Understanding ER+ Breast Cancer Dormancy Using Bioinspired Synthetic Matrices for Long‐Term 3D Culture and Insights into Late Recurrence

Abstract

Late recurrences of breast cancer are hypothesized to originate from disseminated tumor cells that re‐activate after a long period of dormancy, ≥5 years for estrogen‐receptor positive (ER+) tumors. An outstanding question remains as to what the key microenvironment interactions are that regulate this complex process, and well‐defined human model systems are needed for probing this. Here, a robust, bioinspired 3D ER+ dormancy culture model is established and utilized to probe the effects of matrix properties for common sites of late recurrence on breast cancer cell dormancy. Formation of dormant micrometastases over several weeks is examined for ER+ cells (T47D, BT474), where the timing of entry into dormancy versus persistent growth depends on matrix composition and cell type. In contrast, triple negative cells (MDA‐MB‐231), associated with early recurrence, are not observed to undergo long‐term dormancy. Bioinformatic analyses quantitatively support an increased “dormancy score” gene signature for ER+ cells (T47D) and reveal differential expression of genes associated with different biological processes based on matrix composition. Further, these analyses support a link between dormancy and autophagy, a potential survival mechanism. This robust model system will allow systematic investigations of other cell‐microenvironment interactions in dormancy and evaluation of therapeutics for preventing late recurrence.

Platelet Activation In Situ in Breasts at High Risk of Cancer: Relationship with Mammographic Density and Estradiol

“…The levels in breast tissue of premenopausal women were 205 pmol/l (170-234). Similarly, no significant difference was detected of local estradiol levels in abdominal subcutaneous fat, median (25-75 percentiles), between postmenopausal women with dense or nondense breasts; 38 pmol/l (31-49) vs. 36 pmol/l (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46) respectively. The levels in abdominal subcutaneous fat of premenopausal women were 202 pmol/l (155-235).…”

Induction of dormancy by confinement: An agarose‐silica biomaterial for isolating and analyzing dormant cancer cells

“…A similar observation has previously been made in a PEG-based confinement model. 45 Furthermore, encapsulation in agarose microspheres forces cells to maintain a spherical geometry, minimizing surface area to volume ratio and thus minimizing integrin density, while imposing a uniform compressive force on the cell. Therefore, it is plausible that with no integrin binding, a robust actin cytoskeleton network would not form, and cell stiffness would decrease.…”

“…It is known that softer cell phenotypes are more dormancy-capable. 65 And lastly, while cells can manipulate their organic ECM microenvironment enzymatically (through matrix degradation) and mechanically (by applying forces), 44,45 the inability of the cells to enzymatically degrade or apply traction forces on the synthetic agarose-silica biomaterial surrounding them likely contributed to dormancy-inducing stresses cells experienced. Please note that the rheological characterization of the gel provided here is only a limited representation of the complex matrix properties that define cell-gel interactions, which warrants further research.…”

“…22 In vitro models use a wide variety of methods to induce/inhibit dormancy 21,29 by exposing cells to soluble/insoluble cues 16,[30][31][32] (e.g., thrombospondin-1 exposure, 22 inhibitors for integrin β1, 31 and type-I TGF-β, changes in extracellular matrix [ECM] protein composition 33 ), or to stresses (e.g., serum starvation, 34 hypoxia, 35 exposure to chemotherapeutic agents, 36,37 and physical immobilization to inhibit proliferation 38,39 ). In vitro models mostly rely on 3D culture/encapsulation methods 40 using materials of biological origin (e.g., collagen, 30,41 Matrigel, 42 tissue extracts 31,43 ), synthetic materials (e.g., polyethylene glycol [PEG], 44,45 silica-PEG 39,46 ), and organic-synthetic materials (e.g., PEG-collagen, 47 and PEG-collagen/integrin 45 ). For recent comprehensive reviews of in vitro models, please see papers by Pradhan et al, 29 and Montagner et al 21 Shortcomings of 3D matrices that use materials of biological origin include unknown composition, batch-to-batch variability, limited control over chemical and mechanical properties, and inability to recover the dormant cells for downstream analysis.…”

“…47 Ovadia et al, in a detailed study, report a very high dormancy score in dormancy-capable ER+ T47D cell line, with reduction of proliferation, and metabolic activity in matrices of higher stiffness. 45 In this report, we present an agarose-silica immobilization biomaterial and method to induce single cell dormancy. Cells are first individually coated in a thin layer (~8 μm) of agarose using a flow-focusing microfluidic device.…”