The feasibility of using dielectrophoresis for isolation of glioblastoma subpopulations with increased stemness.

Cancer stem cells (CSCs) are aggressive subpopulations with increased stem-like properties. CSCs are usually resistant to most standard therapies and are responsible for tumor repropagation. Similar to normal stem cells, isolation of CSCs is challenging due to the lack of reliable markers. Antigen-based sorting of CSCs usually requires staining with multiple markers, making the experiments complicated, expensive, and sometimes unreliable. Here, we study the feasibility of using dielectrophoresis (DEP) for isolation of glioblastoma cells with increased stemness. We culture a glioblastoma cell line in the form of neurospheres as an in vitro model for glioblastoma stem cells. We demonstrate that spheroid forming cells have higher expression of stem cell marker, nestin. Next, we show that dielectric properties of neurospheres change as a result of changing culture conditions. Our results indicate that spheroid forming cells need higher voltages to experience the same DEP force magnitude compared to normal monolayer cultures of glioblastoma cell line. This study confirms the possibility of using DEP to isolate glioblastoma stem cells.

Spatial composition of prostate cancer spheroids in mixed and static cultures.

Aggregation of neoplastic cells produces multicellular spheroids resembling micrometastases. The objective of this study was to investigate the effects of mixing culture medium on the spatial composition of spheroids prepared from well (LNCaP) and poorly (DU 145) differentiated human prostate cancer cells. Spheroids were cultured in a mixed suspension within a high-aspect rotating wall vessel and static liquid-overlay plate. Results from this study demonstrate that mixed cultures consistently manifested differences in morphology and composition between DU 145 and LNCaP spheroids. For example, 40 +/- 12% of DU 145 cells were Ki-67 positive 100 microm from the surface within mixed spheroids versus 0% for LNCaP cells; there was no significant difference in this spatial profile for static cultures. The results suggest that poorly differentiated spheroids may be more likely to experience a change in composition from mixing culture medium than well-differentiated spheroids, due to low tissue density. Immunostaining for P-glycoprotein is representative of this trend; average staining intensity increased 50% for DU 145 spheroids on mixing but was unchanged for LNCaP spheroids. The effects of mixing on spheroid composition were attributed to faster interstitial mass transport. Applications include drug development and delivery, as well as basic research on drug action and resistance.

Sphere-forming-like cells (squamospheres) with cancer stem-like cell traits from VX2 rabbit buccal squamous cell carcinoma.

Previous studies have demonstrated that spheroid type cells grown under suspension culture conditions have cancer stem cell (CSC) traits in a number of cancers, but this phenomenon has not yet been reported in the VX2 rabbit oral cancer model. Hence, this study aimed to study the spheroid cells from VX2 rabbit buccal squamous cell carcinomas (SCCs) and assess their CSC characteristics. Five adult male New Zealand white outbred rabbits were used to generate VX2 rabbit buccal SCC. Sphere-forming cell culture was performed for the VX2 rabbit buccal SCC specimens. The self-renewal capability; cluster of designation (CD) 44, CD133, acetaldehyde dehydrogenase 1 (ALDH1), B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1), Nestin, octamer-binding transcription factor 4 (Oct4) and reduced expression protein-1 (Rex-1) expression with reverse transcription-polymerase chain reaction (RT-PCR); chemoresistance to cisplatin and 5-fluorouracil; and in vivo tumorigenicity of spheroid cell transplantation in nude mice were evaluated to determine the CSC characteristics of the resulting spheroid cells. We successfully obtained spheroid cells from the VX2 rabbit OSCC tissues. The spheroid cells exhibited CSC traits, including the expression of CSC and stem cell markers (CD44, Bmi-1, Nestin, Oct4 and Rex-1), capacity to generate new spheroid colonies within 1 week of reseeding from single-dissociated spheroid cells, chemoresistance capacity and generation of tumour xenografts (with histological features resembling those of the original VX2 rabbit buccal SCC) from the transplantation of 10(3) undifferentiated spheroid cells into nude mice. In summary, we demonstrated that spheroid cells with CSC cell traits can be derived from VX2 rabbit buccal SCCs, indicating that this animal cancer model is applicable for studying CSCs in human oral cancers.