3.2 Growth of mammary spheroids in A-C hydrogels
We generated mammary spheroids from three different breast cancer cell lines. We focused on luminal estrogen receptor positive cells, MCF-7 and MDA-MB-361, and a triple negative model (MDA-MB-231) for comparative analysis. To start, cells were seeded at the density of 2.5 x 104 per mL inside the three types of hydrogels and time course studies (up to 14 days) were performed to monitor the process of multicellular spheroid formation. MCF-7 and MDA-MB-361 formed spheroids successfully in all the three types of hydrogels. On the other hand, MDA-MB-231 did not form spheroids in any of the hydrogel conditions. As shown in Figure 3S, MDA-MB-231 cells start to replicate during the first days without reaching a defined 3D organization.
As reported, MCF-7 and MDA-MB-361 cells formed spheroids, but the size and the morphology of the 3D structures was not the same in all the conditions tested. Figure 4 and 4S show the spheroids at different days of growth in the three types of hydrogels. In the stiffer ones the spheroids were spherical but smaller (Figure 4S) than those grown in the other two conditions (Figure 4), especially in the case of MDA-MB-361 cells. This effect is more evident as the 3D structure progressively grows over time: the average size of MDA-MB-361 derived spheroids after 12 days growth in hydrogels with 0.5% agarose was around 63.1 ± 7.8 µm, while it reached 81.3 ± 6.3 µm and 94.7 ± 9.5 µm in 0.25%-0.02% and 0.125%-0.02% A-C hydrogels, respectively (Table 1S). In the case of MCF-7 cells, the size gap was of about 7 and 27 µm respectively, being the spheroids at 12 days about 64 µm large in the hydrogels with 0.5% agarose, while they reached a diameter of about 70 µm in the 0.25%-0.02% hydrogel and 91 µm in the softer ones (Table 1S). The images of Figure 4 clearly evidence how the 3D structures evolve from a single cell over time, and the growth curves reported in the lower panels of the Figure show that while in the hydrogel with 0.25% agarose the spheroids look to reach the growth stabilization at 14 days, in the softest environment they still display a positive growth trend. Thus, though this study is focused to the analysis of the spheroids growth within two weeks, that is a time range sufficient for drug testing, we prolonged the observation time up to 28 days to point out the growth curve of the 3D cell structures in the 0.125%-0.02% A-C hydrogels. As shown in lower panels of Figure 5S, in the softest matrix the spheroids continued to grow exceeding the 100 µm diameter in both cell lines at 28 days. On the other hand, in the 0.25%-0.02% A-C hydrogels the spheroids stop their growth and after 4 weeks they started to shrink and appear senescent with dark cellular stuff. In this sense, it looks that the softest hydrogel is capable of sustaining the spheroids growth for longer time, and, especially in the case of MCF-7, the spheroids display irregular contours and looser structure. As a general consideration, we observed that MCF-7 tolerate stiffer environments as compared to MDA-MB-361. Indeed MCF-7 generated small spheroids in hydrogels with 1% agarose too (Figure 6S), while MDA-MB-361 did not.
Therefore, the hydrogel stiffness and the matrix composition regulate the spheroids growth and morphology and, more interestingly, affect the local migration of the outer cells. Indeed, as shown in Figure 5, only the softest matrix is able to induce protrusion of cells from the outer layer and their local dissemination. As already reported, the motility of these cells depends on the interaction with the microenvironment, mainly with collagen (Nguyen-Ngoc et al., 2012). It is likely that the hydrogels with 0.125% agarose promote the protrusive behavior while collagen provides the anchoring point for the spatial dissemination of the cells. The reduced migration of the outer cells in the hydrogels with 0.25% agarose should be related to the tighter pressure that the matrix exerts on the cells, leading, as already described, to more compact spheroids.
Based on these observations and with the aim to study the effects of the mechanical features of the environment on the tumoroids features, the following analyses were carried out comparing the two conditions (0.25% and 0.125% agarose-based hydrogels) in which both cell lines were able to form healthy and stable spheroids up to two weeks.