Three-Dimensional Breast Cancer Models Mimic Hallmarks of Size-Induced Tumor Progression

Abstract

Tumor size is strongly correlated with breast cancer metastasis and patient survival. Increased tumor size contributes to hypoxic and metabolic gradients in the solid tumor and to an aggressive tumor phenotype. Thus, it is important to develop three-dimensional (3D) breast tumor models that recapitulate size-induced microenvironmental changes and consequently, natural tumor progression in real time without the use of artificial culture conditions or gene manipulations. Here, we developed size-controlled multicellular aggregates (“microtumors”) of subtype-specific breast cancer cells by using non-adhesive polyethylene glycol dimethacrylate hydrogel microwells of defined sizes (150–600 μm). These 3D microtumor models faithfully represent size-induced microenvironmental changes such as hypoxic gradients, cellular heterogeneity and spatial distribution of necrotic/proliferating cells. These microtumors acquire hallmarks of tumor progression in the same cell lines within 6 days. Of note, large microtumors of hormone receptor positive cells exhibited an aggressive phenotype characterized by collective cell migration and upregulation of mesenchymal markers at mRNA and protein level, which was not observed in small microtumors. Interestingly, triple negative breast cancer (TNBC) cell lines did not show size-dependent upregulation of mesenchymal markers. In conclusion, size-controlled microtumor models successfully recapitulated clinically observed positive association between tumor size and aggressive phenotype in hormone receptor positive breast cancer while maintaining clinically proven poor correlation of tumor size with aggressive phenotype in TNBC. Such clinically relevant 3D models generated under controlled experimental conditions can serve as precise preclinical models to study mechanisms involved in breast tumor progression as well as antitumor drug effects as a function of tumor progression.

The production of 3D tumor spheroids for cancer drug discovery

“…These non-adhesive microwell arrays enabled generation of uniform, defined size microtumors within 1–2 days using various HNC, breast and cervical cancer cell lines (Fig. 3B, C) [38, 39]. We have recently demonstrated the application of this platform technology to probe activation as well as inhibition of EGFR signaling in 3D HNC microtumors in response to EGF and cetuximab treatments, respectively [38].…”

“…Another advantage of PEGDMA hydrogel microarrays is their ability to precisely control the microtumor size and thus, the physico-chemical factors in the tumor microenvironment [39]. When noninvasive breast cancer cells (MCF7, T47D) were cultured in non-adhesive PEG hydrogel microwells, precise control over microtumor size enabled controlled changes in tumor microenvironment in a reproducible manner recreating spatial distribution of proliferating and necrotic cells, the development of a hypoxic central core, up-regulation of hypoxia-inducible factor-1 alpha (Hif-1α) and pro-angiogenic vascular endothelial growth factor (vegf) and the overexpression of Glut-1 (metabolic stress marker) in large (600 μm) microtumors of breast cancer cell lines (Fig.…”

“…When noninvasive breast cancer cells (MCF7, T47D) were cultured in non-adhesive PEG hydrogel microwells, precise control over microtumor size enabled controlled changes in tumor microenvironment in a reproducible manner recreating spatial distribution of proliferating and necrotic cells, the development of a hypoxic central core, up-regulation of hypoxia-inducible factor-1 alpha (Hif-1α) and pro-angiogenic vascular endothelial growth factor (vegf) and the overexpression of Glut-1 (metabolic stress marker) in large (600 μm) microtumors of breast cancer cell lines (Fig. 3D,E) [39]. Interestingly, large microtumors of these non-invasive breast cancer cell lines consistently acquired mesenchymal phenotype, migratory phenotype (Fig.…”

“…Several methods to generate anchorage-independent 3D tumor spheroids have also been developed; spontaneous aggregation, liquid overlay on agarose, hanging drop cultures, spinner flask cultures, rotary cell culture systems, ultra-low attachment (ULA) plates, and encapsulation in biologically inert hydrogels lacking attachment cues (Table 1) [14, 16–18, 21, 24, 25, 32, 33, 38, 39]. However, not all tumor cell lines form 3D spheroids under these conditions [14, 33], and many of these methods generate irregular 3D cellular aggregates that have a wide range of morphologies and sizes [16, 17].…”

Isolation of spheroid-forming single cells from gastric cancer cell lines: enrichment of cancer stem-like cells

“…The microwell-based spheroid culture method provides the following advantages over the hanging drop method and the other developed spheroid culture platforms: an extended culture period of 1030 days due to easy medium changes; ready for co-culture without direct cell-to-cell contact [30]; easy generation of size-controlled cell spheroids; easy growth medium changes in the device due to the shear-free and stress-free structure [31]; ready application to various cell types; and unlimited throughput, given that 361 cell spheroids can be obtained from a single device. Moreover, the microwell mimics the in vivo environment and contains CSC-secreted factors [32]. One recent study reported that round spheroids showed a signature of high mRNA expression for secreted molecules, which activate cell cycle-related signaling pathways and their downstream transcriptional regulators [33].…”