Abstract
The stromal microenvironment of breast tumors, namely the vasculature, has a key role in tumor development and metastatic spread. Tumor angiogenesis is a coordinated process, requiring the cooperation of cancer cells, stromal cells, such as fibroblasts and endothelial cells, secreted factors and the extracellular matrix (ECM). In vitro models capable of capturing such complex environment are still scarce, but are pivotal to improve success rates in drug development and screening. To address this challenge, we developed a hybrid alginate-based 3D system, combining hydrogel-embedded mammary epithelial cells (parenchymal compartment) with a porous scaffold co-seeded with fibroblasts and endothelial cells (vascularized stromal compartment). For the stromal compartment, we used porous alginate scaffolds produced by freeze-drying with particle leaching, a simple, low-cost and non-toxic approach that provided storable ready-to-use scaffolds fitting the wells of standard 96-well plates. Co-seeded endothelial cells and fibroblasts were able to adhere to the surface, spread and organize into tubular-like structures. For the parenchymal compartment, a designed alginate gel precursor solution load with mammary epithelial cells was added to the pores of pre-vascularized scaffolds, forming a hydrogel in situ by ionic crosslinking. The 3D hybrid system supports epithelial morphogenesis in organoids/tumoroids and endothelial tubulogenesis, allowing heterotypic cell-cell and cell-ECM interactions, while presenting excellent experimental tractability for whole-mount confocal microscopy, histology and mild cell recovery for down-stream analysis. It thus provides a unique 3D in vitro platform to dissect epithelial-stromal interactions and tumor angiogenesis, which may assist in the development of selective and more effective anticancer therapies.
Highlights
Breast cancer is the leading cause of cancer death in women worldwide, accounting for approximately 7% of the nearly 10 millions of cancer deaths in 2018, according to the International Agency for Research on Cancer
We have previously shown that bioengineered alginate hydrogels support mammary epithelial cell morphogenesis into organoids that recapitulate histological and functional features of the mammary gland, the site from which breast cancers emerge (Bidarra et al, 2016; Barros da Silva et al, 2020)
We developed a hybrid alginatebased 3D system, combining a porous scaffold co-seeded with fibroblasts and endothelial cells (EC) with gel-embedded mammary epithelial cells (Figure 1)
Summary
Breast cancer is the leading cause of cancer death in women worldwide, accounting for approximately 7% of the nearly 10 millions of cancer deaths in 2018, according to the International Agency for Research on Cancer. The design of new models that better recapitulate in vivo tumor biology and microenvironmental features is pivotal to improve success rates in drug development and screening. It is well-accepted that three-dimensional (3D) in vitro systems better emulate the in vivo environment than the traditional twodimensional (2D) monolayer cultures (Pape et al, 2019). Due to their superior biological relevance and higher predictive value for the therapeutic outcome, 3D cell cultures are becoming more prominent in drug discovery. 3D cell culture models using human cells can circumvent drawbacks of rodent models that, aside from the high cost and ethical considerations, are often non-representative of humanspecific conditions
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