Morphological Phenotyping of Organotropic Brain- and Bone-Seeking Triple Negative Metastatic Breast Tumor Cells.

Triple negative breast cancer (TNBC) follows a non-random pattern of metastasis to the bone and brain tissue. Prior work has found that brain-seeking breast tumor cells display altered proteomic profiles, leading to alterations in pathways related to cell signaling, cell cycle, metabolism, and extracellular matrix remodeling. Given the unique microenvironmental characteristics of brain and bone tissue, we hypothesized that brain- or bone-seeking TNBC cells may have altered morphologic or migratory phenotypes from each other, or from the parental TNBC cells, as a function of the biochemical or mechanical microenvironment. In this study, we utilized TNBC cells (MDA-MB-231) that were conditioned to metastasize solely to brain (MDA-BR) or bone (MDA-BO) tissue. We quantified characteristics such as cell morphology, migration, and stiffness in response to cues that partially mimic their final metastatic niche. We have shown that MDA-BO cells have a distinct protrusive morphology not found in MDA-P or MDA-BR. Further, MDA-BO cells migrate over a larger area when on a collagen I (abundant in bone tissue) substrate when compared to fibronectin (abundant in brain tissue). However, migration in highly confined environments was similar across the cell types. Modest differences were found in the stiffness of MDA-BR and MDA-BO cells plated on collagen I vs. fibronectin-coated surfaces. Lastly, MDA-BO cells were found to have larger focal adhesion area and density in comparison with the other two cell types. These results initiate a quantitative profile of mechanobiological phenotypes in TNBC, with future impacts aiming to help predict metastatic propensities to organ-specific sites in a clinical setting.

Modeling the Roles of Astrocytes in the Metastatic Tumor Cell Microenvironment.

Astrocytes can play a seemingly contradictory dual role during tumor metastasis to the brain; they can be both protective of the brain and supportive of tumor cells during brain metastasis. The role of astrocytes is further complicated by the fact that metastatic tumor cells arriving in the brain via the circulatory system are separated from the brain perivascular space (in which the astrocytes reside) by the blood-brain barrier (BBB). It is not yet clear how tumor cells cross this highly selective barrier. The BBB can be modeled in vitro using different systems, cell types, and extracellular matrix components to study the interactions of metastatic tumor cells and astrocytes, with the specific aspects of the tumor microenvironment depending on the research questions. Some models focus on the interaction of two cell types while others are more complex and involve the neighboring neural cells and microenvironment. Regardless, these models have pointed to astrocytes as key regulators of tumor cell metastasis into the brain because they can influence tumor cells both directly and indirectly through other cells and/or the extracellular matrix (ECM). It is critical that in vitro models are carefully designed to consider how, and at which point in the metastatic cascade, astrocytes and tumor cells interact, both physically and biochemically. This chapter provides a critical evaluation of the different assays used to study metastatic tumor cell-astrocyte interactions and discusses their physiological implications.

Tumor Cell Mechanosensing During Incorporation into the Brain Microvascular Endothelium.

INTRODUCTION: Tumor metastasis to the brain occurs in approximately 20% of all cancer cases and often occurs due to tumor cells crossing the blood-brain barrier (BBB). The brain microenvironment is comprised of a soft hyaluronic acid (HA)-rich extracellular matrix with an elastic modulus of 0.1-1 kPa, whose crosslinking is often altered in disease states. METHODS: To explore the effects of HA crosslinking on breast tumor cell migration, we developed a biomimetic model of the human brain endothelium, consisting of brain microvascular endothelial cell (HBMEC) monolayers on HA and gelatin (HA/gelatin) films with different degrees of crosslinking, as established by varying the concentration of the crosslinker Extralink. RESULTS AND DISCUSSION: Metastatic breast tumor cell migration speed, diffusion coefficient, spreading area, and aspect ratio increased with decreasing HA crosslinking, a mechanosensing trend that correlated with tumor cell actin organization but not CD44 expression. Meanwhile, breast tumor cell incorporation into endothelial monolayers was independent of HA crosslinking density, suggesting that alterations in HA crosslinking density affect tumor cells only after they exit the vasculature. Tumor cells appeared to exploit both the paracellular and transcellular routes of trans-endothelial migration. Quantitative phenotyping of HBMEC junctions via a novel Python software revealed a VEGF-dependent decrease in punctate VE-cadherin junctions and an increase in continuous and perpendicular junctions when HBMECs were treated with tumor cell-secreted factors. CONCLUSIONS: Overall, our quantitative results suggest that a combination of biochemical and physical factors promote tumor cell migration through the BBB.