posted on 2021-10-11, 16:15authored byVenkanagouda S. Goudar, Manohar Prasad Koduri, Yen-Nhi Ngoc Ta, Yunching Chen, Li-An Chu, Long-Sheng Lu, Fan-Gang Tseng
Three-dimensional (3D) spheroid culture
provides opportunities
to model tumor growth closer to its natural context. The collagen
network in the extracellular matrix supports autonomic tumor cell
proliferation, but its presence and role in tumor spheroids remain
unclear. In this research, we developed an in vitro 3D co-culture model in a microwell 3D (μ-well 3D) cell-culture
array platform to mimic the tumor microenvironment (TME). The modular
setup is used to characterize the paracrine signaling molecules and
the role of the intraspheroidal collagen network in cancer drug resistance.
The μ-well 3D platform is made up of poly(dimethylsiloxane)
that contains 630 round wells for individual spheroid growth. Inside
each well, the growth surface measured 500 μm in diameter and
was functionalized with the amphiphilic copolymer. HCT-8 colon cancer
cells and/or NIH3T3 fibroblasts were seeded in each well and incubated
for up to 9 days for TME studies. It was observed that NIH3T3 cells
promoted the kinetics of tumor organoid formation. The two types of
cells self-organized into core–shell chimeric tumor spheroids
(CTSs) with fibroblasts confined to the shell and cancer cells localized
to the core. Confocal microscopy analysis indicated that a type-I
collagen network developed inside the CTS along with increased TGF-β1
and α-SMA proteins. The results were correlated with a significantly
increased stiffness in 3D co-cultured CTS up to 52 kPa as compared
to two-dimensional (2D) co-culture. CTS was more resistant to 5-FU
(IC50 = 14.0 ± 3.9 μM) and Regorafenib (IC50 = 49.8 ± 9.9 μM) compared to cells grown under
the 2D condition 5-FU (IC50 = 12.2 ± 3.7 μM)
and Regorafenib (IC50 = 5.9 ± 1.9 μM). Targeted
collagen homeostasis with Sclerotiorin led to damaged collagen structure
and disrupted the type-I collagen network within CTS. Such a treatment
significantly sensitized collagen-supported CTS to 5-FU (IC50 = 4.4 ± 1.3 μM) and to Regorafenib (IC50 =
0.5 ± 0.2 μM). In summary, the efficient formation of colon
cancer CTSs in a μ-well 3D culture platform allows exploration
of the desmoplastic TME. The novel role of intratumor collagen quality
as a drug sensitization target warrants further investigation.