Engineering a Vascularized 3D Hybrid System to Model Tumor-Stroma Interactions in Breast Cancer

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.

3D Cancer Models: Depicting Cellular Crosstalk within the Tumour Microenvironment

“…Two antiangiogenic drugs were tested, with no statistically significant differences in EC migration. We have recently reported a strategy for long-term culture of triple heterotypic spheroids of EC (HUVEC), human dermal fibroblasts and a breast cancer cell line (HCC1954), under agitation; in accordance with other authors, we observed that EC were localized in close vicinity to fibroblasts, in the core of the spheroids [310]. In our system, EC maintenance for up a month of culture was dependent on the tumour cell line, the presence of fibroblasts, which secreted collagens I and IV, and agitation but occurred even without formation of hypoxic cores.…”

Establishment of a Pre-vascularized 3D Lung Cancer Model in Fibrin Gel—Influence of Hypoxia and Cancer-Specific Therapeutics

“…Teixeira et al have implemented porous alginate scaffolds produced by freeze-drying for their tri-culture hybrid model with breast cancer cells. However, the in vitro vascularization found in this model was not as extended as in our model ( Teixeira et al, 2021 ). The future model has to balance sufficient elasticity for in vitro vascularization and adequate porosity for cell migration and overcome the limitation of the two-phase hydrogel.…”

“…Furthermore, by using patient cells, 3D models can provide insight into effective, personalised therapy options ( Fong et al, 2017 ). However, it is essential to establish in vitro models that combine vascularized stromal and parenchymal compartments to mimic the natural microenvironment with cell-cell contacts and secretion of angiogenesis related cytokines ( Teixeira et al, 2021 ). Amann et al developed a multicellular 3D cancer model made by hanging drop technology, in which they could observe the tumor mircroenvironment and aggregation of the different cell types, but without in vitro vascularization of the model ( Amann et al, 2017 ).…”

Mesenchymal-endothelial nexus in breast cancer spheroids induces vasculogenesis and local invasion in a CAM model

“…To investigate heterotypic interactions between cells within the TME multicellular systems comprising of cancer cells, fibroblasts, and/or endothelial cells (EC) have been studied in hanging drop spheroids, microfluidic chip and polymer-gel scaffolds 23 – 27 . Many of these studies have employed transformed fibroblasts and/or human umbilical vein EC both of which are not physiologically consistent with tumor microenvironment.…”