Heterotypic 3D breast cancer model based on silk protein fibroin

Introduction

Cancer associated fibroblasts, cancer cells, endothelial and immune system cells are the main players in the tumor progression. The non-reductionist view of tumor microenvironment makes aware researchers and oncologists that flat two-dimensional cell cultures do not fulfil all the requirements for investigating cancer. The 2D cell culture model do not recapitulate the complex interaction between the tumor cells and the surrounding microenvironment. Animal models face ethical challenges and fail to mimic the human cancer microenvironment. Therefore, there is an urgent need to develop new tools able to copycat the tumor microenvironment and allow more realistic drug screening predicting drug efficacy and toxicity. Biomaterials support the growth of cancer cells together with the other cellular and acellular players, which offer different options to recapitulate the tumor microenvironment. [1],[2],[3]

Aim of the work

In this work, we develop a 3D in vitro cancer model based on natural silk protein fibroin biomaterial. Fibroin shows good biocompatibility, suitable mechanical properties and tunable biodegradability. Freeze-dried scaffolds are fabricated in order to recapitulate the tumor microenvironment. Breast cancer cells (MCF-7 and MDAMB-231) and normal mammary fibroblast are seeded onto scaffolds. The proliferation of cell is evaluated in the heterotypic model by mean of Alamar blue assay. The cellular morphology is observed by mean of confocal and scansion electron microscopy. Gene expression modulation is investigated to check the role of the extracellular matrix markers in cancer progression and matrix remodeling. The platform is then challenged with doxorubicin, a conventional cancer chemotherapeutic.

Results

Silk fibroin scaffolds support the proliferation of the cancer cells and fibroblasts. The growth of cells is enhanced when cancer cells and fibroblasts are seeded together (p<0.001). Histological staining shows 3D cell organization. MMP-1, MMP-2, MMP-3, Col-1 and fibronectin expressions are upregulated in co-culture (p<0.001). Doxorubicin treatment reduces the cell proliferation. However, higher doxorubicin concentration is needed to kill the cancer cells when they are in co-culture with fibroblasts.

Conclusion

The platform represents a promising model for understanding the crosstalk between cancer cells, fibroblasts and extracellular matrix. Moreover, the developed 3D in vitro tumor model is also useful as drug screening platform.

Keywords: 3D tumor model, silk fibroin, drug testing, breast cancer

ACKNOWLEDGEMENTS: This work is financially supported by the European Union Framework Programme for Research and Innovation Horizon 2020 under ERA-Chair grant agreement nº 668983 — FoReCaST and the FCT project BREAST-IT (PTDC/BTM-ORG/28168/2017).

REFERENCES

[1] Caballero, D. et al. Biomat. 2017;149:98–115.

[2] Rodrigues, T. et al. Pharmacol Ther. 2018;184:201–211.

[3] Kundu, B. et al. ACS Appl Mater Interfaces 2019;11:14548-14559.