Sbp2l contributes to oligodendrocyte maturation through translational control in Tcf7l2 signaling.

Oligodendrocytes (OLs) are the myelin-forming cells in the CNS that support neurons through the insulating sheath of axons. This unique feature and developmental processes are achieved by extrinsic and intrinsic gene expression programs, where RNA-binding proteins can contribute to dynamic and fine-tuned post-transcriptional regulation. Here, we identified SECIS-binding protein 2-like (Sbp2l), which is specifically expressed in OLs by integrated transcriptomics. Histological analysis revealed that Sbp2l is a molecular marker of OL maturation. Sbp2l knockdown (KD) led to suppression of matured OL markers, but not a typical selenoprotein, Gpx4. Transcriptome analysis demonstrated that Sbp2l KD decreased cholesterol-biosynthesis-related genes regulated by Tcf7l2 transcription factor. Indeed, we confirmed the downregulation of Tcf7l2 protein without changing its mRNA in Sbp2l KD OPCs. Furthermore, Sbp2l KO mice showed the decrease of Tcf7l2 protein and deficiency of OL maturation. These results suggest that Sbp2l contributes to OL maturation by translational control of Tcf7l2.

Potential benefit of the cathepsin S inhibitor, ASP1617, as a treatment for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the dysregulation of various cell types and immunological pathways. Autoantibodies play an important role in its pathogenesis. The presence of autoantibodies suggests that self-antigen presentation through major histocompatibility complex (MHC) class II on antigen presenting cells is involved in the pathogenesis of autoimmune diseases, including SLE. Cathepsin S (CatS) is a key protease for antigen peptide loading onto lysosomal/endosomal MHC class II molecules through invariant chain degradation to promote antigen presentation. Inhibition of CatS is therefore expected to suppress antigen presentation via MHC class II, T and B cell activation, and antibody production from B cells. Here, we report the pharmacological profile of ASP1617, a novel CatS inhibitor. ASP1617 induced invariant chain accumulation and decreased the expression level of MHC class ΙΙ on the cell surface in both mouse and human B cells. Further, ASP1617 prevented DO11.10 mice T cell proliferation to ovalbumin antigen. We investigated the effects of ASP1617 and mycophenolate mofetil (MMF) on the development of lupus-like nephritis in NZB/W F1 mice, a widely used SLE mouse model. Oral administration of ASP1617 suppressed anti-dsDNA IgG, prevented progression of lupus-like glomerulonephritis, and significantly prevented proteinuria excretion. In contrast, MMF did not suppress anti-dsDNA IgG. Further, we found that plasma and/or urine CatS levels were increased in specimens from NZB/W F1 mice and several SLE patients. These results indicate that CatS may be an attractive therapeutic target for the treatment of SLE.

DNA damage stress-induced translocation of mutant FUS proteins into cytosolic granules and screening for translocation inhibitors.

Fused in sarcoma/translated in liposarcoma (FUS) is an RNA-binding protein, and its mutations are associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), through the DNA damage stress response, aberrant stress granule (SG) formation, etc. We previously reported that translocation of endogenous FUS into SGs was achieved by cotreatment with a DNA double-strand break inducer and an inhibitor of DNA-PK activity. In the present study, we investigated cytoplasmic SG formation using various fluorescent protein-tagged mutant FUS proteins in a human astrocytoma cell (U251) model. While the synergistic enhancement of the migration of fluorescent protein-tagged wild-type FUS to cytoplasmic SGs upon DNA damage induction was observed when DNA-PK activity was suppressed, the fluorescent protein-tagged FUSP525L mutant showed cytoplasmic localization. It migrated to cytoplasmic SGs upon DNA damage induction alone, and DNA-PK inhibition also showed a synergistic effect. Furthermore, analysis of 12 sites of DNA-PK-regulated phosphorylation in the N-terminal LC region of FUS revealed that hyperphosphorylation of FUS mitigated the mislocalization of FUS into cytoplasmic SGs. By using this cell model, we performed screening of a compound library to identify compounds that inhibit the migration of FUS to cytoplasmic SGs but do not affect the localization of the SG marker molecule G3BP1 to cytoplasmic SGs. Finally, we successfully identified 23 compounds that inhibit FUS-containing SG formation without changing normal SG formation. Highlights Characterization of DNA-PK-dependent FUS stress granule localization.A compound library was screened to identify compounds that inhibit the formation of FUS-containing stress granules.

Copper-64-Labeled Antibody Fragments for Immuno-PET/Radioimmunotherapy with Low Renal Radioactivity Levels and Amplified Tumor-Kidney Ratios.

Copper-64 (64Cu)-labeled antibody fragments such as Fab are useful for molecular imaging (immuno-PET) and radioimmunotherapy. However, these fragments cause high and persistent localization of radioactivity in the kidneys after injection. To solve this problem, this study assessed the applicability of a molecular design to 64Cu, which reduces renal radioactivity levels by liberating a urinary excretory radiometabolite from antibody fragments at the renal brush border membrane (BBM). Since 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) forms a stable complex with Cu, NOTA-conjugated Met-Val-Lys-maleimide (NOTA-MVK-Mal), which is a radio-gallium labeling agent for antibody fragments, was evaluated for applicability to 64Cu. The MVK linkage was recognized by the BBM enzymes to liberate [64Cu]Cu-NOTA-Met although the recognition of the MVK sequence for the [64Cu]Cu-NOTA-MVK derivative was reduced compared with that of its [67Ga]Ga-counterpart, probably due to the difference in the charge of the metal-NOTA complexes. When injected into mice, [64Cu]Cu-NOTA-MVK-Fab resulted in similar renal radioactivity levels to the 67Ga-labeled counterpart. In addition, [64Cu]Cu-NOTA-MVK-Fab resulted in lower renal radioactivity levels than those from 64Cu-labeled Fab using a conventional method, without a reduction in the tumor radioactivity levels. These findings indicate that our approach to reducing renal radioactivity levels by liberating a radiolabeled compound from antibody fragments at the renal BBM for urinary excretion is applicable to 64Cu-labeled antibody fragments and useful for immuno-PET and radioimmunotherapy.

Effect of Gold Nanoparticle Radiosensitization on Plasmid DNA Damage Induced by High-Dose-Rate Brachytherapy.

PURPOSE: Gold nanoparticles (AuNPs) are candidate radiosensitizers for medium-energy photon treatment, such as γ-ray radiation in high-dose-rate (HDR) brachytherapy. However, high AuNP concentrations are required for sufficient dose enhancement for clinical applications. Here, we investigated the effect of positively (+) charged AuNP radiosensitization of plasmid DNA damage induced by 192Ir γ-rays, and compared it with that of negatively (-) charged AuNPs. METHODS: We observed DNA breaks and reactive oxygen species (ROS) generation in the presence of AuNPs at low concentrations. pBR322 plasmid DNA exposed to 64 ng/mL AuNPs was irradiated with 192Ir γ-rays via HDR brachytherapy. DNA breaks were detected by observing the changes in the form of the plasmid and quantified by agarose gel electrophoresis. The ROS generated by the AuNPs were measured with the fluorescent probe sensitive to ROS. The effects of positively (+) and negatively (-) charged AuNPs were compared to study the effect of surface charge on dose enhancement. RESULTS: +AuNPs at lower concentrations promoted a comparable level of radiosensitization by producing both single-stranded breaks (SSBs) and double-stranded breaks (DSBs) than those used in cell assays and Monte Carlo simulation experiments. The dose enhancement factor (DEF) for +AuNPs was 1.3 ± 0.2 for SSBs and 1.5 ± 0.4 for DSBs. The ability of +AuNPs to augment plasmid DNA damage is due to enhanced ROS generation. While -AuNPs generated similar ROS levels, they did not cause significant DNA damage. Thus, dose enhancement using low concentrations of +AuNPs presumably occurred via DNA binding or increasing local +AuNP concentration around the DNA. CONCLUSION: +AuNPs at low concentrations displayed stronger radiosensitization compared to -AuNPs. Combining +AuNPs with 192Ir γ-rays in HDR brachytherapy is a candidate method for improving clinical outcomes. Future development of cancer cell-specific +AuNPs would allow their wider application for HDR brachytherapy.

Positron emission tomography of cerebral angiogenesis and TSPO expression in a mouse model of chronic hypoxia.

The present study aimed to examine whether positron emission tomography (PET) could evaluate cerebral angiogenesis. Mice were housed in a hypoxic chamber with 8-9% oxygen for 4, 7, and 14 days, and the angiogenic responses were evaluated with a radiotracer, 64Cu-cyclam-RAFT-c(-RGDfK-)4, which targeted αVß3 integrin and was imaged with PET. The PET imaging results showed little uptake during all of the hypoxic periods. Immunofluorescence staining of the ß3 integrin, CD61, revealed weak expression, while the microvessel density assessed by CD31 staining increased with the hypoxic duration. These observations suggest that the increased vascular density originated from other types of vascular remodeling, unlike angiogenic sprouting. We then searched for any signs of vascular remodeling that could be detected using PET. PET imaging of 11C-PK11195, a marker of the 18-kDa translocator protein (TSPO), revealed a transient increase at day 4 of hypoxia. Because the immunofluorescence of glial markers showed unchanged staining over the early phase of hypoxia, the observed upregulation of TSPO expression probably originated from non-glial cells (e.g. vascular cells). In conclusion, a transient increase in TSPO probe uptake was detected with PET at only the early phase of hypoxia, which indicates an early sign of vascular remodeling induced by hypoxia.

Combined treatment of pancreatic cancer xenograft with 90Y-ITGA6B4-mediated radioimmunotherapy and PI3K/mTOR inhibitor.

AIM: To investigate the therapeutic effect of combined integrin α6ß4-targeted radioimmunotherapy (RIT) and PI3K/mTOR inhibitor BEZ235 in a pancreatic cancer model. METHODS: Phosphorylation of Akt, mTOR, the downstream effectors eukaryotic initiation factor 4E binding protein 1 (4EBP1) and S6 ribosomal protein (S6) were evaluated in BxPC-3 human pancreatic cancer cells treated with Yttrium-90 (90Y) labeled anti-integrin α6ß4 antibody (ITGA6B4) and BEZ235 by western blotting. The cytotoxic effect of BEZ235 was investigated using a colony formation assay. Therapeutic efficacy enhancement by oral BEZ235 administration was assessed using mice bearing BxPC-3 xenograft tumors. Tumor volume measurements and immunohistochemical analyses (cell proliferation marker Ki-67, DNA damage marker p-H2AX and p-4EBP1 staining) of tumors were performed for evaluation of combined treatment with 90Y-ITGA6B4 plus BEZ235, or each arm alone. RESULTS: We found that phosphorylation of Akt (p-Akt), 4EBP1 (p-4EBP1) and S6 (p-S6) was inhibited by BEZ235. Colony formation in BxPC-3 cells was additively suppressed by the combination of 90Y-ITGA6B4 and BEZ235. Pretreatment with BEZ235 before 90Y-ITGA6B4 exposure resulted in significant reduction of cells plating efficiency (PE) (0.54 ± 0.11 vs 2.81 ± 0.14 with 185 kBq/mL 90Y-ITGA6B4 exposure, P < 0.01; 0.39 ± 0.08 vs 1.88 ± 0.09 with 370 kBq/mL 90Y-ITGA6B4 exposure, P < 0.01) when 5 × 103 cells per dish were plated. In vivo, the combined treatment with 90Y-ITGA6B4 plus BEZ235 enhanced the inhibition of tumor growth and statistically significant differences of relative tumor volume were observed for 27 d after the treatment start date when compared with the 90Y-ITGA6B4 single injection treatment (1.03 ± 0.38 vs 1.5 ± 0.15 at Day 27, P < 0.05), and for 41 d when compared with the BEZ235 treatment alone (1.8 ± 0.7 vs 3.14 ± 1.19 at Day 41, P < 0.05). Tumors from treatment groups showed reduction in volumes, decreased Ki-67-positive cells, increased p-H2AX-positive cells and decreased p-4EBP1 expression. CONCLUSION: The therapeutic efficacy of 90Y-ITGA6B4-RIT can be improved by combining with dual PI3K and mTOR inhibitor, BEZ235, in a pancreatic cancer model suggesting potential clinical application.

64Cu-ATSM internal radiotherapy to treat tumors with bevacizumab-induced vascular decrease and hypoxia in human colon carcinoma xenografts.

Bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody, is an antiangiogenic agent clinically used for various cancers. However, repeated use of this agent leads to tumor-decreased vascularity and hypoxia with activation of an HIF-1 signaling pathway, which results in drug delivery deficiency and induction of malignant behaviors in tumors. Here, we developed a novel strategy to treat tumors with bevacizumab-induced vascular decrease and hypoxia using 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM), a potential theranostic agent, which possesses high tissue permeability and can target over-reduced conditions under hypoxia in tumors, with a human colon carcinoma HT-29 tumor-bearing mouse model. The long-term treatment with bevacizumab caused decreased blood vessel density and activation of an HIF-1 signaling pathway; increased uptake of 64Cu-ATSM was also observed despite limited blood vessel density in HT-29 tumors. In vivo high-resolution SPECT/PET/CT imaging confirmed reduced vascularity and increased proportion of 64Cu-ATSM uptake areas within the bevacizumab-treated tumors. 64Cu-ATSM therapy was effective to inhibit tumor growth and prolong survival of the bevacizumab-treated tumor-bearing mice without major adverse effects. In conclusion, 64Cu-ATSM therapy effectively enhanced anti-tumor effects in tumors with bevacizumab-induced vascular decrease and hypoxia. 64Cu-ATSM therapy could represent a novel approach as an add-on to antiangiogenic therapy.

An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states.

67Cu-Radiolabeling of a multimeric RGD peptide for αVß3 integrin-targeted radionuclide therapy: stability, therapeutic efficacy, and safety studies in mice.

OBJECTIVE: Copper-67 (Cu) is one of the most promising radionuclides for internal radiation therapy. Globally, several projects have recently been initiated for developing innovative approaches for the large-scale production of Cu. Encouraged by these, we performed Cu-radiolabeling of a tetrameric cyclic Arg-Gly-Asp (cRGD) peptide conjugate, cyclam-RAFT-c(-RGDfK-)4, which selectively targets αVß3 integrin (αVß3), the transmembrane receptor involved in tumor invasion, angiogenesis, and metastasis. We also evaluated the therapeutic potential and safety of this radiocompound. MATERIALS AND METHODS: Cu, produced through the Ni(α, p)Cu reaction, was used for the radiolabeling of cyclam-RAFT-c(-RGDfK-)4 at 70°C for 10 min. The radiolabeling efficiency and product stability were assessed using reversed-phase high-performance liquid chromatography and/or thin-layer chromatography. Mice with subcutaneous αVß3-positive U87MG-glioblastoma xenografts received a single intravenous injection of one of the following: Cu-cyclam-RAFT-c(-RGDfK-)4 (11.1 MBq), peptide control, or vehicle solution. The tumor volumes were measured, side effects were assessed in terms of body weight, routine hematology, and hepatic and renal functions, and the mouse radiation dosimetry was estimated. RESULTS: Cu-cyclam-RAFT-c(-RGDfK-)4 was produced with a radiochemical purity of 97.9±2.4% and a specific activity of 2.7±0.6 MBq/nmol and showed high in-vitro and in-vivo plasma stability. The administration of a single dose of Cu-cyclam-RAFT-c(-RGDfK-)4 resulted in significant tumor growth delay in comparison with that observed upon vehicle or peptide control administration, with an estimated tumor-absorbed dose of 0.712 Gy. No significant toxicity was observed in Cu-cyclam-RAFT-c(-RGDfK-)4-treated mice. CONCLUSION: Cu-cyclam-RAFT-c(-RGDfK-)4 would be a promising therapeutic agent for αVß3 integrin-targeted internal radiotherapy.

αVß3 Integrin-Targeted Radionuclide Therapy with 64Cu-cyclam-RAFT-c(-RGDfK-)4.

The transmembrane cell adhesion receptor αVß3 integrin (αVß3) has been identified as an important molecular target for cancer imaging and therapy. We have developed a tetrameric cyclic RGD (Arg-Gly-Asp) peptide-based radiotracer (64)Cu-cyclam-RAFT-c(-RGDfK-)4, which successfully captured αVß3-positive tumors and angiogenesis by PET. Here, we subsequently evaluated its therapeutic potential and side effects using an established αVß3-positive tumor mouse model. Mice with subcutaneous U87MG glioblastoma xenografts received single administrations of 37 and 74 MBq of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 (37 MBq/nmol), peptide control, or vehicle solution and underwent tumor growth evaluation. Side effects were assessed in tumor-bearing and tumor-free mice in terms of body weight, routine hematology, and hepatorenal functions. Biodistribution of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 with ascending peptide doses (0.25-10 nmol) and with the therapeutic dose of 2 nmol were determined at 3 hours and at various time points (2 minutes-24 hours) postinjection, respectively, based on which radiation-absorbed doses were estimated. The results revealed that (64)Cu-cyclam-RAFT-c(-RGDfK-)4 dose dependently slowed down the tumor growth. The mean tumor doses were 1.28 and 1.81 Gy from 37 and 74 MBq of (64)Cu-cyclam-RAFT-c(-RGDfK-)4, respectively. Peptide dose study showed that the tumor uptake of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 dose dependently decreased at doses ≥1 nmol, indicating a saturation of αVß3 with the administered therapeutic doses (1 and 2 nmol). Combined analysis of the data from tumor-bearing and tumor-free mice revealed no significant toxicity caused by 37-74 MBq of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 Our study demonstrates the therapeutic efficacy and safety of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 for αVß3-targeted radionuclide therapy. (64)Cu-cyclam-RAFT-c(-RGDfK-)4 would be a promising theranostic drug for cancer imaging and therapy. Mol Cancer Ther; 15(9); 2076-85. ©2016 AACR.

Radioimmunotherapy of pancreatic cancer xenografts in nude mice using 90Y-labeled anti-α6ß4 integrin antibody.

The contribution of integrin α6ß4 (α6ß4) overexpression to the pancreatic cancer invasion and metastasis has been previously shown. We have reported immunotargeting of α6ß4 for radionuclide-based and near-infrared fluorescence imaging in a pancreatic cancer model. In this study, we prepared yttrium-90 labeled anti-α6ß4 antibody (90Y-ITGA6B4) and evaluated its radioimmunotherapeutic efficacy against pancreatic cancer xenografts in nude mice. Mice bearing xenograft tumors were randomly divided into 5 groups: (1) single administration of 90Y-ITGA6B4 (3.7MBq), (2) double administrations of 90Y-ITGA6B4 with once-weekly schedule (3.7MBq x 2), (3) single administration of unlabeled ITGA6B4, (4) double administrations of unlabeled ITGA6B4 with once-weekly schedule and (5) the untreated control. Biweekly tumor volume measurements and immunohistochemical analyses of tumors at 2 days post-administration were performed to monitor the response to treatments. To assess the toxicity, body weight was measured biweekly. Additionally, at 27 days post-administration, blood samples were collected through cardiac puncture, and hematological parameters, hepatic and renal functions were analyzed. Both 90Y-ITGA6B4 treatment groups showed reduction in tumor volumes (P < 0.04), decreased cell proliferation marker Ki-67-positive cells and increased DNA damage marker p-H2AX-positive cells, compared with the other groups. Mice treated with double administrations of 90Y-ITGA6B4, exhibited myelosuppression. There were no significant differences in hepatic and renal functions between the 2 treatment groups and the other groups. Our results suggest that 90Y-ITGA6B4 is a promising radioimmunotherapeutic agent against α6ß4 overexpressing tumors. In the future studies, dose adjustment for fractionated RIT should be considered carefully in order to get the optimal effect while avoiding myelotoxicity.

Immunotargeting of Integrin α6ß4 for Single-Photon Emission Computed Tomography and Near-Infrared Fluorescence Imaging in a Pancreatic Cancer Model.

To explore suitable imaging probes for early and specific detection of pancreatic cancer, we demonstrated that α6ß4integrin is a good target and employed single-photon emission computed tomography (SPECT) or near-infrared (NIR) imaging for immunotargeting. Expression levels of α6ß4were examined by Western blotting and flow cytometry in certain human pancreatic cancer cell lines. The human cell line BxPC-3 was used for α6ß4-positive and a mouse cell line, A4, was used for negative counterpart. We labeled antibody against α6ß4with Indium-111 ((111)In) or indocyanine green (ICG). After injection of(111)In-labeled probe to tumor-bearing mice, biodistribution, SPECT, autoradiography (ARG), and immunohistochemical (IHC) studies were conducted. After administration of ICG-labeled probe, in vivo and ex vivo NIR imaging and fluorescence microscopy of tumors were performed. BxPC-3 tumor showed a higher radioligand binding in SPECT and higher fluorescence intensity as well as a delay in the probe washout in NIR imaging when compared to A4 tumor. The biodistribution profile of(111)In-labeled probe, ARG, and IHC confirmed the α6ß4specific binding of the probe. Here, we propose that α6ß4is a desirable target for the diagnosis of pancreatic cancer and that it could be detected by radionuclide imaging and NIR imaging using a radiolabeled or ICG-labeled α6ß4antibody.

(64)Cu-ATSM therapy targets regions with activated DNA repair and enrichment of CD133(+) cells in an HT-29 tumor model: Sensitization with a nucleic acid antimetabolite.

(64)Cu-diacetyl-bis (N(4)-methylthiosemicarbazone) ((64)Cu-ATSM) is a potential theranostic agent targeting the over-reduced state under hypoxia within tumors. Recent clinical Cu-ATSM positron emission tomography studies have revealed a correlation between uptake and poor prognosis; however, the reason is unclear. Here, using a human colon carcinoma HT-29 model, we demonstrated that the intratumoral (64)Cu-ATSM high-uptake regions exhibited malignant characteristics, such as upregulated DNA repair and elevated %CD133(+) cancer stem-like cells. Based on this evidence, we developed a strategy to enhance the efficacy of (64)Cu-ATSM internal radiotherapy (IRT) by inhibiting DNA repair with a nucleic acid (NA) antimetabolite. The results of the analyses showed upregulation of pathways related to DNA repair along with NA incorporation (bromodeoxyuridine uptake) and elevation of %CD133(+) cells in (64)Cu-ATSM high-uptake regions. In an in vivo(64)Cu-ATSM treatment study, co-administration of an NA antimetabolite and (64)Cu-ATSM synergistically inhibited tumor growth, with little toxicity, and effectively reduced %CD133(+) cells. (64)Cu-ATSM therapy targeted malignant tumor regions with activated DNA repair and high concentrations of CD133(+) cells in the HT-29 model. NA antimetabolite co-administration can be an effective approach to enhance the therapeutic effect of (64)Cu-ATSM IRT.

Regorafenib as a potential adjuvant chemotherapy agent in disseminated small colon cancer: Drug selection outcome of a novel screening system using nanoimprinting 3-dimensional culture with HCT116-RFP cells.

Colon cancer is one of the leading causes of cancer death worldwide. Adjuvant chemotherapy following primary surgical treatment is suggested to be beneficial in eradicating invisible disseminated small tumors in colon cancer; however, an effective drug remains to be developed. Recently, we reported a novel drug screening system using a nanoimprinting 3-dimensional (3D) culture that creates multicellular spheroids, which simulate in vivo conditions and, thereby, predict effective drugs in vivo. This study aimed to perform drug selection using our recently developed 3D culture system in a human colon cancer HCT116 cell line stably expressing red fluorescent protein (HCT116-RFP), to determine the most effective agent in a selection of clinically used antitumor agents for colon cancer. In addition, we confirmed the efficacy of the selected drug regorafenib, in vivo using a mouse model of disseminated small tumors. HCT116-RFP cells were cultured using a nanoimprinting 3D culture and in vitro drug selection was performed with 8 clinically used drugs [bevacizumab, capecitabine, cetuximab, 5-fluorouracil (5-FU), irinotecan, oxaliplatin, panitumumab and regorafenib]. An in vivo study was performed in mice bearing HCT116-RFP intraperitoneally disseminated small tumors using 3'-[18F]-fluoro-3'-deoxythymidine-positron emission tomography and fluorescence microscopy imaging to evaluate the therapeutic effects. Regorafenib was determined to be the most effective drug in the 3D culture, and significantly inhibited tumor growth in vivo, compared to the untreated control and 5-FU-treated group. The drug 5-FU is commonly used in colon cancer treatment and was used as a reference. Our results demonstrate that regorafenib is a potentially efficacious adjuvant chemotherapeutic agent for the treatment of disseminated small colon cancer and, therefore, warrants further preclinical and clinical studies.

Immuno-PET Imaging of HER3 in a Model in which HER3 Signaling Plays a Critical Role.

HER3 is overexpressed in various carcinomas including colorectal cancer (CRC), which is associated with poor prognosis, and is involved in the development of therapy resistance. Thus, an in vivo imaging technique is needed to evaluate the expression of HER3, an important therapeutic and diagnostic target. Here, we report successful HER3 PET imaging using a newly generated anti-human HER3 monoclonal antibody, Mab#58, and a mouse model of a HER3-overexpressing xenograft tumor. Furthermore, we assessed the role of HER3 signaling in CRC cancer tissue-originated spheroid (CTOS) and applied HER3 imaging to detect endogenous HER3 in CTOS-derived xenografts. Cell binding assays of 89Zr-labeled Mab#58 using the HER3-overexpressing cell line HER3/RH7777 demonstrated that [89Zr]Mab#58 specifically bound to HER3/RH7777 cells (Kd = 2.7 nM). In vivo biodistribution study in mice bearing HER3/RH7777 and its parent cell xenografts showed that tumor accumulation of [89Zr]Mab#58 in HER3/RH7777 xenografts was significantly higher than that in the control from day 1 to day 4, tending to increase from day 1 to day 4 and reaching 12.2 ± 4.5%ID/g. Radioactivity in other tissues, including the control xenograft, decreased or remained unchanged from day 1 to day 6. Positron emission tomography (PET) in the same model enabled clear visualization of HER3/RH7777 xenografts but not of RH7777 xenografts. CTOS growth assay and signaling assay revealed that CRC CTOS were dependent on HER3 signaling for their growth. In PET studies of mice bearing a CRC CTOS xenograft, the tumor was clearly visualized with [89Zr]Mab#58 but not with the 89Zr-labeled control antibody. Thus, tumor expression of HER3 was successfully visualized by PET with 89Zr-labeled anti-HER3 antibody in CTOS xenograft-bearing mice, a model that retains the properties of the patient tumor. Non-invasive targeting of HER3 by antibodies is feasible, and it is expected to be useful for cancer diagnosis and treatment.

Transcriptomic Signatures of Auger Electron Radioimmunotherapy Using Nuclear Targeting (111)In-Trastuzumab for Potential Combination Therapies.

(111)In-labeled trastuzumab modified with nuclear localizing signal (NLS) peptides ((111)In-trastuzumab-NLS) efficiently delivers an Auger electron (AE) emitter (111)In into the cell nucleus and is thus a promising radiopharmaceutical in AE radioimmunotherapy (AE-RIT) for targeted killing of HER2-positive cancer. However, further improvement of its therapeutic efficacy is required. In this study, the authors show a transcriptomic approach to identify potential targets for enhancing the cytotoxic effects of (111)In-trastuzumab-NLS. They generated two types of (111)In-trastuzumab-NLS harboring different numbers of NLS peptides, (111)In-trastuzumab-NLS-S and -L. These radioimmunoconjugates (230 and 460 kBq) showed a significant higher cytotoxicity to SKBR3 human breast cancer cells overexpressing HER2 compared to (111)In-trastuzumab. Microarray analysis revealed that NF-kB-related genes (38 genes) were significantly changed in transcription by (111)In trastuzumab-NLS-L (230 kBq) treatment. Quantitative reverse transcription polymerase chain reaction confirmed the microarray data by showing transcriptional alternation of selected NF-κB target genes in cells treated with (111)In-trastuzumab-NLS-L. Interestingly, bortezomib, a drug known as a NF-κB modulator, significantly enhanced the cytotoxicity of (111)In-trastuzumab-NLS-L in SKBR3 cells. Taken together, the transcriptome data suggest the possibility that the modulation of NF-kB signaling activity is a molecular signature of (111)In-trastuzumab-NLS and coadministration of bortezomib may be efficacious in enhancement of AE-RIT with (111)In-trastuzumab-NLS.

Validation of 64Cu-ATSM damaging DNA via high-LET Auger electron emission.

Radioactive copper (II) (diacetyl-bis N4-methylthiosemicarbazone) (Cu-ATSM) isotopes were originally developed for the imaging of hypoxia in tumors. Because the decay of a (64)Cu atom is emitting not only positrons but also Auger electrons, this radionuclide has great potential as a theranostic agent. However, the success of (64)Cu-ATSM internal radiation therapy would depend on the contribution of Auger electrons to tumor cell killing. Therefore, we designed a cell culture system to define the contributions to cell death from Auger electrons to support or refute our hypothesis that the majority of cell death from (64)Cu-ATSM is a result of high-LET Auger electrons and not positrons or other low-LET radiation. Chinese hamster ovary (CHO) wild type and DNA repair-deficient xrs5 cells were exposed to (64)Cu-ATSM during hypoxic conditions. Surviving fractions were compared with those surviving gamma-radiation, low-LET hadron radiation, and high-LET heavy ion exposure. The ratio of the D(10) values (doses required to achieve 10% cell survival) between CHO wild type and xrs5 cells suggested that (64)Cu-ATSM toxicity is similar to that of high-LET Carbon ion radiation (70 keV/µm). γH2AX foci assays confirmed DNA double-strand breaks and cluster damage by high-LET Auger electrons from (64)Cu decay, and complex types of chromosomal aberrations typical of high-LET radiation were observed after (64)Cu-ATSM exposure. The majority of cell death was caused by high-LET radiation. This work provides strong evidence that (64)Cu-ATSM damages DNA via high-LET Auger electrons, supporting further study and consideration of (64)Cu-ATSM as a cancer treatment modality for hypoxic tumors.

Development of the Fibronectin-Mimetic Peptide KSSPHSRN(SG)5RGDSP as a Novel Radioprobe for Molecular Imaging of the Cancer Biomarker α5ß1 Integrin.

α5ß1 Integrin, a fibronectin receptor, is becoming a pertinent therapeutic target and a promising prognostic biomarker for cancer patients. The aim of this study was to functionalize an α5ß1-specific fibronectin-mimetic peptide sequence KSSPHSRN(SG)5RGDSP (called PR_b) as a positron emission tomography (PET) probe. PR_b was modified by addition of a ß-alanine residue, conjugated with 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA), and radiolabeled with (18)F based on the chelation of (18)F-aluminum fluoride. A control probe was produced by glycine to alanine substitution in the RGD motif of PR_b. Cell binding and blocking assays, autoradiographic evaluation of tissue binding and blocking, dynamic PET scans, and a biodistribution study were conducted using cell lines and murine tumor models with determined expression levels of α5ß1 and other related integrins. (18)F-PR_b was produced with a labeling yield of 22.3±1.9% based on (18)F-F(-), a radiochemical purity of >99%, and a specific activity of 30-70 GBq/µmol; it exhibited α5ß1-binding activity and specificity in vitro, ex vivo, and in vivo, and had a rapid blood clearance and a predominant renal excretion pathway. In vivo α5ß1-positive tumors could be clearly visualized by (18)F-PR_b PET imaging. Both imaging and biodistribution studies suggested higher uptake of (18)F-PR_b in α5ß1-positive tumors than in α5ß1-negative tumors and higher α5ß1-positive tumor uptake of (18)F-PR_b than the control probe. In contrast, there was no significant difference seen in the contralateral muscle uptake. A PET radioprobe, (18)F-PR_b, was developed de novo and potentially can be used for noninvasive detection of α5ß1 expression in tumors.

A limited overlap between intratumoral distribution of 1-(5-fluoro-5-deoxy-α-D-arabinofuranosyl)-2-nitroimidazole and copper-diacetyl-bis[N(4)-methylthiosemicarbazone].

Positron emission tomography (PET) imaging of tumor hypoxia provides valuable information for cancer treatment planning. Two types of PET tracers, nitroimidazole compounds and [62,64Cu] copper-diacetyl-bis[N(4)-methylthio- semicarbazone] (Cu-ATSM), have been used for imaging hypoxic tumors. High accumulation of these tracers in tumors was shown to predict poor prognosis. Both similar and different intratumoral distributions of these PET tracers have been reported with some studies questioning the dependence of the Cu-ATSM accumulation on hypoxia. In the present study, we compared the intratumoral distribution and cellular uptake of 1-(5-fluoro-5-deoxy-α-D-arabinofuranosyl)-2-nitroimidazole (FAZA) and Cu-ATSM. Intratumoral distributions of FAZA and Cu-ATSM compared by double tracer autoradiography in xenografts of 8 cancer cell lines and 3 cancer tissue originated spheroids (CTOSs) showed that only a limited overlap was observed between the regions with high levels of FAZA and Cu-ATSM accumulation in all the xenografts. Immunohistochemistry in the regions enriched with FAZA and Cu-ATSM in xenografts demonstrated that pimonidazole adducts were in regions that accumulated high levels of FAZA, while HIF-1α was in areas enriched with either tracer. In addition, we examined the cellular uptake of FAZA and Cu-ATSM at different levels of oxygen concentration in 4 cell lines and revealed that cellular uptake of FAZA was increased with the decrease of oxygen concentration from 20 to 2 and from 2 to 1%, while the Cu-ATSM uptake increased with the decrease of oxygen concentration from 20 to 2%, but did not increase with the decrease from 2 to 1%. Our findings indicate that intratumoral distributions of FAZA and Cu-ATSM were essentially non-overlapping and although hypoxia affects the buildup of both tracers, the accumulation of Cu-ATSM occurred at milder hypoxia compared to the conditions required for the accumulation of FAZA. Therefore, accumulation levels of FAZA and Cu-ATSM may be considered as independent biomarkers.