4. Discussion
In this work blended hydrogels composed of agarose (variable weight
amount from 0.125 to 0.5%) and collagen (fixed weight amount equal to
0.02%) were prepared as enabling matrices for the growth of 3D cellular
structures. They combine the biomechanical properties of agarose and the
bioadhesivity of collagen. The amount of collagen is 6.25-, 12.5- and
25- times lower than that of agarose, and the formation of the hydrogel
is ascribed only to agarose and not to collagen. Indeed, although the
self-assembling capability of collagen molecules in vitro under
physiological conditions is well known, reconstituted collagen fibrils,
that are held together by non-covalent interactions (hydrogen bonding,
hydrophobic and electrostatic interactions), are free to slide and do
not form a stable 3D network (Tian, Liu,
& Li, 2016).
The characterization of the hydrogels evidenced that the agarose
percentage governs the morphological, structural and mechanical features
of the matrix. Reasonably, higher the agarose amount, stiffer the
hydrogel results. On the other hand, the lowest agarose amount
corresponds to the fastest degradation and diffusion of molecules
through the matrix. The higher degree of degradation of the softest
hydrogel is also associated to a higher release of collagen. Noteworthy,
the collagen is simply entrapped within the hydrogel mesh and not
chemically linked to the agarose backbone. Thus, it can be expected that
a less compact hydrogel facilitate the release of collagen, as
determined by the protein quantification assay. These findings are in
accordance with the compression tests that evidenced a significant
reduction of the E modulus as the agarose amount shifted from 0.5 (5.7 ±
0.5 kPa) to 0.25 (1.6 ± 0.4 kPa) up to 0.125 (0.7 ±0.2 kPa). Presumably,
a higher amount of agarose allows, during the cooling down phase after
synthesis of the hydrogels, the formation of more and tighter hydrogen
bonds that reflect the higher structural stability observed visually
because of the increase in the compression modulus.
Similarly, the entry and movement of biomolecules through the blended
hydrogel looks to be associated to the agarose percentage. The bigger
the biomolecule and the stronger the type of non-covalent interactions
it can establish with the matrix the slower they are, and that the total
amount that may reach the cells embedded into the matrix is only a
fraction of the feeding solution. In this sense, the hydrogel may act as
a physical barrier to the diffusive transport of specific nutrients and
drugs to the tumoroid, similarly to what occurs in vivo(Monteiro, Gaspar, Ferreira, & F.,
2020).
The formation and the growth of tumoroids from three breast cancer cell
lines, namely MCF-7, MDA-MB-361 and MDA-MB-231 cells, were investigated
upon variation of the agarose concentration of the matrix. While MCF-7
and MDA-MB-361 formed 3D structures, whose size and compactness is
strictly related to the stiffness and thus to the percentage of agarose
of the surrounding matrix, MDA-MB-231 cells, a triple negative breast
cell line, did not, in accordance with previous findings
(Iglesias et al., 2013;
Tasdemir, Bossart, Li, & Zhu, 2018).
The inability of this cell line to form spontaneously tumor spheroids is
probably consistent with their lack of adherens junctions.
In detail, we observed that after lading individual cells into each of
the three blended hydrogels, both cell lines formed spheroids. By
analysing the size and morphology of the structures, we realized that
breast cancer cells prefer hydrogels with a stiffness from 1.5 to 0.7
kPa, while stiffer matrices, as those with 0.5% agarose,did not result
suitable to support the growth of the spheroids. We reasoned that a
different tissue-specific tropism of the cellular models probably
contribute to this result. In this case, MCF-7 cells have a low
metastatic potential, and are not tissue-specific, while MDA-MB-361
cells were derived from a brain metastasis. The growth of MDA-MB-361
cells on a softer hydrogel matches more closely their in vivometastatic microenvironment. This was in part confirmed when we examined
the growth of a neural cell model. In A-C 0.125%-0.02% hydrogel, the
neuroblastoma SH-SY5Y cells generated spheroids in a time-dependent
manner and their size was bigger than when grown in the stiffer
0.25%-0.02% hydrogels (Figure 11S). As a general remark, SH-SY5Y
spheroids reached larger diameters (121.7 ± 14.2 µm) than those obtained
with MCF-7 and MDA-MB-361 grown in the same matrix, and this evidence
could be related to the different origin of the cell line.
The Live/Dead assay and the mitochondrial staining showed that the
spheroids are viable up to 14 days in both types of hydrogels. Notably,
we monitored the 3D cultures up to one-month: the spheroids continued to
grow in the softer hydrogel, reaching an average size of around 100 µm.
On the other hand, in the 0.25%-0.02% hydrogels the spheroids stopped
their growth after 2 weeks and started to senesce resulting definitely
dead after 4 weeks. This different behavior might be related to the
interactions between the cells and the surrounding environment as the 3D
structure grows over time: the limited degradation and capability of the
stiffer hydrogels to accommodate the spheroids induce their slow aging
and death.
These results confirm that soft agarose-collagen hydrogels allow 3D
long-term cell growth although cells derived from different tissues
sense the change in stiffness of the substrate and significantly modify
their behaviour. It has been shown, in this respect, that cells respond
to ECM environment by regulating a plethora of transcription factors and
other signals that affect cytoskeleton, cellular uptake, cell cycle that
in turn determine their morphology, proliferation, differentiation,
tumor invasion and metastasis (Kruger et
al., 2019; Ranamukhaarachchi et al.,
2019; Trappmann et al., 2012;
Wang, Gong, Zhang, & Fu, 2017). Among
them, it is worth mentioning the transmembrane glycoprotein E-cadherin
that is expressed in epithelial cells and connect them through lateral
adherent junctions. It has been demonstrated that the level of
expression of cadherin represents a crucial feature in cancer
progression as it is involved in the epithelial-mesenchymal transition
(Wong, Fang, Chuah, Leong, & Ngai,
2018). Loss of E-cadherin expression is generally associated to the
lack of intercellular contacts and to increased tumor cell invasiveness
via activation of the signalling pathways that regulate metastatic
progression (Vergara et al., 2016).
E-cadherin expression is also associated to the formation of
multicellular tumor spheroids, as already demonstrated
(Cui et al., 2017). Indeed, the spheroids
derived from MCF-7 and MDA-MB-361 displayed the expression of
E-cadherin, as shown in Figure 7.
As a general consideration, softer hydrogels allow for the establishment
of long-term culture of large and irregular breast tumor spheroids,
while stiffer environment condition the growth of small and compact 3D
cell strictures for shorter periods (Figure 12S).
Finally, we showed the possibility to recover the tumoroids from the
hydrogel after treatment with Agarase enzyme. The yield of the recovery
process is quantitative and highly reproducible in the softest gel that
undergoes spontaneously to degradation, while in the matrix with 0.25%
agarose only a small percentage of 3D structures can be successfully
recovered for further biological and biomolecular studies.