Our aim was to synthesize Zr-labeled trastuzumab-emtansine (Zr-DFO-T-DM1) to probe the delivery of trastuzumab-emtansine (T-DM1) to HER2-positive breast cancer (BC) by positron emission tomography (PET). We further aimed to compare the tumor and normal tissue uptake of Zr-DFO-T-DM1 withZr-DFO-trastuzumab. T-DM1 was modified with 3.0 ± 0.2 desferrioxamine (DFO) chelators for complexing Zr by reaction with a 14-fold molar excess of p-NCS-Bz-DFO. The number of DFO chelators per T-DM1 molecule was quantified spectrophotometrically at 430 nm after the reaction with FeCl. SDS-PAGE and SE-HPLC demonstrated a pure and homogeneous immunoconjugate. DFO-T-DM1 and DFO-trastuzumab were labeled to high efficiency (>97%) with Zr at a specific activity of 0.55 MBq/μg in a 2 M NaCO/0.5 M HEPES buffer, pH 7.0, at RT for 60-90 min. The labeling efficiency was measured by instant thin layer-silica gel chromatography (ITLC-SG) and SE-HPLC. HER2 immunoreactivity was measured in a saturation binding assay using SK-BR-3 human BC cells. Zr-DFO-T-DM1 exhibited high affinity HER2 binding ( K = 3.7 ± 0.4 nM) that was not significantly different than Zr-DFO-trastuzumab (4.4 ± 0.5 nM; P = 0.06). The optimal time for tumor imaging withZr-DFO-T-DM1 was 96 h post-injection in NOD-scid mice with s.c. HER2 overexpressing (HER2 3+) BT-474 human BC xenografts. Tumor uptake was dependent on the level of HER2 expression in mice with s.c. BT-474 (HER2 3+), MDA-MB-231/H2N (HER2 2+), MDA-MB-231 (HER2 0-1+), or MDA-MB-468 (HER2 0) human BC xenografts injected with Zr-DFO-T-DM1 (10 μg, 5.2 MBq). All tumors were visualized by microPET/CT, but the tumor intensity was greatest for BT-474 and MDA-MB-231/H2N xenografts. The tumor uptake ofZr-DFO-T-DM1 was 4.1-fold significantly higher than Zr-DFO-trastuzumab in mice with s.c. BT-474 (HER2 3+) xenografts (43.5 ± 4.3%ID/g vs 10.6 ± 5.4%ID/g, respectively; P< 0.001). Tumor uptake of Zr-DFO-T-DM1 in MDA-MB-231/H2N xenografts (HER2 2+) was 3.7-fold significantly higher thanZr-DFO-trastuzumab (10.1 ± 3.6%ID/g vs 2.7 ± 0.5%ID/g; P < 0.001). The higher tumor uptake of Zr-DFO-T-DM1 compared toZr-DFO-trastuzumab was not due to a higher HER2 binding affinity or to differences in the residence time in the blood or tumor size. We conclude that Zr-DFO-T-DM1 is a useful probe to assess the delivery of T-DM1 to HER2-positive BC. PET withZr-DFO-trastuzumab has been studied clinically to predict response to T-DM1, but our results suggest that Zr-DFO-T-DM1 may be more accurate due to the differences in the tumor uptake observed in the preclinical BC xenograft mouse models.

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In this study, we investigated convection-enhanced delivery (CED) of 23 ± 3 nm gold nanoparticles (AuNPs) labeled with the β-particle-emitting radionuclide 177 Lu ( 177 Lu-AuNPs) for treatment of orthotopic U251-Luc human glioblastoma multiforme (GBM) tumors in NRG mice. The cytotoxicity in vitro of 177 Lu-AuNPs (0.0−2.0 MBq, 4 × 10 11 AuNPs) on U251-Luc cells was also studied by a clonogenic survival assay, and DNA double-strand breaks (DSBs) caused by β-particle emissions of 177 Lu were measured by confocal immunofluorescence microscopy for γH2AX. NRG mice with U251-Luc tumors in the right cerebral hemisphere of the brain were treated by CED of 1.1 ± 0.2 MBq of 177 Lu-AuNPs (4 × 10 11 AuNPs). Control mice received unlabeled AuNPs or normal saline. Tumor retention of 177 Lu-AuNPs was assessed by single-photon emission computed tomography/ computed tomography (SPECT/CT) imaging and biodistribution studies. Radiation doses were estimated for the tumor, brain, and other organs. The effectiveness for treating GBM tumors was determined by bioluminescence imaging (BLI) and T2-weighted magnetic resonance imaging (MRI) and by Kaplan−Meier median survival. Normal tissue toxicity was assessed by monitoring body weight and hematology and blood biochemistry analyses at 14 d post-treatment. 177 Lu-AuNPs (2.0 MBq, 4 × 10 11 AuNPs) decreased the clonogenic survival of U251-Luc cells to 0.005 ± 0.002 and increased DNA DSBs by 14.3-fold compared to cells treated with unlabeled AuNPs or normal saline. A high proportion of 177 Lu-AuNPs was retained in the U251-Luc tumor in NRG mice up to 21 d with minimal re-distribution to the brain or other organs. The radiation dose in the tumor was high ( 599Gy). The dose in the normal right cerebral hemisphere of the brain excluding the tumor was 93-fold lower (6.4 Gy), and 2000−3000-fold lower doses were calculated for the contralateral left cerebral hemisphere (0.3 Gy) or cerebellum (0.2 Gy). The doses in peripheral organs were <0.1 Gy. BLI revealed almost complete tumor growth arrest in mice treated with 177 Lu-AuNPs, while tumors grew rapidly in control mice. MRI at 28 d post-treatment and histological staining showed no visible tumor in mice treated with 177 Lu-AuNPs but large GBM tumors in control mice. All control mice reached a humane endpoint requiring sacrifice within 39 d (normal saline) or 45 d post-treatment (unlabeled AuNPs), while 5/8 mice treated with 177 Lu-AuNPs survived up to 150 d. No normal tissue toxicity was observed in mice treated with 177 Lu-AuNPs. We conclude that CED of 177 Lu-AuNPs was highly effective for treating U251-Luc human GBM tumors in the brain in NRG mice at amounts that were non-toxic to normal tissues. These 177 Lu-AuNPs administered by CED hold promise for treating patients with GBM to prevent recurrence and improve long-term outcome.