3.3 Mammary spheroids viability and epithelial markers expression
The viability of the spheroids obtained from the MCF-7 and MDA-MB-361 cell lines in both type of hydrogels was qualitatively analyzed through a Live/Dead fluorescence assay that evidence the cell viability even after 14 days of growth (Figure 7S). Indeed, at 8 days the homogeneous green fluorescence evidenced that all the cells of the spheroids were viable; after 14 days few red spots were visible in the 3D structure grown in the matrix with 0.25% agarose, showing their initial aging, while the spheroids embedded in the softer hydrogel resulted absolutely viable. These data are in accordance with the previous analysis of the growth curves of the 3D structures in the two systems. To further confirm this trend, the assay was also performed after 28 days. Notably, while the spheroids grown in the 0.125% agarose-based hydrogels were still viable, those prepared in the stiffer matrix were dead. Furthermore, Mitotracker red, an indicator of mitochondrial membrane potential able to selectively stain live mitochondria, was used to gain deeper insights into the status of cells and the porosity of the hydrogels (Figure 6). Mitotracker staining showed the presence of live mitochondria in the periphery as well as in the center of the spheroids, thus proving the permeability to oxygen of the A-C hydrogels, since mitochondrial function critically depends on its availability.
We then determined by immunofluorescence the expression of E cadherin, a typical epithelial marker in 3D spheroids. Fig 7 clearly shows that mammary spheroids maintain the expression of the transmembrane glycoprotein E-cadherin and clearly demonstrate the presence of tight cell-cell interactions, both typical features of an epithelial phenotype (Figure 7). Overall, these data suggest that our hydrogel is a suitable method for the generation of mammary spheroids.
To further elucidate the morphology of the spheroids and their arrangement into the hydrogel, ultrastructural analysis of MCF-7 spheroids was performed showing their 3D structure that looks enveloped into the hydrogel matrix: the protrusion of the spheroids from the hydrogel can be clearly appreciated and they can be compared to cocoons anchored to a branch (Figure 8S).