Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 µm in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Quantum Dots-siRNA Nanoplexes for Gene Silencing in Central Nervous System Tumor Cells.

RNA interfering (RNAi) using short interfering RNA (siRNA) is becoming a promising approach for cancer gene therapy. However, owing to the lack of safe and efficient carriers, the application of RNAi for clinical use is still very limited. In this study, we have developed cadmium sulphoselenide/Zinc sulfide quantum dots (CdSSe/ZnS QDs)-based nanocarriers for in vitro gene delivery. These CdSSe/ZnS QDs are functionalized with polyethyleneimine (PEI) to form stable nanoplex (QD-PEI) and subsequently they are used for siRNA loading which specially targets human telomerase reverse transcriptase (TERT). High gene transfection efficiency (>80%) was achieved on two glioblastoma cell lines, U87 and U251. The gene expression level (49.99 ± 10.23% for U87, 43.28 ± 9.66% for U251) and protein expression level (51.58 ± 7.88% for U87, 50.69 ± 7.59% for U251) of TERT is observed to decrease substantially after transfecting the tumor cells for 48 h. More importantly, the silencing of TERT gene expression significantly suppressed the proliferation of glioblastoma cells. No obvious cytotoxicity from these QD-PEI nanoplexes were observed over at 10 times of the transfected doses. Based on these results, we envision that QDs engineered here can be used as a safe and efficient gene nanocarrier for siRNA delivery and a promising tool for future cancer gene therapy applications.