Bioengineering multicellular tumor spheroids with tunable extracellular matrix deposition.

Area of Science:

• Biomedical Engineering
• Cancer Biology
• 3D Cell Culture Models

Background:

• Conventional 2D cell culture assays inadequately model the complex tumor microenvironment.
• Three-dimensional multicellular tumor spheroids (MCTS) offer a promising platform for studying tumor biology and drug responses.
• Standardization of MCTS formation methodologies is lacking, particularly regarding stromal content.

Purpose of the Study:

• To systematically investigate how varying cancer cell-fibroblast ratios impact MCTS architecture, molecular profiles, and functionality.
• To establish a reproducible strategy for engineering MCTS with tunable stromal content and desmoplastic activity.
• To enhance the accuracy and relevance of 3D in vitro tumor models for drug screening and biological research.

Main Methods:

• Co-culture of four cancer cell lines with fibroblasts at defined ratios to create MCTS with increasing stromal content.
• Analysis of MCTS using histology, live fluorescence microscopy, immunofluorescence, flow cytometry, and gene expression assays.
• Quantification of growth kinetics, cell organization, proliferation, ECM deposition, and phenotypic states.

Main Results:

• Cancer cell identity and fibroblast proportion significantly influenced spheroid compactness, internal architecture, desmoplastic activity, and proliferation.
• Fibroblast-rich MCTS exhibited increased extracellular matrix (ECM) deposition and upregulation of genes related to fibroblast activation and ECM remodeling.
• Increasing stromal content in MCTS reduced sensitivity to chemotherapeutics, more accurately reflecting in vivo drug responses.

Conclusions:

• A reproducible strategy was established for engineering MCTS with tunable stromal content, enhancing their resemblance to in vivo tumors.
• These findings provide a standardized framework for generating MCTS with defined stromal properties, improving the reproducibility of 3D in vitro tumor models.
• The developed MCTS platform enables controlled interrogation of tumor-stroma interactions and offers a foundation for studying stromal modulation of therapy response.