Glioma is the most common malignant cancer affecting the central nerve system, with dismal prognosis. Differentiationinducing therapy is a novel strategy that has been preliminarily proved effective against malignant glioma. We have reported previously that activation of cAMP/protein kinase A (PKA) pathway is capable of inducing glioma cell differentiation, characterized by astrocyte-like shape and dramatic induction of astrocyte biomarker glial fibrillary acidic protein (GFAP). However, little progress has been made on molecular mechanisms related. Here we demonstrate that microRNA 335 (miR-335) is responsible for the glioma cell differentiation stimulated by activation of cAMP/PKA pathway. In the cAMP elevator cholera toxin-induced differentiation model of rat C6 glioma cells, miR-335 was significantly up-regulated, which was mimicked by other typical cAMP/PKA pathway activators (e.g., forskolin, dibutyryl-cAMP) and abolished by PKA-specific inhibitor (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3Ј,2Ј,1Ј-kl]pyrrolo[3,4-i] [1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT5720). In an assay measuring gain and loss of miR-335 function, exogenetic miR-335 resulted in induction of GFAP, whereas miR-335 specific inhibitor antagomir-335 violently blocked cholera toxin-induced GFAP up-regulation. It is noteworthy that in human U87-MG glioma cells and human primary culture glioma cells, miR-335 also mediated cholera toxin-induced differentiation. Taken together, our findings suggest that miR-335 is potently required for differentiation of malignant glioma cells induced by cAMP/PKA pathway activation, and a single microRNA may act as an important fate determinant to control the differentiation status of malignant gliomas, which has provided a new insight into differentiation-inducing therapy against malignant gliomas.

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As a novel co-receptor for vascular endothelial growth factor (VEGF), neuropilin receptor type-1 (NRP-1) is overexpressed in several cancers and metastases, and serves as an attractive target for cancer molecular imaging and therapy. Previous single photon emission computerized tomography (SPECT) studies demonstrated that the small NRP-1-targeting peptides 99mTc-MA-ATWLPPR and 99mTc-CK3 showed poor tumor imaging quality, because of their rapid blood clearance and very low tumor uptake. Compared with small peptides, monoclonal antibodies (mAbs) can improve imaging of NRP-1-expression, due to their high affinity, specificity and slow extraction. A6-11-26 is a novel monoclonal antibody against NRP-1 b1b2 domain that exhibits inhibition of tumor growth in NPR-1-expressing preclinical models. The aim of the present study was to develop the 131I-labeled anti-NRP-1 monoclonal antibody A6-11-26 as a SPECT probe for imaging of NRP-1-positive tumor. An anti-NRP-1 monoclonal antibody (A6-11-26) was produced by hybridomas and was labeled with iodine-131 by the iodogen method. In vitro, the radiolabeling efficiency, radiochemical purity, immunoreactive fraction and stability were assessed. Binding affinity and specificity of 131I‑A6-11-26 to NRP-1 were evaluated using human glioblastoma U87MG cells. In vivo, biodistribution and SPECT/CT studies were conducted on mice bearing U87MG xenografts after the injection of 131I-A6-11-26 with or without co-injection of unlabeled A6-11-26 antibody. A6-11-26 was generated successfully by hybridoma with high purity (>95%) and was labeled with iodine-131 within 60 min with high labelling efficiency (95.46±3.34%), radiochemical purity (98.23±1.41%). 131I-A6-11-26 retained its immunoreactivity and also displayed excellent stability in mouse serum and PBS solution during 1 to 96 h. Cell uptake assays showed high NRP-1-specific uptake (15.80±1.30% applied activity at 6 h) in U87MG cells. 131I-A6-11-26 bound to NRP-1 with low nanomolar affinity (KD=1.67±0.14 nM) in U87MG cells. In vivo, biodistribution study demonstrated targeting of U87MG glioma xenografts was NRP-1 specific. The tumor uptake was 6.00±1.24%ID/g at 24 h, and the tumor to muscle ratio was 3.20±0.30 at 24 h, and reached the highest level of 6.13±0.24 at 120 h after injection. SPECT imaging studies revealed that 131I-A6-11-26 could clearly identify U87MG tumors with good contrast, especially at 72-120 h after injection. The present study demonstrates that 131I-A6-11-26 is capable of detecting lesions in an NRP-1-expressing tumor with high target selectivity, and may offer a promising SPECT agent for NRP-1 expression positive tumor and encourage further investigation.

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