Development and characterisation of a three-dimensional breast cancer organotypic model of obesity associated adipose inflammation

Abstract

Obesity is an established risk factor for breast cancer (BC) in postmenopausal women. However, the underlying biological mechanisms of how obesity mediates BC progression remain unclear, which may in part be due to a lack of suitable models. Therefore, the main aim of this study was to develop a 3D in vitro model that recapitulates the inflammatory environment associated with obesity in BC patients. To model the complexity of the breast tumour microenvironment, a 3D organotypic quadraculture system was first developed containing BC cells, myoepithelial cells, macrophages, and fibroblasts in a collagen type I matrix. Tumour cell lines of different disease subtypes formed distinct morphologies in 3D culture, recapitulating aspects of the tissue structure observed in patients. To develop a novel model of obesity-associated BC, hypertrophic adipocyte spheroids were generated by treating mature adipocytes with palmitic acid for 12 days. These were then embedded within the collagen matrix alongside the other cell populations as a penta-culture system. Adipocyte hypertrophy was characterised by downregulation of PPARγ and FABP4 mRNA levels and increased lipolytic activity. Penta-cultures containing hypertrophic adipocytes showed increased tumour cell proliferation and the formation of large tumour islands. Infiltration of BC cells and macrophages was observed within and surrounding hypertrophic adipocyte spheroids, forming crown-like structures. Immunohistochemical staining of organotypic cultures revealed that macrophages surrounding adipocytes exhibited both CD32b and CD16 expression, suggestive of a dysregulated phenotype, replicating the adipose-inflamed border observed in BC patients with obesity. ‘Obese’ organotypic penta-cultures containing hypertrophic adipocytes had increased mRNA expression levels of key lipid receptors and binding proteins, including CD36 and FABP4. Uptake of fatty acids was observed in tumour cells in penta-cultures with mature adipocytes, and this was further enhanced in penta-cultures containing hypertrophic adipocytes. Cell metabolic phenotype analysis showed that hypertrophic adipocyte penta-cultures exhibited increased mitochondrial respiration, reduced reliance on glycolysis and reduced metabolic plasticity. Thus, ‘obese’ pentacultures displayed increased proliferation and altered metabolism. This organotypic system was then explored for its potential utility as a drug testing platform. Organotypics were treated with three commonly used therapeutics for luminal disease; Paclitaxel, Doxorubicin, Tamoxifen, or Metformin for 48 hours and assessed for cell viability. Penta-cultures containing hypertrophic adipocytes exhibited increased sensitivity to Metformin and resistance to Paclitaxel, compared to those containing mature adipocytes. Furthermore, Metformin could be used in combination with Paclitaxel to alleviate chemoresistance in ‘obese’ penta-cultures. This study proposes a 3D organotypic model which recapitulates several aspects of the adipose environment associated with obesity in BC patients. This model could provide a useful tool to interrogate the metabolic crosstalk between adipocytes and breast tumour cells and to investigate mechanisms underpinning obesity-related chemoresistance.

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Identifiers

ORCID for Rhianna Rachael Romany Blyth: orcid.org/0000-0001-9640-404X

ORCID for Stephen Beers: orcid.org/0000-0002-3765-3342

ORCID for Charlie Birts: orcid.org/0000-0002-0368-8766

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