Establishment of a bead-based duplex assay for the simultaneous quantitative detection of Neuropilin-1 and Neuropilin-2 using xMAP technology and its clinical application.

BACKGROUND: Neuropilins (Nrps) are a new type of broad-spectrum tumor marker. Currently, a method for accurate simultaneous quantification of Nrps is not available. We aimed to develop a bead-based and duplexed flow cytometric assay that could be used for accurate and simultaneous quantification of Nrp1 and Nrp2 for scientific research or clinical diagnosis. METHODS: We coupled anti-human Nrp1-11# mAb and anti-human Nrp2-C3 mAb to magnetic beads 18# and 25#, respectively. Capturing antibodies and detecting antibodies were then combined to detect Nrps by a bead-based Luminex assay, which was subsequently applied to quantify Nrps in clinical serum samples. RESULTS: The results showed that the detection value of Nrps ranged from 10 to 100 000 pg/mL for Nrp1 and from 25 to 100 000 pg/mL for Nrp2. The detection sensitivity reached 10 pg/mL for Nrp1 and 24.8 pg/mL for Nrp2. Intra-assay variances ranged from 1.0% to 2.6% for Nrp1 and from 2.9% to 4.0% for Nrp2, and interassay variances ranged from 1.5% to 6.4% for Nrp1 and from 4.2% to 8.1% for Nrp2. The Nrp1 and Nrp2 recoveries were 96.6%-103.6% and 95.6%-102.3%, respectively. Irrelevant antigens had no interference in the paired-detection system, and the mean fluorescence intensity (MFI) values were stable for months. CONCLUSION: A bead-based, duplexed flow cytometric assay (xMAP® technology) was developed to detect Nrp1 and Nrp2. The assay provided rapid, high-throughput results and was much more sensitive, specific, reproducible, and stable than existing assays. In addition, this assay could be applied in early-stage cancer screening, tumor malignancy analysis, and prognosis assessment.

Self-assembly of Au nanoparticles on PMMA template as flexible, transparent, and highly active SERS substrates.

We report a simple and rapid method for fabricating a surface-enhanced Raman scattering (SERS) substrate, which offers good flexibility, excellent optical transparency, and high SERS activity. Specifically, the SERS substrate (AuNPs/PMMA film) was obtained through self-assembly of gold nanoparticles (AuNPs) on newborn poly(methyl methacrylate) (PMMA) template. The UV-vis spectroscopy analysis and scanning electron microscopy observation revealed that the gold nanoparticles were closely assembled on the flexible and transparent PMMA template. The fabricated AuNPs/PMMA film SERS substrate allowed detection of model molecule, malachite green isothiocyanate, at a concentration as low as 0.1 nM, and exhibited good reproducibility in the SERS measurement. The Raman enhancement factor (EF) of the AuNPs/PMMA film was found to be as high as (2.4 ± 0.3) × 10(7). In addition, measure of residual malachite green on fish surface was carried out, and the result indicated that the AuNPs/PMMA film had great potential in the in situ ultrasensitive detection of analyte on irregular objects.

Optimal treatment opportunity for mTHPC-mediated photodynamic therapy of liver cancer.

Photodynamic therapy (PDT) has been clinically used for liver cancer. The pharmacokinetics of a photosensitizer needs to be monitored so that PDT can be performed at the most favorable time and with the proper dose to increase the cure rate. As mTHPC is a fluorescent compound, we investigate its pharmacokinetics, distribution, and elimination in the rat orthotropic liver cancer model in order to confirm an optimal treatment opportunity of liver cancer PDT. After intravenous administration at a single dose of 300 µg/kg, mTHPC was extracted from tissue homogenates or plasma. Then, mTHPC concentrations were assessed by fluorescence spectroscopy and the data were processed with PK-GRAPH pharmacokinetic procedure. The plasma concentration-time profile of mTHPC showed a short distribution half-life (T½α = 0.082 h) and a relatively longer elimination half-life (T½ß = 28.23 h), which quite fitted with a two-compartment model. The results of mTHPC tissue distributions showed that the highest drug accumulation was in tumor tissue, and successively decreased in liver, heart, spleen, muscle, and skin tissues. The drug distribution ratio of tumor to normal tissue reached the peak at 24 h after mTHPC administration. mTHPC was eliminated at a suitable rate in rat orthotropic liver cancer model, and there was no long-term accumulation of mTHPC in rat tissues. For PDT of orthotropic liver cancer, 24 h after mTHPC intravenous injection may be the optimal treatment time point, which might provide higher clinical efficacy and reduce side effects.

Discrimination of ground-glass nodular lung adenocarcinoma pathological subtypes via transfer learning: A multicenter study.

BACKGROUND: The surgical approach and prognosis for invasive adenocarcinoma (IAC) and minimally invasive adenocarcinoma (MIA) of the lung differ. However, they both manifest as identical ground-glass nodules (GGNs) in computed tomography images, and no effective method exists to discriminate them. METHODS: We developed and validated a three-dimensional (3D) deep transfer learning model to discriminate IAC from MIA based on CT images of GGNs. This model uses a 3D medical image pre-training model (MedicalNet) and a fusion model to build a classification network. Transfer learning was utilized for end-to-end predictive modeling of the cohort data of the first center, and the cohort data of the other two centers were used as independent external validation data. This study included 999 lung GGN images of 921 patients pathologically diagnosed with IAC or MIA at three cohort centers. RESULTS: The predictive performance of the model was assessed using the area under the receiver operating characteristic curve (AUC). The model had high diagnostic efficacy for the training and validation groups (accuracy: 89%, sensitivity: 95%, specificity: 84%, and AUC: 95% in the training group; accuracy: 88%, sensitivity: 84%, specificity: 93%, and AUC: 92% in the internal validation group; accuracy: 83%, sensitivity: 83%, specificity: 83%, and AUC: 89% in one external validation group; accuracy: 78%, sensitivity: 80%, specificity: 77%, and AUC: 82% in the other external validation group). CONCLUSIONS: Our 3D deep transfer learning model provides a noninvasive, low-cost, rapid, and reproducible method for preoperative prediction of IAC and MIA in lung cancer patients with GGNs. It can help clinicians to choose the optimal surgical strategy and improve the prognosis of patients.

[Pharmacokinetics of photosensitizer m-THPC in rat models of liver cancer via orthotropic implantation using Walker-256].

OBJECTIVE: To study the pharmacokinetics, distribution and excretion of m-THPC in rat models of liver cancer via orthotropic implantation using Walker-256. METHODS: After an intravenous injection of m-THPC with 0.3 mg/kg, the concentrations of m-THPC in biological specimens were determined by a fluorescence method. The data obtained were processed with PK-GRAPH pharmacokinetic procedure. RESULTS: The disposition of m-THPC in rat models of liver cancer Walker-256 was conformed to a two compartment model with T(1/2)α = 1.18 h, T(1/2)ß = 22.57 h at the dose of 0.3 mg/kg.m-THPC was shown to be widely distributed to the various tissues. There was a highest drug accumulation in liver and liver cancer, and lowest in skin and muscle. Ratio of m-THPC concentration in the Walker-256 tumor compared to normal tissue reach the peak 24 h after m-THPC administration. CONCLUSIONS: m-THPC is distributed widely and eliminated at a rapid rate in Walker-256 rats. Twenty four hours after m-THPC administration may be the best time for photodynamic therapy of liver cancer.

Preparation, Purification, and Identification of a Monoclonal Antibody Against the C-Terminal Domain of Semaphorin3F.

Class three semaphorins were originally identified as mediators of axon guidance, which repelled axons and collapsed growth cones. As a member of class three semaphorins, semaphorin3F (Sema3F) has been found to have similar effects on tumor cells and endothelial cells and also is implicated in the signaling of tumor metastasis by forming a complex with neuropilins and plexins. In this study, our laboratory produced a monoclonal antibody against the C-terminal domain of Sema3F (Sema3Fc mAb) using the hybridoma method, expecting to explore the potential role of the antibody and its application in the detection of Sema3F. The capture enzyme-linked immunosorbent assay (ELISA) method indicated that mAb belonged to the IgM subclass and purified Sema3Fc mAb had a titer of 5.12 × 105 against Sema3Fc by indirect ELISA. In addition, results showed that the Sema3Fc mAb could be applied in such experiments as Western blotting, flow cytometry, immunofluorescence, and immunocytochemical staining. It indicates the Sema3Fc mAb is available in the detection of Sema3F with specificity and will help further study the role and mechanism of Sema3F among tumor cells.

Programmed Nanococktail Based on pH-Responsive Function Switch for Self-Synergistic Tumor-Targeting Therapy.

Tumor-targeting combination chemotherapy is an important way to improve the therapeutic index and reduce the side effects as compared to traditional cancer treatments. However, one of the major challenges in surface functionalization of nanoparticle (NP) is accomplishing multiple purposes through one single ligand. Upon such consideration, methotrexate (MTX), an anticancer drug with a targeting moiety inspired by the similar structure of folate, could be used to covalently link with lipid-polymer conjugate (DSPE-PEG) via a pH-sensitive dynamic covalent imine (CH═N) bond to synthesize the acid-induced function "targeting-anticancer" switching DSPE-PEG-CH═N-MTX. We hypothesize that using this kind of MTX prodrug to functionalize NP's surface would be conductive to combine the early phase active targeting function and the late-phase anticancer function in one nanosystem. Herein, a nanococktail is programmed for codelivery of epirubicin (EPI) and MTX by co-self-assembly of acid-dissociated EPI-phospholipid (PC) complex and acid-cleavable DSPE-PEG-CH═N-MTX conjugate. The obtained nanococktail (MTX-PEG-EPI-PC NPs) could not only actively target folate receptors-overexpressing tumor cells but also respond to acidic endo/lysosomes for triggering the on-demand release of pharmaceutically active EPI/MTX. The intracellular drug distribution also demonstrated that the system could codeliver two drugs to individual target sites of action, inducing the significant synergistic anticancer efficiency based on different anticancer mechanisms. More importantly, the in vivo tumor accumulation and anticancer efficacy of MTX-PEG-EPI-PC NPs (via cleavable imine bond) were significantly enhanced as compared to the individual free drug, both free drugs, PEG-EPI-PC NPs, and MTX-PEG-EPI-PC NPs (via the uncleavable amide bond). This self-synergistic tumor-targeting therapy might represent a promising strategy for cancer treatment.

Preparation, Purification, and Identification of a Monoclonal Antibody Against NRP2 b1b2 Domain.

First identified as a high-affinity kinase-deficient receptor for class-3 semaphorins and vascular endothelial growth factor (VEGF) families, Neuropilin2 (NRP2) is a transmembrane non-tyrosine-kinase glycoprotein that has a vital function in neuronal patterning. Furthermore, NRP2 expression is often upregulated in cancer tissues and correlated with poor prognosis. In the present study, we report the establishment of a monoclonal antibody specific for NRP2b1b2 domain (NRP2 MAb) through hybridoma method. NRP2 MAb is measured to have a titer of 5.12 × 10(5) against NRP2b1b2 in indirect ELISA. Western blotting, flow cytometry, and immunofluorescence analysis indicate that NRP2 MAb can combine full-length NRP2 in LoVo and SW480 cells. Besides helping further understand NRP2-related pathological mechanisms and cell-signaling pathways, NRP2 MAb may act as a therapeutic agent for cancer in the future.

Development of monoclonal antibody-based sandwich ELISA for detection of dextran.

Dextran as anti-nutritional factor is usually a result of bacteria activity and has associated serial problems during the process stream in the sugar industry and in medical therapy. A sensitive method is expected to detect dextran quantitatively. Here we generated four monoclonal antibodies (MAbs) against dextran using dextran T40 conjugated with bovine serum albumin (BSA) as immunogen in our lab following hybridoma protocol. Through pairwise, an MAb named D24 was determined to be conjugated with horseradish peroxidase (HRP) and was used in the establishment of a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) method for determination of dextran, in which MAb D9 was chosen as a capture antibody. The detection limit and working scope of the developed sandwich ELISA method were 3.9 ng/mL and 7.8-500 ng/mL with a correlation coefficient of 0.9909. In addition, the cross-reaction assay demonstrated that the method possessed high specificity with no significant cross-reaction with dextran-related substances, and the recovery rate ranged from 96.35 to 102.00%, with coefficient of variation ranging from 1.58 to 6.94%. These results indicated that we developed a detection system of MAb-based sandwich ELISA to measure dextran and this system should be a potential tool to determine dextran levels.