EpCAM is a putative stem marker in retinoblastoma and an effective target for T-cell-mediated immunotherapy.

PURPOSE: The molecular markers cluster of differentiation (CD)24, CD44, adenosine tri-phosphate (ATP) binding cassette protein G2 (ABCG2), and epithelial cell adhesion molecule (EpCAM) are widely used, individually or in combination, to characterize some types of cancer stem cells. In this study we characterized the EpCAM+ retinoblastoma (RB) cells for their cancer stem-like properties in vitro. Additionally, we targeted RB tumor cells via redirecting T cells using bispecific EpCAM×CD3 antibody. METHODS: Flow cytometry was used to study the co-expression of EpCAM with putative cancer stem cell markers, such as CD44, CD24, and ABCG2, in RB primary tumors. In vitro methyl thiazol tetrazolium (MTT) assay, invasion assay, and neurosphere formation assay were performed to characterize EpCAM+ cells for their cancer stem/progenitor cell-like properties. We assessed the in vitro efficacy of bispecific EpCAM×CD3 antibody on RB tumor cell proliferation and validated the results by evaluating effector cytokine production in the culture medium with the ELISA method. RESULTS: EpCAM was co-expressed with all cancer stem cell markers (CD44, CD24, and ABCG2) in primary RB tumors. EpCAM+ cells showed significantly higher proliferative invasive potential and neurosphere formation in vitro compared to EpCAM⁻ Y79 cells. EpCAM+ cells showed higher ß-catenin expression compared to EpCAM- cells. EpCAM×CD3 significantly retarded proliferation of RB primary tumor cells. EpCAM×CD3 effectively induced the secretion of effector cytokines, such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-10, IL-2, and transforming growth factor (TGF)-ß1, and also perforin levels by pre-activated lymphocytes. CONCLUSIONS: EpCAM might be a novel cancer stem cell marker in RB. EpCAM×CD3 antibody redirecting T cells to attack RB tumor cells may prove effective in RB management. Further preclinical studies are needed to confirm the initial findings of our study.

Toxicogenomics of nanoparticulate delivery of etoposide: potential impact on nanotechnology in retinoblastoma therapy.

To develop a suitable formulation with high entrapment efficiency, etoposide-loaded poly(lactide-co-glycolide) nanoparticles (NPs) were formulated by single emulsion-solvent evaporation method by changing different formulation parameters such as drug loading, choice of organic solvent and percentage of emulsifier polyvinyl alcohol. The NPs showed higher entrapment efficiency, ~86% (with 15% (w/w) drug loading). The physicochemical parameters revealed smooth topology with size range (240-320 nm), a negative zeta potential (~19 mV) and in vitro sustained-release activity (~60% drug release in 40 days). Greater anti-proliferative activity ~100 times was observed with NPs (IC50 = 0.002 µg/ml) than that of native etoposide (IC50 = 0.2 µg/ml) in retinoblastoma cell line (Y-79). These NPs demonstrated greater (G1/S) blocking and decreased mitochondrial membrane potential as measured by flow cytometry. There was upregulation of apoptotic gene activity in NPs than native etoposide, as revealed through microarray analysis. However, this is the first ever report demonstrating the intricate modulation of genetic network affected by NPs. Collectively, these results suggest that etoposide-loaded NPs could be potentially useful as a novel drug delivery system for retinoblastoma in the future. FigureNanoparticle-mediated etoposide delivery promotes apoptosis through upregulating several apoptotic inducer genes.

Reversal of stathmin-mediated microtubule destabilization sensitizes retinoblastoma cells to a low dose of antimicrotubule agents: a novel synergistic therapeutic intervention.

PURPOSE: To explore the possibility of stathmin as an effective therapeutic target and to evaluate the synergistic combination of stathmin RNAi and the antimicrotubule agents paclitaxel and vincristine to retinoblastoma Y79 cells. METHODS: RNAi-mediated specific inhibition of stathmin expression in Y79 cells was shown by real-time quantitative RT-PCR (RT-Q-PCR), its effect on cell proliferation by MTT assay, cell invasion using matrigel, microtubule polymerization by immunohistochemistry, apoptosis, cell cycle analysis by flow cytometry analysis, and the changes in FOXM1 protein expression were studied by Western blot. The effect of combination treatment of stathmin siRNA and paclitaxel/vincristine was studied by assessing cell viability and apoptosis. RESULTS: Short interfering RNA-mediated transient stathmin downregulation resulted in a marked inhibition of retinoblastoma cell proliferation and cell invasion in vitro. Stathmin inhibition promoted Y79 cells to G2/M phase, and ultimately there were increased apoptotic events as evidenced by higher caspase-3 activation and cleaved poly(ADP-ribose) polymerase expression. Cells transfected with stathmin siRNA showed long and bundled microtubule polymers and sensitized the Y79 cells significantly to paclitaxel and vincristine. CONCLUSIONS: Stathmin may be a pivotal determinant for retinoblastoma tumorigenesis and chemosensitivity. Strategies to inhibit stathmin will help to enhance the cytotoxic effect of paclitaxel while reducing toxicity (or side effects) to normal cells caused by high doses.