# A 3D Collagen-Based In Vitro Cancer Model Created Through Modular Tissue Engineering

## TL;DR

Researchers created a 3D cancer model using collagen hydrogels that better mimics real tumors and shows drug resistance, offering a promising alternative to traditional models.

## Contribution

A novel 3D cancer model using modular tissue engineering that reproduces tumor features like hypoxia and drug resistance.

## Key findings

- Cells in microtissues showed sustained viability and natural development of tumor properties like hypoxia.
- The model demonstrated drug resistance at clinically relevant concentrations.
- The 3D environment influenced stem cell regulation, as shown by CD44+/CD24− phenotype analysis.

## Abstract

Most new cancer drugs fail in clinical trials, largely because preliminary drug studies do not adequately reflect the complexity of this human pathology. In this context, three-dimensional culture systems have advanced cancer research, but many existing models are difficult to fabricate or fail to reproduce important tumour characteristics. In this study, we developed cancer microtissues, which are small, free-floating collagen hydrogels containing cancer cells. They are produced through an efficient process known as modular tissue engineering and naturally replicate key features of real tumours, including low oxygen regions, stemness phenotypes and resistance to treatment. Our findings highlight the potential of microtissues as a practical model that can be used as an alternative to other three-dimensional cancer culture systems.

Background: An emerging tool to better simulate the complexity of tumour biology in vitro is 3D culture models. Several approaches have been introduced, yet many face challenges such as technical complexity or limited ability to reproduce critical tumour traits. Modular tissue engineering is a well-known method in tissue transplantation, where it has been used to develop various healthy tissue constructs. In this study, we set out to adapt this established approach to fabricate cancer microtissues and to assess their effectiveness as a tumour model that can capture essential features of cancer biology and drug-treatment response. Methods: Two triple-negative breast cancer (TNBC) cell lines, HCC1806 and MDA-MB-231, were cultured in microtissues and assessed for viability, cell death, generation of hypoxia and response to chemotherapy. To benchmark our model, we utilized flow cytometry to analyze the CD44+/CD24− cancer stem cell (CSC) phenotype across microtissues, 2D monolayers, and established 3D models, including spheroids, collagen domes, and laminin-rich domes. Results: The cells showed sustained cell viability with minimal cell death, along with natural development of tumour properties, such as hypoxia. Crucially, flow cytometry revealed a cell-line-dependent regulation of the CD44+/CD24− phenotype, underscoring the complex influence of the 3D microenvironment on stem cell regulation. Furthermore, by screening the model with standard anti-breast cancer chemotherapeutics, we observed drug resistance at concentrations comparable to those used in the clinic. Conclusions: Our model offers the unique ability to spontaneously reproduce fundamental features of tumours in vitro, capturing the cellular heterogeneity and reprogramming that drive clinical drug resistance.