In Vivo MR Imaging of Glioma Recruitment of Adoptive T‐Cells Labeled with NaGdF4‐TAT Nanoprobes

Adoptive T lymphocyte immunotherapy is one of the most promising methods to treat residual lesions after glioma surgery. However, the fate of the adoptively transferred T‐cells in vivo is unclear, hampering the understanding of this emerging therapy. Thus, it is highly desirable to develop noninvasive and quantitative in vivo tracking of these T‐cells to glioma for better identification of the migratory fate and to provide objective evaluation of outcomes of adoptive T‐cell immunotherapy targeting glioma. In this work, ultrasmall T₁ MR‐based nanoprobes, NaGdF₄‐TAT, as molecular probes with high longitudinal relaxivity (8.93 mm ^?1 s^?1) are designed. By means of HIV‐1 transactivator (TAT) peptides, nearly 95% of the adoptive T‐cells are labeled with the NaGdF₄‐TAT nanoprobes without any measurable side effects on the labeled T‐cells, which is remarkably superior to that of the control fluorescein isothiocyanate‐NaGdF₄ concerning labeling efficacy. Labeled adoptive T‐cell clusters can be sensitively tracked in an orthotopic GL261‐glioma model 24 h after intravenous infusion of 10⁷ labeled T‐cells by T₁‐weighted MR imaging. Both in vitro and in vivo experiments show that the NaGdF₄‐TAT nanoprobes labeling of T‐cells may be a promising method to track adoptive T‐cells to improve our understanding of the pathophysiology in adoptive immunotherapy for gliomas.     

Specific Capture of Peptide‐Receptive Major Histocompatibility Complex Class I Molecules by Antibody Micropatterns Allows for a Novel Peptide‐Binding Assay in Live Cells

Binding assays with fluorescently labeled ligands and recombinant receptor proteins are commonly performed in 2D arrays. But many cell surface receptors only function in their native membrane environment and/or in a specific conformation, such as they appear on the surface of live cells. Thus, receptors on live cells should be used for ligand binding assays. Here, it is shown that antibodies preprinted on a glass surface can be used to specifically array a peptide receptor of the immune system, i.e., the major histocompatibility complex class I molecule H‐2K^b, into a defined pattern on the surface of live cells. Monoclonal antibodies make it feasible to capture a distinct subpopulation of H‐2K^b and hold it at the cell surface. This patterned receptor enables a novel peptide‐binding assay, in which the specific binding of a fluorescently labeled index peptide is visualized by microscopy. Measurements of ligand binding to captured cell surface receptors in defined confirmations apply to many problems in cell biology and thus represent a promising tool in the field of biosensors.     

Selective Capture and Quick Detection of Targeting Cells with SERS‐Coding Microsphere Suspension Chip

Circulating tumor cells (CTCs) captured from blood fluid represent recurrent cancers and metastatic lesions to monitor the situation of cancers. We develop surface‐enhanced Raman scattering (SERS)‐coding microsphere suspension chip as a new strategy for fast and efficient capture, recovery, and detection of targeting cancer cells. Using HeLa cells as model CTCs, we first utilize folate as a recognition molecule to be immobilized in magnetic composite microspheres for capturing HeLa cells and attaining high capturing efficacy (up to 95%). After capturing cells, the composite microsphere, which utilizes a disulfide bond as crosslinker in the polymer shell and as a spacer for linking folate, can recycle 90% cells within 20 min eluted by glutathion solution. Taking advantage of the SERS with fingerprint features, we characterize captured/recovered cells with the unique signal of report‐molecule 4‐aminothiophenol through introducing the SERS‐coding microsphere suspension chip to CTCs. Finally, the exploratory experiment of sieving cells shows that the magnetic composite microspheres can selectively capture the HeLa cells from samples of mixed cells, indicating that these magnetic composite microspheres have potential in real blood samples for capturing CTCs.     

Tracking the Down‐Regulation of Folate Receptor‐α in Cancer Cells through Target Specific Delivery of Quantum Dots Coupled with Antisense Oligonucleotide and Targeted Peptide

Based on the multivalent binding capability of streptavidin (SA) to biotin, a multifunctional quantum dot probe (QD‐(AS‐ODN+p160)) coupled with antisense oligonucleotide (AS‐ODN) and peptide p160 is designed for real‐time tracking of targeted delivery of AS‐ODN and regulation of folate receptor‐α (hFR‐α) in MCF‐7 breast cancer cells. Fluorescence spectra, capillary electrophoresis (CE) and dynamic light scattering (DLS) are used to characterize the conjugation of AS‐ODN and p160 with quantum dots (QDs), DLS results confirm the well stability of the probe in aqueous media. Confocal imaging and quantitative flow cytometry show that QD‐(AS‐ODN+p160) is able to specifically target human breast cancer MCF‐7 cells. Low temperature and ATP depletion treatments reveal the cellular uptake of QD‐(AS‐ODN+p160) is energy‐dependent, and the effects of inhibition agents and co‐localization imaging further confirm the endocytic pathway is mainly receptor‐mediated. Transmission electron microscopy (TEM) shows the intracellular delivery and endosomal escape of QD probe along with incubation time extended. Two transfection concentrations of QD probe (10 nM and 50 nM) below half inhibitory concentration (IC₅₀) value are chosen according to MTT assay. Real‐time PCR shows at these two concentration cases the relative mRNA expression levels of hFR‐α reduce to 72.5 ± 3.9% and 17.6 ± 1.0%, respectively. However, western blot and quantitative ELISA analysis show the expression level of hFR‐α protein has a significant decrease only at 50 nM, indicating that gene silence is concentration‐dependent. These results demonstrate that the QD‐(AS‐ODN+p160) probe not only achieves gene silence in a cell‐specific manner but also achieves real‐time tracking during AS‐ODN intracellular delivery.