Innovative experimental approach for the morphological characterization of cancer stem cells spheroids

Abstract

It’s widely accepted the involvement of EMT in the onset of cancer metastasis given its abil- ity to enable both cancer cells dissemination and self-renewal. Many researches have showed that the generation of circulating tumor cells (CTCs) with stem cells-like properties is respon- sible for metastasis successful; despite the advances in CTCs detection systems, the molecular characterization appears tough since their existence in a very small amount. For this purpose, increasing numbers of studies have developed multiple methodological tools for the culture of cancer cells exhibiting stem cells-like properties, including 3D-spheroids propagation [1]. Our group have previously showed that ferritin heavy chain (FHC) exerts a negative role on both ovarian cancer stem cells expansion and EMT, via the application of the in vitro 3D spheroid assay [2]. Here, we applied an innovative experimental approach for the characterization of cancer stem cell spheroids. FHC-silenced (shFHC) and control shScr SKOV3-cells were cultured in ultra-low attachment plates and maintained with RPMI supplemented with 10% FBS. The first generation of spheroids derived from a 10-day cultures of FHC-silenced and control shScr SKOV3-cells were characterized for their number, size and morphology. The expression of pro- liferative markers, extracellular matrix components and stem cells markers was evaluated in both adherent and spheroids cells using the cell block technique. First, shFHC cells showed a significant greater number and larger 3D spheroids than control shScr SKOV3-cells in 10-day cultures. Proliferative activity as determined by Ki-67 immunoreactivity showed an even distri- bution in adherent cells. Otherwise, in FHC-silenced spheroids, proliferation was predominant in the peripheral areas. The spheroid cell cultures also exhibited a distinct network of CD44 and CD56. We suggest our experimental approach as a useful tool for testing the role of FHC in the acquisition of stem cells-like properties.