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.
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