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.