Dissecting the interplay between biomechanics and ERK signaling during tumor invasion

乳腺的导管形成一个广泛的分支网络，允许牛奶的运输。乳腺上皮组织也会导致乳腺癌，这是女性中最常见的癌症类型。乳腺癌相关的死亡通常包括肿瘤侵入周围组织的转移形成。为了阻止侵袭性转移，了解驱动侵袭的机制至关重要。近年来，越来越明显的是，细胞间异质性和信号动力学可以深刻影响基因表达（Davies et al. 2020）和细胞行为，如增殖、侵袭和集体迁移（Hallou et al. 2017; Bugaj et al. 2018）。细胞外信号调节激酶（ERK）信号通路的失调已广泛涉及乳腺癌（McCain 2013），但在一些患者和动物模型中，改变ERK信号的新辅助化疗与转移增加有关（Perelmuter et al. 2019）。在侵入性三维组织的单细胞水平上表征ERK信号动力学在技术上具有挑战性。在这里，我们假设组织几何和ERK信号通过机械反馈来调节入侵。为了解决这一假设，我们将生物工程工具与最近开发的荧光标记，信号报告，光遗传学工具和3D细胞跟踪相结合，以获得关于细胞如何根据其在组织中的定位调节其动态信号行为的宝贵见解。在Aim 1中，我们将创建具有预定义几何形状的3D生物工程组织，以确定几何约束如何影响乳腺上皮组织入侵的能力。在目标2中，我们将使用荧光报告器进行延时共聚焦显微镜分析，以跟踪这些生物工程组织中所有细胞的ERK信号动力学，并定义细胞形状和定位在入侵过程中如何调节ERK信号。我们还将通过使用允许空间和时间控制的光遗传学工具来测试ERK通路的激活是否足以诱导或抑制入侵。在Aim 3中，我们将应用最近开发的光遗传学工具来诱导细胞收缩，特别是在3D组织内的某些细胞中，以确定机械力对ERK信号传导和侵袭的影响。总之，这项工作将揭示组织几何、机械力和ERK信号动力学如何相互作用以驱动肿瘤侵袭，从而确定早期乳腺癌转移级联的关键步骤，这可以通过治疗来中断。此外，这项工作将为监测和操纵肿瘤细胞内的信号提供新的途径，这将广泛适用于其他类型的癌症。

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.