Bioprinted cancer-stromal in-vitro models in a decellularized ECM-based bioink exhibit progressive remodeling and maturation

Abstract

Continuous extracellular matrix (ECM) remodeling and cellular heterogeneity are key contributors to cancer development and can both profoundly affect treatment efficacy. Developing in-vitro models that recapitulate matrix and cellular heterogeneity of the tumor microenvironment (TME) can aid in observations that are currently challenging to acquire with conventional 2D cultures and preclinical animal models. Here we report an extrusion bioprinted co-culture model of head and neck cancer and stromal fibroblasts using a composite bioink containing a reinforced decellularized extracellular matrix hydrogel. Fibroblasts have a significant role in remodeling and matrix deposition. When cultured in the bioactive extracellular matrix ink, they provide the cellular elements typically found in the tumor stroma. Head and neck squamous carcinoma cells (UM-SCC-38) were integrated into the bioink, and in the presence of fibroblasts (HVFFs), they began to proliferate into cell-cell interactive spheroids. As the co-culture model is capable of remodeling, we evaluated the ultrastructure of the bioink. We observed a fibrous collagenous network retained from the ECM of the source tissue containing nanometer-scale pores. Following the deposition of the co-culture model, we observed UM-SCC-38 spheroid formation that began during the first week in culture and continued over a three-week period in which the fibroblasts migrated to regions directly surrounding each spheroid. Using a Luminex assay to quantify matrix metalloproteases in co-cultures compared to monocultures, we observed significant differences in the presence of MMP-9 and MMP-10 expression corresponding to periods of the culture in which collagen underwent remodeling. Time-dependent characterization of collagen synthesis, protease activity, and spheroid growth rates are developed to characterize the system as an advanced co-culture model to evaluate tumor-stromal interactions and remodeling.

Contribution of the ELRs to the development of advanced in vitro models

“…This capability is pivotal for generating in vitro models that accurately mimic the intricate features of native tissues, facilitating regenerative medical endeavors ( Nam et al, 2015 ; Langhans, 2018 ; Bédard et al, 2020 ; Hwang et al, 2021 ). Recent examples have highlighted the potential of 3D bioprinting in creating in vitro models of human tissues and diseases, including notable applications such as: accurate modeling of the complex tumor microenvironments ( Kort-Mascort et al, 2023 ; Mu et al, 2023 ; Zhou et al, 2023 ); physiologically relevant models for the entire respiratory tract ( Moura et al, 2023 ); replication of native skin features for wound healing studies ( Norahan et al, 2023 ); engineered cardiac tissues such as in vitro cardiac models and vascular channels ( Lu et al, 2023 ); or normal and disease hepatic tissue models ( Sun et al, 2023 ).…”

In Vitro Models of Head and Neck Cancer: From Primitive to Most Advanced

“…The authors continued their work in this direction and recently published data on an improved 3D model of HNSCC, in which the cellular component was represented by two types of cells-UM-SCC-38 and A8-HVFFs (immortalized human vocal fold fibroblasts) in a ratio of 1:2. In the process of culturing the model, spheroid development and growth over time with cancer cells in the core and fibroblasts in the periphery were observed [104].…”

“…It can be concluded that the 3D bioprinted model of HNSCC is essentially an improved tissue-engineered model, where a hydrogel is used as a scaffold, and the use of a printing method instead of the usual layering of the gel on a substrate allows for more accurate control of the compliance of the tissue architecture with the specified parameters. So far, bioprinted HNSCC models have been obtained only for immortalized lines, which is due to the need to use a large number of cells to obtain the desired result (in [104] cells were encapsulated in gel at a final concentration of 10 million cells per ml). Obviously, an important task for researchers is to obtain such 3D models with HNSCC patient-derived cells to closely match more biological features and potentially use this technique for personalized medicine.…”