR

This study investigated the inhibitory action of (131)I-recombinant human EGF ((131)I-rhEGF) on MCF-7 human breast cancer tumor development in nude mice. The activity and tumor uptake of (131)I-rhEGF was measured by tissue distribution assay, and its effect on tumor growth was measured by monitoring tumor size after treatment with (131)I-rhEGF. Changes in tumor cell ultrastructure were observed by transmission electron microscopy (TEM), and pathological changes in tumor tissue were observed by light microscopy. The tissue distribution assay revealed that (131)I-rhEGF was markedly absorbed by the tumor and reached its maximal uptake rate (16.73%ID. g(-1)) at 120 hours at which point the drug concentration in the tumor was 11.1-fold, 8.1-fold, and 6.6-fold higher than that in blood, liver, and kidneys, respectively. Tumor size measurements showed that tumor development was significantly inhibited by intravenously and intratumorally injected (131)I-rhEGF. Tumor inhibition rates (82.0% and 80.7%, respectively) were significantly higher than those of tumors treated with (131)I (7.49%) and (131)I-HSA (6.91%; P < 0.05). TEM and light microscopy revealed that intravenous and intratumoral injection of (131)I-rhEGF could significantly damage and ultimately kill tumor cells. Our results suggest that (131)I-rhEGF suppresses development of xenografted breast cancer cells in nude mice, providing a novel candidate for receptor-mediated targeted radiotherapy.     

Antitumor effects and normal tissue toxicity of 111In-labeled epidermal growth factor administered to athymic mice bearing epidermal growth factor receptor-positive human breast cancer xenografts.

The epidermal growth factor receptor (EGFR) is an attractive target for the design of radiotherapeutic agents for breast cancer because it is present on almost all estrogen receptor-negative, hormone-resistant tumors with a poor prognosis. In this study, we describe the antitumor effects and normal tissue toxicity of the novel Auger electron-emitting radiopharmaceutical (111)In-labeled diethylenetriaminepentaacetic acid-human epidermal growth factor ((111)In-DTPA-hEGF) administered to athymic mice bearing EGFR-positive human breast cancer xenografts. METHODS: Mice bearing subcutaneous MDA-MB-468 or MCF-7 human breast cancer xenografts were treated with 5 weekly doses of (111)In-DTPA-hEGF (total, 27.7-92.5 MBq or 5-17 micro g). Treatment was commenced 6 wk after tumor cell implantation (established tumors) or 1 wk after implantation (nonestablished tumors). Antitumor effects were assessed by use of the slope of the tumor growth curve. Normal tissue toxicity was assessed by use of plasma alanine transaminase and creatinine levels, hematologic indices (leukocytes, platelets, erythrocytes, and hemoglobin), histopathologic examination of the liver and kidneys, and changes in body weight. The uptake of (111)In-DTPA-hEGF in tumors of different sizes (<5-200 mm(3)) was investigated, and microdosimetry estimates were calculated. RESULTS: (111)In-DTPA-hEGF exhibited strong antitumor effects against established MDA-MB-468 xenografts, decreasing their growth rate 3-fold compared with that in normal saline-treated mice (slopes, 0.0225 and 0.0737 d(-1), respectively; P = 0.002). The antitumor effects of (111)In-DTPA-hEGF were much more profound in mice with small, nonestablished MDA-MB-468 tumors, which regressed, than in saline-treated mice (slopes, -0.009 and 0.0297 d(-1), respectively; P < 0.001). The growth of MCF-7 xenografts, with a 100-fold-lower level of EGFR expression, was modestly inhibited by (111)In-DTPA-hEGF compared with that in saline-treated mice (slopes, 0.0250 and 0.0488 d(-1), respectively; P = 0.051). There was a 1.4- to 2-fold decrease in leukocyte and platelet counts with (111)In-DTPA-hEGF treatment, but these counts remained in the normal ranges. There was no change in other biochemical or hematologic parameters or body weight. There was no evidence of morphologic damage to the liver or kidneys. A strong inverse relationship was observed between radiopharmaceutical uptake and tumor size, with small tumors (<5 mm(3)) accumulating >30% of the injected dose (%ID) per gram, compared with 5 %ID/g for tumors measuring 6-30 mm(3). Exceptionally high uptake (>80 %ID/g) was achieved in tumors measuring 1-2 mm(3). Microdosimetry estimates indicated that the nucleus of an MDA-MB-468 cell would receive 90-1,400 cGy, depending on the level of radiopharmaceutical uptake. CONCLUSION: (111)In-DTPA-hEGF exhibited strong antitumor effects against MDA-MB-468 breast cancer xenografts overexpressing EGFR. The highest tumor localization, radiation-absorbed doses, and growth inhibition were achieved for small, nonestablished tumors, suggesting that the radiopharmaceutical may be most valuable for the treatment of small-volume metastatic breast cancer or occult micrometastases in an adjuvant setting.     

111In-labeled galectin-3-targeting peptide as a SPECT agent for imaging breast tumors.

Galectin-3 is a member of the galectin family of beta-galactoside-binding animal lectins. Galectin-3 is overexpressed in a wide range of neoplasms and is associated with tumor growth and metastases. Given this fact, radiolabeled galectin-3-targeting molecules may be useful for the noninvasive imaging of tumors expressing galectin-3, as well as for targeted radionuclide therapy. In this study, the tumor cell-targeting and SPECT properties of a galectin-3-avid peptide identified from bacteriophage display were evaluated in human breast carcinoma cells and in human breast tumor-bearing mice. METHODS: The galectin-3-avid peptide G3-C12 (ANTPCGPYTHDCPVKR) was synthesized with a Gly-Ser-Gly (GSG) linker at the amino terminus. After conjugation with 1,4,7,10-tetra-azacyclododecane-N,N',N'N'-tetraacetic acid (DOTA), the peptide was labeled with (111)In. The radiochemical purity and stability of the compound was assessed by high-performance liquid chromatography. MDA-MB-435 human breast carcinoma cells expressing galectin-3 were used to characterize the in vitro binding properties of the radiolabeled compound. SCID mice bearing MDA-MB-435 xenografts were used as an in vivo model for biodistribution and imaging studies with the (111)In-labeled peptide. RESULTS: (111)In-DOTA(GSG)-G3-C12 bound specifically to galectin-3-expressing MDA-MB-435 cells. The radiolabeled peptide was stable in serum and was found intact in excreted urine for at least 1 h. Competitive binding experiments indicated that the radiolabeled peptide exhibited an inhibitory concentration of 50% of 200.00+/-6.70 nM for cultured breast carcinoma cells. In vivo biodistribution studies revealed that tumor uptake was 1.2+/-0.24, 0.75+/-0.05, and 0.6+/-0.04 (mean +/- SD) percentage injected dose per gram at 30 min, 1.0 h, and 2.0 h after injection of the radiotracer, respectively. SPECT/CT studies with (111)In-DOTA(GSG)-G3-C12 showed excellent tumor uptake and contrast in the tumor-bearing mice. Specificity of peptide binding was demonstrated by successful blocking (52%) of in vivo tumor uptake of (111)In-DOTA(GSG)-G3-C12 in the presence of its nonradiolabeled counterpart at 2 h after injection. CONCLUSION: This study demonstrated the successful use of a new radiolabeled peptide for the noninvasive imaging of galectin-3-positive breast tumors. This peptide may be a promising candidate for future clinical applications.     

Novel series of 111In-labeled bombesin analogs as potential radiopharmaceuticals for specific targeting of gastrin-releasing peptide receptors expressed on human prostate cancer cells.

Gastrin-releasing peptide (GRP) receptors have been shown to be expressed with high densities on several types of cancer cells including prostate, breast, small cell lung, and pancreas cancers. Bombesin (BBN) has been known to bind to GRP receptors with high affinity and specificity. The aim of these studies was to develop new (111)In-labeled BBN analogs having high tumor uptake and optimal pharmacokinetics for specific targeting of human prostate cancers. METHODS: A novel series of dodecanetetraacetic acid (DOTA)-X-BBN[7-14]NH(2) (X = 0, beta-Ala, 5-Ava, 8-Aoc, or 11-Aun) conjugates and their In(III)/(111)In complexes exhibiting high GRP-receptor-binding affinities were synthesized and characterized. RESULTS: In vitro competitive binding assays, using PC-3 androgen-independent human prostate cancer cells, demonstrated values of <2.5 nmol/L for inhibitory concentration of 50% for analogs with beta-Ala, 5-Ava, and 8-Aoc spacers. In vivo biodistribution studies of the (111)In-DOTA-X-BBN[7-14]NH(2) conjugates performed on CF-1 mice at 1 h after injection have revealed that the uptake of radioactivity in the pancreas, a GRP-receptor-expressing tissue, increased as a function of hydrocarbon spacer length (i.e., from 0.20 +/- 0.04 percentage injected dose [%ID] per gram for the analog with no spacer to a maximum of 26.97 +/- 3.97 %ID/g for the analog with 8-Aoc spacer). The radioactivity was cleared efficiently from the blood pool by excretion mainly through the renal/urinary pathway (e.g., 71.6 +/- 1.8 %ID at 1 h after injection for 8-Aoc spacer analog). In vivo pharmacokinetic studies of the (111)In-DOTA-8-Aoc-BBN[7-14]NH(2) conjugate conducted on PC-3 human prostate cancer-derived xenografts in SCID mice showed a specific uptake of radioactivity in tumor, with 3.63 +/- 1.11 %ID/g observed at 1 h after injection. High tumor-to-blood and tumor-to-muscle ratios of approximately 6:1 and 45:1, respectively, were achieved at 1 h after injection. Relative to the radioactivity observed in the tumor at 1 h after injection, 43%, 19%, and 9% of the radioactivity was retained at, respectively, 24, 48, and 72 h after injection. CONCLUSION: These studies showed that radiometallated DOTA-X-BBN[7-14]NH(2) constructs with hydrocarbon spacers ranging from 5 to 8 carbon atoms are feasible candidates for further development as diagnostic and therapeutic radiopharmaceuticals for patients with GRP-positive cancers.     

111In-labeled CD34+ hematopoietic progenitor cells in a rat myocardial infarction model.

Transplantation of progenitor cells (PCs) has been shown to improve neovascularization and left ventricular function after myocardial ischemia. The fate of transplanted PCs has been monitored by fluorescence labeling or by genetic modifications introducing reporter genes. However, these techniques are limited by the need to kill the experimental animal. The aim of this study was to radiolabel CD34(+) hematopoietic PCs (HPCs) with (111)In-oxine and to evaluate the feasibility of this in vivo method for monitoring myocardial homing of transplanted cells in a rat myocardial infarction model. METHODS: Human HPCs were isolated from mobilized peripheral blood and labeled with (111)In-oxine. Labeled HPCs were injected into the cavity of the left ventricle in nude rats 24 h after induction of myocardial infarction (n = 4) or sham operation (n = 4). Scintigraphic images were acquired up to 96 h after HPC injection. After animals were killed, tissue samples of various organs were harvested to calculate tissue-specific activity and for immunostaining. RESULTS: Labeling efficiency of HPCs was 32% +/- 11%. According to trypan-blue staining, viability of radiolabeled HPCs was impaired by 30% after 48 and 96 h in comparison with unlabeled cells, whereas proliferation and differentiation of HPCs was nullified after 7 d, as assessed by colony-forming assays. After injection of HPCs, the specific activity ratio of heart to peripheral muscle tissue increased from 1.10 +/- 0.32 in sham-operated rats to 2.47 +/- 0.92 (P = 0.020) in infarcted rats. However, the overall radioactivity detected in the heart was only about 1%. A transient high lung uptake of 17% +/- 6% was observed within the first hour after infusion of HPCs. At 24 h after injection, the initial lung activity had shifted toward liver, kidneys, and spleen, resulting in an increase of radioactivity in these organs from 37% +/- 6% to 57% +/- 5%. CONCLUSION: Radiolabeling with (111)In-oxine is a feasible in vivo method for monitoring transplanted HPCs in a rat myocardial infarction model. The potential to detect differences in myocardial homing between infarcted and normal hearts suggests that this method may provide a noninvasive imaging approach for clinical trials using transplanted HPCs in patients. Our findings, however, also demonstrated a negative effect of (111)In-oxine on cellular function, which resulted in complete impairment of HPC proliferation and differentiation. For future trials in stem cell imaging with (111)In-oxine, therefore, it will be mandatory to carefully check for radiation-induced cell damage.     

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 gamma-radiation. The cell lines (followed by number of EGFR per cell in parentheses) were MDA-MB-468 (1.3 x 10(6)), MDA-MB-231 (1.3 x 10(5)), and MCF-7 (1.5 x 10(4)). The proportion of radioactivity partitioning into the nucleus was measured by cell fractionation. DNA double-strand breaks were evaluated using the gamma-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 gamma-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 gamma-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 gamma-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 gamma-H2AX assay may be a useful biomarker to predict and monitor the outcome of treatment with 111In-DTPA-hEGF.     

(111)In-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 (111)In were evaluated. METHODS: Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) for reaction with NLS-peptides and labeled with (111)In using diethylenetriaminepentaacetic acid (DTPA). The immunoreactivity of (111)In-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 (111)In-trastuzumab and (111)In-NLS-trastuzumab was studied by clonogenic assays, and DNA damage was assessed by probing for phosphorylated histone H2AX (gammaH2AX) foci. RESULTS: The dissociation constant for binding of (111)In-NLS-trastuzumab to SK-BR-3 cells was reduced <3-fold compared with that of (111)In-trastuzumab, demonstrating relatively preserved receptor-binding affinity. The receptor-mediated internalization of (111)In-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 (111)In-NLS-trastuzumab harboring 6 NLS-peptides, respectively. NLS-trastuzumab localized in the nuclei of BC cells, whereas unmodified trastuzumab remained surface-bound. Conjugation of (111)In-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 (111)In-NLS-trastuzumab and 1.1 %ID/g for (111)In-trastuzumab). (111)In-NLS-trastuzumab was 5- and 2-fold more potent at killing SK-BR-3 and MDA-MB-361 cells than (111)In-trastuzumab, respectively, whereas toxicity toward MDA-MB-231 cells was minimal. (111)In-NLS-trastuzumab was 6-fold more effective at killing SK-BR-3 cells than unlabeled trastuzumab. Formation of gammaH2AX foci occurred in a greater proportion of BC cells after incubation with (111)In-NLS-trastuzumab compared with (111)In-trastuzumab or unlabeled trastuzumab. CONCLUSION: NLS-peptides routed (111)In-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 (111)In-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.     

Effect of the EGFR density of breast cancer cells on nuclear importation, in vitro cytotoxicity, and tumor and normal-tissue uptake of [111In]DTPA-hEGF

INTRODUCTION: Our objective was to evaluate the effect of epidermal growth factor receptor(s) (EGFR) density on the importation and nuclear localization of 111In-labeled diethylenetriaminepentaacetic acid human epidermal growth factor ([111In]DTPA-hEGF) in breast cancer (BC) cells in vitro and in tumor xenografts and normal tissues in vivo in athymic mice, as well as on its cytotoxicity and tumor and normal-tissue distribution. METHODS: The internalization and nuclear importation of [111In]DTPA-hEGF were measured in MCF-7, MDA-MB-231, BT-474 and MDA-MB-468 BC cells (10(4), 2 x 10(5), 6 x 10(5) and 10(6) EGFR/cell, respectively). The molecular size (Mr) distribution and immunoreactivity of nuclear radioactivity were characterized. Tumor and normal-tissue uptake of [111In]DTPA-hEGF in athymic mice implanted subcutaneously with BC xenografts were compared. Nuclear radioactivity in the tumor, lungs, liver, kidneys, spleen and colon was measured. RESULTS: There was a direct association between EGFR density and the nuclear localization of [111In]DTPA-hEGF in BC cells; nuclear importation approached saturation at 6 x 10(5) EGFR/cell. Almost all nuclear radioactivities exhibited an Mr of >100 kDa; immunoreactivity with anti-hEGF, anti-EGFR and anti-importin beta 1 antibodies was detected. The efflux of nuclear radioactivity was slowest for MDA-MB-468 cells. Cytotoxicity was correlated with EGFR expression. Uptake was greater in MDA-MB-468 than in MCF-7 xenografts and improved with preinjection of a 100-fold excess of unlabeled DTPA-hEGF. Nuclear importation was higher in liver, kidney and spleen cells than in tumor cells. CONCLUSION: [111In]DTPA-hEGF is translocated to the nucleus of BC cells complexed with EGFR and importin beta1. Nuclear importation and cytotoxicity are effected by EGFR density. The absence of hepatic and renal toxicities in [111In]DTPA-hEGF cannot be explained by a low efficiency of nuclear importation.     

乙醇降低人乳腺癌MCF-7细胞放射敏感性的研究

目的 研究乙醇对人乳腺癌MCF-7细胞放射敏感性的影响及相关机制.方法 将人乳腺癌MCF-7细胞分为4组,对照组(不做任何处理)、乙醇组(乙醇处理)、单纯照射组(6 GyX射线处理)和联合组(乙醇与X射线联合作用).克隆形成法检测50或100 mmol/L乙醇对MCF-7细胞放射敏感性的影响;流式细胞术检测细胞周期变化;Annexin V-FITC法检测细胞凋亡.结果 50和100 mmol/L乙醇处理MCF-7细胞50 h,对生长无明显影响(t--0.82和1.15,P＞0.05);乙醇预处理2h,可明显增加X射线照射后MCF-7细胞克隆形成的能力(t=4.15和10.28,P＜0.05).相比于单纯照射组,乙醇降低了X射线照射诱导的细胞G2/M期阻滞(t=7.18,P＜0.05),以及sub-G1 峰的比例(t =5.39,P＜0.05).而且,Annexin V-FITC检测结果示,乙醇联合X射线照射的细胞晚期和早期凋亡减少(t=4.86和7.59,P＜0.05).结论 乙醇可以增加MCF-7细胞的辐射抗性,其机制可能与降低辐射诱导的细胞G2/M期阻滞及早、晚期凋亡的发生相关.     

Thermal dosimetry predictive of efficacy of 111In-ChL6 nanoparticle AMF--induced thermoablative therapy for human breast cancer in mice.

Antibody (mAb)-linked iron oxide nanoparticles (bioprobes) provide the opportunity to develop tumor specific thermal therapy (Rx) for metastatic cancer when inductively heated by an externally applied alternating magnetic field (AMF). To evaluate the potential of this Rx, in vivo tumor targeting, efficacy, and predictive radionuclide-based heat dosimetry were studied using (111)In-ChL6 bioprobes (ChL6 is chimeric L6) in a human breast cancer xenograft model. METHODS: Using carbodiimide, (111)In-DOTA-ChL6 (DOTA is dodecanetetraacetic acid) was conjugated to polyethylene glycol-iron oxide-impregnated dextran 20-nm particles and purified as (111)In-bioprobes. (111)In doses of 740-1,110 kBq (20-30 muCi) (2.2 mg of bioprobes) were injected intravenously into mice bearing HBT3477 human breast cancer xenografts. Pharmacokinetic (PK) data were obtained at 1, 2, 3, and 5 d. AMF was delivered 72 h after bioprobe injection at amplitudes of 1,410 (113 kA/m), 1,300 (104 kA/m), and 700 (56 kA/m) oersteds (Oe) at 30%, 60%, and 90% "on" time (duty), respectively, and at 1,050 Oe (84 kA/m) at 50% and 70% duty over the 20-min treatment. Treated and control mice were monitored for 90 d. Tumor total heat dose (THD) from activated tumor bioprobes was calculated for each Rx group using (111)In-bioprobe tumor concentration and premeasured particle heat response to AMF amplitudes. Tumor growth delay was analyzed by Wilcoxon rank sum comparison of time to double, triple, and quintuple tumor volume in each group, and all groups were compared with the controls. RESULTS: Mean tumor concentration of (111)In-bioprobes at 48 h was 14 +/- 2 percentage injected dose per gram; this concentration 24 h before AMF treatment was used to calculate THD. No particle-related toxicity was observed. Toxicity was observed at the highest AMF amplitude-duty combination of 1,300 Oe and 60% over 20 min; 6 of 10 mice died acutely. Tumor growth delay occurred in all of the other groups, correlated with heat dose and, except for the lowest heat dose group, was statistically significant when compared with the untreated group. Electron microscopy showed (111)In-bioprobes on tumor cells and cell death by necrosis at 24 and 48 h after AMF. CONCLUSION: mAb-guided bioprobes (iron oxide nanoparticles) effectively targeted human breast cancer xenografts in mice. THD, calculated using empirically observed (111)In-bioprobe tumor concentration and in vitro nanoparticle heat induction by AMF, correlated with tumor growth delay.     

p53-dependent radiobiological responses to internalised indium-111 in human cells

IntroductionThe p53 tumour suppressor protein plays a pivotal role in the response of mammalian cells to DNA damage. It regulates cell cycle progression, apoptosis and DNA repair mechanisms and is therefore likely to influence response to targeted radionuclide therapy. This study investigated the role of p53 in the cellular response to the Auger-emitting radionuclide indium-111.MethodsTwo stable clones of a HT1080 fibrosarcoma cell line, differing only in p53 status due to RNAi-mediated knockdown of p53 expression, were incubated for 1 h with [¹¹¹In]-oxinate (0–10 MBq/ml). Radiopharmaceutical uptake into HT1080 cells was measured in situ using a non-contact phosphorimager method. Cellular sensitivity and DNA damage were measured by, respectively, clonogenic survival analysis and the single cell gel electrophoresis (Comet) assay.ResultsMean uptake of [¹¹¹In]-oxinate in HT1080 cells was unaffected by p53 status, reaching a maximum of 9 Bq/cell. [¹¹¹In]-oxinate-induced cytotoxicity was also identical in both clones, as measured by IC50 (0.68 MBq/ml). However the formation of DNA damage, measured immediately after treatment with [¹¹¹In]-oxinate, was found to be up to 2.5-fold higher in the p53-deficient HT1080 clone.ConclusionsThe increased DNA damage induced in p53-deficient HT1080 cells suggests an early deficiency in the repair of DNA damage during the treatment period. However, the similarity in cellular sensitivity, irrespective of p53 status, suggests that reduced p53 leads to a concomitant reduction in p53-dependent cytotoxicity despite the persistence of DNA damage. The results may provide insight into how tumours that differ in p53 status respond to therapeutic radionuclides.     

Specific nanomarkers fluorescence: in vitro analysis for EGFR overexpressed cells in triple-negative breast cancer and malignant glioblastoma

BackgroundEpidermal Growth Factor Receptor (EGFR receptor) is encoded by the EGFR gene. EGFR receptor signaling pathways are activated by EGF protein, regulating cell actions. Overexpression of EGFR receptor may be linked to malignancies with a poor prognosis. As a result, EGFR receptor is being studied for a variety of tumor diagnostics, spurring the development of innovative approaches to increase quality and efficiency. Nanomaterials can recognize cancer cells by specifically targeting of molecular pathways, underscoring the importance of nanomedicine. In this study, we synthesized EGFR-specific nanomarkers by functionalizing EGF protein and Chlorin e6 in gold nanoparticles. These nanoparticles use active targeting to deliver EGF protein to EGFR receptor, and Chlorin e6 serves as a fluorescent marker moleculeMethodsNanomarkers were examined in vitro in MDA-MB-468 and M059J cell lines. Confocal microscopy and flow cytometry were used to examine the distribution, uptake, internalization, and fluorescence intensity of nanomarkers in vitroResultsThe results show that both lines examined accumulate nanomarkers. However, MDA-MB-468 had the highest intensity due to its EGFR receptor overexpression propertiesConclusionThe findings point to ideal properties for detecting EGFR receptor overexpressed cells.