Dissecting the interplay between biomechanics and ERK signaling during tumor invasion

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The ducts of the mammary gland form an extensive branched network that allows the transport of milk. Breast epithelial tissue can also give rise to breast cancer, the most common cancer type in women. Breast cancer-related deaths typically involve the formation of metastases from tumors that invade the surrounding tissue. To block invasive metastasis, it is crucial to understand the mechanisms that drive invasion. In recent years, it has become evident that intercellular heterogeneity and signaling dynamics can profoundly impact gene expression (Davies et al. 2020) and cell behavior, such as proliferation, invasion, and collective migration (Hallou et al. 2017; Bugaj et al. 2018). Dysregulation of the extracellular-signal regulated kinase (ERK) signaling pathway has been widely implicated in breast cancer (McCain 2013), but neoadjuvant chemotherapy, which alters ERK signaling, paradoxically is associated with an increase in metastasis in some patients and animal models (Perelmuter et al. 2019). Characterization of ERK signaling dynamics at the single-cell level in invasive 3D tissues has been technically challenging. Here, we hypothesize that tissue geometry and ERK signaling are integrated via mechanical feedback to regulate invasion. To address this hypothesis, we will combine bioengineering tools with recently developed fluorescent markers, signaling reporters, optogenetic tools, and 3D cell tracking to gain valuable insights into how cells regulate their dynamic signaling behavior according to their positioning within a tissue. In Aim 1, we will create 3D bioengineered tissues with predefined geometries to determine how geometrical constraints affect the ability of mammary epithelial tissues to invade. In Aim 2, we will perform time-lapse confocal microscopy analysis using fluorescent reporters to track ERK signaling dynamics in all cells within these bioengineered tissues and define how cell shape and positioning regulate ERK signaling during invasion. We will also test whether activation of the ERK pathway is sufficient to induce or inhibit invasions by using optogenetic tools that permit spatial and temporal control. In Aim 3, we will apply recently developed optogenetic tools to induce cell contractions specifically in certain cells within 3D tissues to define the effects of mechanical forces on ERK signaling and invasion. In conclusion, this work will reveal how tissue geometry, mechanical forces, and ERK signaling dynamics interact to drive tumor invasion, and thereby define the key steps of the early breast cancer metastatic cascade, which could be interrupted therapeutically. Furthermore, this work will provide new avenues to monitor and manipulate signaling within tumor cells, which is broadly applicable to other types of cancer.