Oxygen-Independent Synchronized ROS Generation and Hypoxia Prodrug Activation with Z-Scheme Heterostructure Sonosensitizer.

Combination therapy has emerged as a promising approach for effective tumor treatment. However, the combination of sonodynamic therapy (SDT) and hypoxia-activated prodrugs (HAPs) has not been explored due to the contradictory requirement of oxygen (O2 ) for reactive oxygen species (ROS) generation and the necessity to avoid O2 for the activation of HAPs. In this study, this challenge is addressed by developing BiOCl-Au-Ag2 S Z-scheme heterostructure nanoparticles loaded with tirapazamine (TPZ) to achieve O2 -independent therapy. These nanoparticles demonstrate efficient electron-hole separation under ultrasound irradiation while maintaining a high redox potential. The generated holes react with water to efficiently produce hydroxyl radicals, while the electrons autonomously activate TPZ, negating the need for O2 . In vitro and in vivo assessments validate the effective tumor elimination by these Z-scheme nanoparticles without disrupting the hypoxic environment. This innovative design overcomes the limitations associated with O2 requirement in SDT and introduces a novel strategy for HAP activation and synergistic therapy between ROS and HAPs-based therapy.

Generative modeling of time-dependent densities via optimal transport and projection pursuit.

Motivated by the computational difficulties incurred by popular deep learning algorithms for the generative modeling of temporal densities, we propose a cheap alternative that requires minimal hyperparameter tuning and scales favorably to high-dimensional problems. In particular, we use a projection-based optimal transport solver [Meng et al.,Advances in Neural Information Processing Systems (Curran Associates, 2019), Vol. 32] to join successive samples and, subsequently, use transport splines (Chewi et al., 2020) to interpolate the evolving density. When the sampling frequency is sufficiently high, the optimal maps are close to the identity and are, thus, computationally efficient to compute. Moreover, the training process is highly parallelizable as all optimal maps are independent and can, thus, be learned simultaneously. Finally, the approach is based solely on numerical linear algebra rather than minimizing a nonconvex objective function, allowing us to easily analyze and control the algorithm. We present several numerical experiments on both synthetic and real-world datasets to demonstrate the efficiency of our method. In particular, these experiments show that the proposed approach is highly competitive compared with state-of-the-art normalizing flows conditioned on time across a wide range of dimensionalities.

Copper indium selenium nanomaterials for photo-amplified immunotherapy through simultaneously enhancing cytotoxic T lymphocyte recruitment and M1 polarization of macrophages.

Photoactivated immunotherapy has promising therapeutic efficacy for treating malignancies, especially metastatic tumors. In this study, an erythrocyte membrane-encapsulated copper indium selenium (RCIS) semiconductor nanomaterial was developed to eliminate primary and metastatic tumors, in which copper ions can induce chemodynamic performance, and the narrow band gap endows RCIS with the properties of near-infrared (NIR) light-activated photothermal and photodynamic amplified immunotherapy. Furthermore, RCIS can be used as a nanocarrier to form RNCIS nanoparticles (NPs) by loading NLG919, which blocks the indoleamine 2,3-dioxygenase-1. Under NIR light irradiation, RNCIS NPs release NLG919 at tumor sites via photothermal properties, thereby promoting the recruitment of cytotoxic T lymphocytes and M1 polarization of macrophages, targeting the activation and amplification of immune responses. Herein, in vitro and in vivo studies showed that RNCIS NPs effectively kill cancer cells and eliminate primary and metastatic tumors. Therefore, this study suggests that semiconductor nanomaterials with narrow bandgaps have great potential as photoimmunotherapy agents and NIR light-responsive nanocarriers for controlled release, providing a great paradigm for synergetic tumor photoimmunotherapy. STATEMENT OF SIGNIFICANCE: The Erythrocyte membrane-coated, NLG919-loaded copper indium selenium (RNCIS) semiconductor was designed for eliminating primary and metastatic tumors. RNCIS exhibits chemodynamic, photodynamic, and photothermal activated immunotherapy by inhibiting indoleamine 2,3-dioxygenase-1. This can enhance the recruitment of cytotoxic T lymphocyte and M1 polarization of macrophage, leading to higher synergetic photo-immune therapeutic efficacy.

Identifying the specific pathways to improve nitrogen fixation of different straw biochar during chicken manure composting based on its impact on the microbial community.

The application of straw biochar to chicken manure composting mitigated nitrogen loss. However, the impact of biochar derived from different types of straw on nitrogen fixation in chicken manure composting is discrepant, and the specific pathways remain unclear. Therefore, this study aimed to clarify the specific pathways of maize straw biochar (M) and rice straw biochar (R) to improve nitrogen fixation during chicken manure composting. The nitrogen losses in control (no addition, CK), M, and R composting were 51.84 %, 33.47 %, and 38.24 %, respectively, suggesting that adding straw biochar effectively improved nitrogen fixation. Microbial community analysis suggested that inhibiting denitrification and NH4+-N transformation by microorganisms was the primary means of improving nitrogen fixation. Meanwhile, biochar addition reduced the number of bacteria participating in nitrogen transformation and strengthened the NO3--N and total organic nitrogen transformation processes, among which the effect of M composting was stronger. The stronger effect was attributed to the significant role of the core microorganisms in M composting in shifting the transformation processes of the nitrogen components (P < 0.05). Therefore, the function of different straw biochar was determined by its different impacts on the microbial community, highlighting the important role of microbial community variability.

Learning dynamics on invariant measures using PDE-constrained optimization.

We extend the methodology in Yang et al. [SIAM J. Appl. Dyn. Syst. 22, 269-310 (2023)] to learn autonomous continuous-time dynamical systems from invariant measures. The highlight of our approach is to reformulate the inverse problem of learning ODEs or SDEs from data as a PDE-constrained optimization problem. This shift in perspective allows us to learn from slowly sampled inference trajectories and perform uncertainty quantification for the forecasted dynamics. Our approach also yields a forward model with better stability than direct trajectory simulation in certain situations. We present numerical results for the Van der Pol oscillator and the Lorenz-63 system, together with real-world applications to Hall-effect thruster dynamics and temperature prediction, to demonstrate the effectiveness of the proposed approach.

Natural variation of Alfin-like family affects seed size and drought tolerance in rice.

The Alfin-like (AL) family is a group of small plant-specific transcriptional factors involved in abiotic stresses in dicotyledon. In an early study, we found an AL gene in rice that was associated with grain yield under drought stress. However, little information is known about the AL family in rice. In this study, AL genes in the rice genome were identified, and the OsAL proteins were found to locate in the nucleus and have no transcriptional self-activation activity. The expression of the OsALs was regulated by different environmental stimulations and plant hormones. Association and domestication analysis revealed that natural variation of most OsALs was significantly associated with yield traits, drought resistance and divergence in grain size in indica and japonica rice varieties. Hap1 of OsAL7.1 and Hap7 of OsAL11 were favorable haplotypes of seed weight and germination under osmotic stress. Furthermore, osal7.1 and osal11 mutants have larger seeds and are more sensitive to abscisic acid and mannitol during germination stage. Overexpressing of OsAL7.1 and OsAL11 in rice weakened the tolerance to drought in the adult stage. Thus, our work provides informative knowledge for exploring and harnessing haplotype diversity of OsALs to improve yield stability under drought stress.

Overexpressing PpBURP2 in Rice Increases Plant Defense to Abiotic Stress and Bacterial Leaf Blight.

Mosses are one of the earliest diverging land plants that adapted to living on land. The BURP domain-containing proteins (BURP proteins) are plant-specific proteins that appeared when plants shifted from aquatic environments to land. Phylogenetic analysis revealed that the BURP domain of higher plants is originated from lower land plants and divergent because of motif conversion. To discover the function of BURP protein in moss, rice transgenics with ectopic expression of PpBURP2 were subjected to different abiotic stresses treatments. The results revealed that the ectopic expression of PpBURP2 enhanced the tolerance to osmotic and saline stresses at the seedling stage and drought stress at the adult stage. Further ectopic expression of PpBURP2 improved the cadmium (2+) (Cd2+) tolerance and reduced Cd2+ accumulation in rice leaves. Transcriptomic analysis of the transgenic PpBURP2 plants showed that the differentially expressed genes were involved in the metabolism of secondary metabolites, energy, oxidation-reduction process, and defense-related genes. Further experiments showed that the photosynthetic efficiency and resistance against bacterial leaf blight were obviously improved in transgenic plants. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays revealed the physical interaction of BURP domain protein from rice and moss with mitogen-activated protein kinase kinase (MKK) from rice. Therefore, our findings demonstrate that overexpressing PpBURP2 in rice confers resistance to abiotic stresses and bacterial leaf blight. They also suggested that the regulatory role of BURP-like proteins across lower and higher plants was evolutionary conservation of responses of different classes of plants to different environmental challenges.

ImmunoPET of the differential expression of CD146 in breast cancer.

With advancement in antibody engineering, the development and characterization of new cancer-specific molecular targets are in the forefront of this PET-antibody combination "revolution". Overexpression of CD146 in different types of tumors, including breast tumor, has been associated with tumor progression and poor prognosis. Non-invasive detection of CD146 with a monoclonal antibody may provide a noninvasive diagnostic tool with high specificity and accountability. METHODS: Herein, we have developed a CD146-specific monoclonal antibody (YY146), radiolabeled it with 52Mn and 89Zr and identified its capability in acting as a non-invasive imaging agent that specific targets CD146 in different murine breast cancer models. CD146 expression was first screened in different breast tumor cell lines through Western Blot and confirmed its binding ability to YY146 using Flow Cytometry. Serial immunoPET images were carried out after intravenous administration of 52Mn or 89Zr labeled YY146. In addition, we also performed in vivo fluorescence imaging in animals injected with YY146 conjugated with Cy5.5. RESULTS: Western Blot results show that MDA-MB-435 cell line had greater levels of CD146 expression when compared to the other cell lines investigated. Flow cytometry confirmed binding ability of YY146. PET images revealed well correlated uptake between tumor uptake and CD146 expression levels, confirmed by biodistribution studies and fluorescence imaging. CONCLUSION: PET imaging, for up to 7 days, of mice bearing three different breast tumors were carried out and revealed radiotracer uptake in tumors that strongly (r2 = 0.98, P < 0.01), correlated with CD146 expression levels, as confirmed by in vitro and ex vivo studies.

ImmunoPET of CD146 in Orthotopic and Metastatic Breast Cancer Models.

The overexpression of CD146 in breast cancer is considered a hallmark of tumor progression and metastasis, particularly in triple negative breast cancer. Aimed at imaging differential CD146 expressions in breast cancer, a noninvasive method for predictive prognosis and diagnosis was investigated using a 64Cu-labeled CD146-specific monoclonal antibody, YY146. CD146 expression was screened in human breast cancer cell lines using Western blotting. Binding ability was evaluated using flow cytometry and immunofluorescent staining. YY146 was conjugated with 1,4,7-triazacyclononane-triacetic acid (NOTA) and radiolabeled with 64Cu following standard procedures. Serial PET or PET/CT imaging was performed in orthotopic and metastatic breast cancer tumor models. Biodistribution was performed after the final time point of imaging. Finally, tissue immunofluorescent staining and hematoxylin and eosin (H&E) staining were performed on tumor tissues. The MDA-MB-435 cell line showed the highest CD146 expression level, whereas MCF-7 had the lowest level at the cellular level. ImmunoPET showed that MDA-MB-435 orthotopic tumors had high and clear radioactive accumulation after the administration of 64Cu-NOTA-YY146. The tumor uptake of 64Cu-NOTA-YY146 in MDA-MB-435 was significantly higher than that in MCF-7 and nonspecific IgG control groups (P < 0.01). Biodistribution verified the PET imaging results. For metastatic models, 64Cu-NOTA-YY146 allowed for the visualization of high radioactivity accumulation in metastatic MDA-MB-435 tumors, which was confirmed by ex vivo biodistribution of lung tissues. H&E staining proved the successful building of metastatic tumor models. Immunofluorescent staining verified the differential expression of CD146 in orthotopic tumors. Therefore, 64Cu-NOTA-YY146 could be used as an immunoPET probe to visualize CD146 in the breast cancer model and is potentially useful for cancer diagnosis, prognosis prediction, and monitoring therapeutic response.

Spatial distributions, source apportionment and ecological risks of C9-C17 chlorinated paraffins in mangrove sediments from Dongzhai Harbor, Hainan Island.

The spatial distributions, possible sources of C9-C17 chlorinated paraffins (CPs), and the ecological risks posed in mangrove sediment in Dongzhai Harbor (Hainan Island, China) were investigated. Comprehensive two-dimensional gas chromatography combined with electron capture negative ionization mass spectrometry was used to determine 50 C9-C17 CP congener groups. The concentrations of C9-CPs, short-chain CPs (SCCPs), and medium-chain CPs (MCCPs) in the mangrove sediment samples were 8.28-79.7, 89.2-931, and 58.8-834 ng g-1 dry weight, respectively. The CPs concentrations in the mangrove sediment samples were moderate compared with those found in other regions worldwide. The spatial distributions and congener patterns of the CPs indicated that the CP concentrations were mainly controlled by local emissions and that wastewater discharged from livestock and shrimp breeding facilities and domestic sewage were the main sources of CPs in mangrove sediment in Dongzhai Harbor. C10Cl6-7 and C14Cl7-8 were the dominant SCCP and MCCP congener groups, respectively. The MCCP concentrations and total organic carbon contents significantly correlated (R2 = 0.607, P < 0.05). Hierarchical cluster analysis and principal component analysis indicated that the SCCP and MCCP congeners were from different commercial CP formulations and sources. Risk assessments suggested that SCCPs and MCCPs in mangrove sediment in Dongzhai Harbor do not currently pose marked risks to sediment-dwelling organisms.

Dual-cell structure digitally controlled oscillator with portability for clock generation applications.

This paper presents a dual-cell structure and cell-based design digitally controlled oscillator (DCO) for high-performance clock generation applications. The proposed dual-cell DCO not only can reduce the intrinsic delay to increase the maximum output frequency significantly but also extend the controllable range. In addition, the proposed digitally controlled delay cell uses the cascading structure to realize the wide output frequency range and high timing resolution at the same time. A DCO chip is fabricated in 0.18 µm CMOS technology, and the core area is 0.003 mm2. Measurement results show that operation range is from 169 MHz to 522 MHz and the finest delay resolution is 2.2 ps. Furthermore, because the proposed DCO is implemented with the digital standard cells, it is a portable design to migrate to different processes easily and reduce design time significantly.

CD146-Targeted Multimodal Image-Guided Photoimmunotherapy of Melanoma.

For melanoma resistant to molecularly targeted therapy and immunotherapy, new treatment strategies are urgently needed. A molecularly targeted theranostic pair may thus be of importance, where the diagnostic probe facilitates patient stratification and the therapeutic companion treats the selected cases. For this purpose, flow cytometry is used to assess the CD146 level in melanoma cells. Based on YY146, a CD146-specific monoclonal antibody, an imaging probe 89Zr-Df-YY146 is synthesized and its diagnostic performance is evaluated by positron emission tomography (PET) imaging. Furthermore, a photoimmunotherapy (PIT) agent IR700-YY146 is developed and the therapeutic effect of IR700-YY146 PIT is assessed comprehensively. CD146 is highly expressed in A375 and SK-MEL-5 cells. 89Zr-Df-YY146 PET readily detects CD146-positive A375 melanomas. Tumor accumulation of 89Zr-Df-YY146 peaks at 72 h with an uptake value of 26.48 ± 3.28%ID g-1, whereas the highest uptake of the nonspecific 89Zr-Df-IgG is 4.80 ± 1.75%ID g-1. More importantly, IR700-YY146 PIT effectively inhibits the growth of A375 tumors, owing to production of reactive oxygen species, decreased glucose metabolism, and reduced expression of CD146. To conclude, 89Zr-Df-YY146 and IR700-YY146 are a promising theranostic pair with the former revealing CD146 expression in melanoma as a PET probe and the latter specifically treating CD146-positive melanoma as an effective PIT agent.

ImmunoPET of CD146 in a Murine Hindlimb Ischemia Model.

Peripheral arterial disease (PAD) consists of a persistent obstruction of lower-extremity arteries further from the aortic bifurcation attributable to atherosclerosis. PAD is correlated with an elevated risk of morbidity and mortality as well as of deterioration of the quality of life with claudication and chronic leg ischemia being the most frequent complications. Therapeutic angiogenesis is a promising therapeutic strategy that aims to restore the blood flow to the ischemic limb. In this context, assessing the efficacy of pro-angiogenic treatment using a reliable noninvasive imaging technique would greatly benefit the implementation of this therapeutic approach. Herein, we describe the angiogenesis and perfusion recovery characteristics of a mouse model of PAD via in vivo positron emission tomography (PET) imaging of CD146 expression. For that, ischemia was generated by ligation and excision of the right femoral artery of Balb/C mice and confirmed through laser Doppler imaging. The angiogenic process, induced by ischemia, was noninvasively monitored and quantified through PET imaging of CD146 expression in the injured leg using a 64Cu-labeled anti-CD146 monoclonal antibody, 64Cu-NOTA-YY146, at post-operative days 3, 10, and 17. The CD146-specific character of 64Cu-NOTA-YY146 was verified via a blocking study performed in another cohort at day 10 after surgery. Tracer uptake was correlated with in situ CD146 expression by histological analysis. PET scan results indicated that 64Cu-NOTA-YY146 uptake in the injured leg was significantly higher, with the highest uptake with a value of 14.1 ± 2.0 %ID/g at post-operative day 3, compared to the normal contralateral hindlimb, at all time points (maximum uptake of 2.2 ± 0.2 %ID/g). The pre-injection of a blocking dose resulted in a significantly lower tracer uptake in the ischemic hindlimb on day 10 after surgery, confirming tracer specificity. CD146/CD31 immunofluorescent co-staining showed an excellent correlation between the high uptake of the tracer with in situ CD146 expression levels and a marked co-localization of CD146 and CD31 signals. In conclusion, persistent and CD146-specific tracer accumulation in the ischemic hindlimb was observed, confirming the feasibility of 64Cu-NOTA-YY146 to be used as an imaging agent to monitor the progression of angiogenesis and recovery in future PAD research.

ImmunoPET Imaging of CD146 in Murine Models of Intrapulmonary Metastasis of Non-Small Cell Lung Cancer.

CD146 has been identified as an excellent biomarker for lung cancer as its overexpression in solid tumors has been linked to disease progression, invasion, and metastasis. Previously, our group described a positive correlation between 64Cu-labeled YY146 uptake and increased expression of CD146 in six human lung cancer cell lines using subcutaneous tumor models. In this study, we investigate a monoclonal antibody called YY146 for immunoPET imaging of CD146 in two intrapulmonary metastasis models of non-small cell lung cancer (NSCLC). The binding and immunoreactivity of the tracer were assessed by in vitro assays. Radiolabeling of YY146 with positron emitting Cu-64 (64Cu-NOTA-YY146) enabled PET imaging of intrapulmonary metastasis. Mice were intravenously injected with two million tumor cells, and CT imaging was used to verify the presence of lung metastases. 64Cu-NOTA-YY146 was injected into tumor-bearing mice, and animals were subjected to PET/CT imaging at 4, 24, and 48 h postinjection. Both the average and maximum lung PET signal intensities were quantified and compared between high and low CD146-expressing metastases. Further validation was accomplished through immunofluorescence imaging of resected tissues with CD31 and CD146. In flow cytometry, YY146 revealed strong binding to CD146 in H460 cells due to its high expression with minimal binding to CD146-low expressing H358 cells. Both YY146 and NOTA-YY146 showed similar binding, suggesting that NOTA conjugation did not elicit any negative effects on its binding affinity. Imaging of 64Cu-NOTA-YY146 in H460 tumor-bearing mice revealed rapid, persistent, and highly specific tracer accumulation. Uptake of 64Cu-NOTA-YY146 in the whole lung was calculated for H460 and H358 as 7.43 ± 0.38 and 3.95 ± 0.47% ID/g at 48 h postinjection (n = 4, p < 0.05), and the maximum lung signals were determined to be 13.85 ± 1.07 (H460) and 6.08 ± 0.73% ID/g (H358) at equivalent time points (n = 4, p < 0.05). To ensure the specificity of the tracer, a nonspecific antibody was injected into H460 tumor-bearing mice. Ex vivo biodistribution and immunofluorescence imaging validated the PET findings. In summary, 64Cu-NOTA-YY146 allowed for successful imaging of CD146-expressing intrapulmonary metastases of NSCLC in mice. This preliminary study provides evidence supporting the future clinical utilization of 64Cu-NOTA-YY146 for possible treatment monitoring of CD146-targeted therapy or improving patient stratification.

ImmunoPET imaging of tissue factor expression in pancreatic cancer with 89Zr-Df-ALT-836.

Overexpression of tissue factor (TF) has been associated with increased tumor growth, tumor angiogenesis, and metastatic potential in many malignancies, including pancreatic cancer. Additionally, high TF expression was shown to strongly correlate with poor prognoses and decreased survival in pancreatic cancer patients. Herein, we exploited the potential targeting of TF for positron emission tomography (PET) imaging of pancreatic cancer. The TF-targeted tracer was developed through radiolabeling of the anti-human TF monoclonal antibody (ALT-836) with 89Zr. The tracer was characterized by fluorescence microscopy and flow cytometry assays in BXPC-3 and PANC-1 cells, two pancreatic cancer cell lines with high and low TF expression levels, respectively. Non-invasive PET scans were acquired in tumor-bearing mice injected with 89Zr-Df-ALT-836. Additionally, ex vivo biodistribution, blocking, and histological studies were performed to establish the affinity and specificity of 89Zr-Df-ALT-836 for TF in vivo. 89Zr-labeling of Df-ALT-836 was achieved in high yield and good specific activity. Flow cytometry and microscopy studies revealed no detectable difference in TF-binding affinity between ALT-836 and Df-ALT-836 in vitro. Longitudinal PET scans unveiled a lasting and prominent 89Zr-Df-ALT-836 uptake in BXPC-3 tumors (peak at 31.5±6.0%ID/g at 48h post-injection; n=3), which was significantly abrogated (2.3±0.5%ID/g at 48h post-injection; n=3) when mice were pre-injected with a blocking dose (50mg/kg) of unlabeled ALT-836. Ex vivo biodistribution data confirmed the accuracy of the PET results, and histological analysis correlated high tumor uptake with in situ TF expression. Taken together, these results attest to the excellent affinity and TF-specificity of 89Zr-Df-ALT-836. With elevated, persistent, and specific accumulation in TF-positive BXPC-3 tumors, PET imaging using 89Zr-Df-ALT-836 promises to open new avenues for improving future diagnosis, stratification, and treatment response assessment in pancreatic cancer patients.

CD146-targeted immunoPET and NIRF Imaging of Hepatocellular Carcinoma with a Dual-Labeled Monoclonal Antibody.

Overexpression of CD146 has been correlated with aggressiveness, recurrence rate, and poor overall survival in hepatocellular carcinoma (HCC) patients. In this study, we set out to develop a CD146-targeting probe for high-contrast noninvasive in vivo positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging of HCCs. YY146, an anti-CD146 monoclonal antibody, was employed as a targeting molecule to which we conjugated the zwitterionic near-infrared fluorescence (NIRF) dye ZW800-1 and the chelator deferoxamine (Df). This enabled labeling of Df-YY146-ZW800 with (89)Zr and its subsequent detection using PET and NIRF imaging, all without compromising antibody binding properties. Two HCC cell lines expressing high (HepG2) and low (Huh7) levels of CD146 were employed to generate subcutaneous (s.c.) and orthotopic xenografts in athymic nude mice. Sequential PET and NIRF imaging performed after intravenous injection of (89)Zr-Df-YY146-ZW800 into tumor-bearing mice unveiled prominent and persistent uptake of the tracer in HepG2 tumors that peaked at 31.65 ± 7.15 percentage of injected dose per gram (%ID/g; n=4) 72 h post-injection. Owing to such marked accumulation, tumor delineation was successful by both PET and NIRF, which facilitated the fluorescence image-guided resection of orthotopic HepG2 tumors, despite the relatively high liver background. CD146-negative Huh7 and CD146-blocked HepG2 tumors exhibited significantly lower (89)Zr-Df-YY146-ZW800 accretion (6.1 ± 0.5 and 8.1 ± 1.0 %ID/g at 72 h p.i., respectively; n=4), demonstrating the CD146-specificity of the tracer in vivo. Ex vivo biodistribution and immunofluorescent staining corroborated the accuracy of the imaging data and correlated tracer uptake with in situ CD146 expression. Overall, (89)Zr-Df-YY146-ZW800 showed excellent properties as a PET/NIRF imaging agent, including high in vivo affinity and specificity for CD146-expressing HCC. CD146-targeted molecular imaging using dual-labeled YY146 has great potential for early detection, prognostication, and image-guided surgical resection of liver malignancies.

ImmunoPET Imaging of CD146 Expression in Malignant Brain Tumors.

Recently, the overexpression of CD146 and its potential as a therapeutic target in high-grade gliomas, the most lethal type of brain cancer, was uncovered. In this study, we describe the generation of (89)Zr-Df-YY146, a novel (89)Zr-labeled monoclonal antibody (mAb) for the targeting and quantification of CD146 expression in a mouse model of glioblastoma, using noninvasive immunoPET imaging. YY146, a high affinity anti-CD146 mAb, was conjugated to deferoxamine (Df) for labeling with the long-lived positron emitter (89)Zr (t1/2: 78.4 h). In vitro assays, including flow cytometry, immunofluorescence microscopy, and Western blot, were performed with two glioblastoma cell lines, U87MG and U251, to determine their CD146 expression levels. Also, YY146 and Df-YY146's CD146-binding affinities were compared using flow cytometry. In vivo CD146-targeting of (89)Zr-Df-YY146 was evaluated by sequential PET imaging, in athymic nude mice bearing subcutaneously implanted U87MG or U251 tumors. CD146 blocking, ex vivo biodistribution, and histological studies were carried out to confirm (89)Zr-Df-YY146 specificity, as well as the accuracy of PET data. In vitro studies exposed elevated CD146 expression levels in U87MG cells, but negligible levels in U251 cells. Flow cytometry revealed no differences in affinity between YY146 and Df-YY146. (89)Zr labeling of Df-YY146 proceeded with excellent yield (∼80%), radiochemical purity (>95%), and specific activity (∼44 GBq/µmol). Longitudinal PET revealed prominent and persistent (89)Zr-Df-YY146 uptake in mice bearing U87MG tumors that peaked at 14.00 ± 3.28%ID/g (n = 4), 48 h post injection of the tracer. Conversely, uptake was significantly lower in CD146-negative U251 tumors (5.15 ± 0.99%ID/g, at 48 h p.i.; n = 4; P < 0.05). Uptake in U87MG tumors was effectively blocked in a competitive inhibition experiment, corroborating the CD146 specificity of (89)Zr-Df-YY146. Finally, ex vivo biodistribution validated the accuracy of PET data and histological examination successfully correlated tracer uptake with in situ CD146 expression. Prominent, persistent, and specific uptake of (89)Zr-Df-YY146 was observed in brain tumors, demonstrating the potential of this radiotracer for noninvasive PET imaging of CD146 expression. In a future clinical scenario, (89)Zr-Df-YY146 may serve as a tool to guide intervention and assess response to CD146-targeted therapies.

ImmunoPET for assessing the differential uptake of a CD146-specific monoclonal antibody in lung cancer.

PURPOSE: Overexpression of CD146 in solid tumors has been linked to disease progression, invasion, and metastasis. We describe the generation of a 64Cu-labeled CD146-specific antibody and its use for quantitative immunoPET imaging of CD146 expression in six lung cancer models. METHODS: The anti-CD146 antibody (YY146) was conjugated to 1,4,7-triazacyclononane-triacetic acid (NOTA) and radiolabeled with 64Cu. CD146 expression was evaluated in six human lung cancer cell lines (A549, NCI-H358, NCI-H522, HCC4006, H23, and NCI-H460) by flow cytometry and quantitative western blot studies. The biodistribution and tumor uptake of 64Cu-NOTA-YY146 was assessed by sequential PET imaging in athymic nude mice bearing subcutaneous lung cancer xenografts. The correlation between CD146 expression and tumor uptake of 64Cu-NOTA-YY146 was evaluated by graphical software while ex vivo biodistribution and immunohistochemistry studies were performed to validate the accuracy of PET data and spatial expression of CD146. RESULTS: Flow cytometry and western blot studies showed similar findings with H460 and H23 cells showing high levels of expression of CD146. Small differences in CD146 expression levels were found among A549, H4006, H522, and H358 cells. Tumor uptake of 64Cu-NOTA-YY146 was highest in CD146-expressing H460 and H23 tumors, peaking at 20.1 ± 2.86 and 11.6 ± 2.34 %ID/g at 48 h after injection (n = 4). Tumor uptake was lowest in the H522 model (4.1 ± 0.98 %ID/g at 48 h after injection; n = 4), while H4006, A549 and H358 exhibited similar uptake of 64Cu-NOTA-YY146. A positive correlation was found between tumor uptake of 64Cu-NOTA-YY146 (%ID/g) and relative CD146 expression (r 2 = 0.98, p < 0.01). Ex vivo biodistribution confirmed the accuracy of the PET data. CONCLUSION: The strong correlation between tumor uptake of 64Cu-NOTA-YY146 and CD146 expression demonstrates the potential use of this radiotracer for imaging tumors that elicit varying levels of CD146. In the future, this tool may promote enhanced monitoring of therapeutic response and improved patient stratification.

Targeting CD146 with a 64Cu-labeled antibody enables in vivo immunoPET imaging of high-grade gliomas.

Given the highly heterogeneous character of brain malignancies and the associated implication for its proper diagnosis and treatment, finding biomarkers that better characterize this disease from a molecular standpoint is imperative. In this study, we evaluated CD146 as a potential molecular target for diagnosis and targeted therapy of glioblastoma multiforme (GBM), the most common and lethal brain malignancy. YY146, an anti-CD146 monoclonal antibody, was generated and radiolabeled for noninvasive positron-emission tomography (PET) imaging of orthotopic GBM models. (64)Cu-labeled YY146 preferentially accumulated in the tumors of mice bearing U87MG xenografts, which allowed the acquisition of high-contrast PET images of small tumor nodules (∼ 2 mm). Additionally, we found that tumor uptake correlated with the levels of CD146 expression in a highly specific manner. We also explored the potential therapeutic effects of YY146 on the cancer stem cell (CSC) and epithelial-to-mesenchymal (EMT) properties of U87MG cells, demonstrating that YY146 can mitigate those aggressive phenotypes. Using YY146 as the primary antibody, we performed histological studies of World Health Organization (WHO) grades I through IV primary gliomas. The positive correlation found between CD146-positive staining and high tumor grade (χ(2) = 9.028; P = 0.029) concurred with the GBM data available in The Cancer Genome Atlas (TCGA) and validated the clinical value of YY146. In addition, we demonstrate that YY146 can be used to detect CD146 in various cancer cell lines and human resected tumor tissues of multiple other tumor types (gastric, ovarian, liver, and lung), indicating a broad applicability of YY146 in solid tumors.

Noninvasive brain cancer imaging with a bispecific antibody fragment, generated via click chemistry.

Early diagnosis remains a task of upmost importance for reducing cancer morbidity and mortality. Successful development of highly specific companion diagnostics targeting aberrant molecular pathways of cancer is needed for sensitive detection, accurate diagnosis, and opportune therapeutic intervention. Herein, we generated a bispecific immunoconjugate [denoted as Bs-F(ab)2] by linking two antibody Fab fragments, an anti-epidermal growth factor receptor (EGFR) Fab and an anti-CD105 Fab, via bioorthogonal "click" ligation of trans-cyclooctene and tetrazine. PET imaging of mice bearing U87MG (EGFR/CD105(+/+)) tumors with (64)Cu-labeled Bs-F(ab)2 revealed a significantly enhanced tumor uptake [42.9 ± 9.5 percentage injected dose per gram (%ID/g); n = 4] and tumor-to-background ratio (tumor/muscle ratio of 120.2 ± 44.4 at 36 h postinjection; n = 4) compared with each monospecific Fab tracer. Thus, we demonstrated that dual targeting of EGFR and CD105 provides a synergistic improvement on both affinity and specificity of (64)Cu-NOTA-Bs-F(ab)2. (64)Cu-NOTA-Bs-F(ab)2 was able to visualize small U87MG tumor nodules (<5 mm in diameter), owing to high tumor uptake (31.4 ± 10.8%ID/g at 36 h postinjection) and a tumor/muscle ratio of 76.4 ± 52.3, which provided excellent sensitivity for early detection. Finally, we successfully confirmed the feasibility of a ZW800-1-labeled Bs-F(ab)2 for near-infrared fluorescence imaging and image-guided surgical resection of U87MG tumors. More importantly, our rationale can be used in the construction of other disease-targeting bispecific antibody fragments for early detection and diagnosis of small malignant lesions.