Effects of Microenvironmental Stiffness on Epigenetic Regulation

肿瘤硬度已被认为是肿瘤进展和转移的驱动力。矩阵刚度 已经显示出将乳腺上皮细胞的表型从在柔软条件下生长停滞改变为 在僵硬的条件下是恶性的和侵入性的。然而，目前尚不清楚入侵细胞是否保留了对僵硬的“记忆”。 从原发肿瘤扩散后的环境，如果是这样，通过什么机制。这项建议旨在 确定乳腺上皮细胞中刚性微环境的机械记忆的程度和基础。 使用一种新的3D模型，其中基质刚度可以独立于配体密度进行调节，极化， 生长停滞的乳腺腺泡可以在软凝胶中产生，而硬凝胶引起恶性的、侵入性的乳腺癌。 表型在软或硬条件下培养后，将改变凝胶硬度以确定凝胶的硬度。 最初建立的表型的可逆性。这些实验将证明， 记忆通常存在于乳腺上皮细胞或癌症环境中。下一篇染色质可及性和 将使用基于转座的测定（ATAC-seq）在全基因组范围内评估转录因子占用率 适用于3D培养所需的低细胞数。将根据候选人筛选表观遗传修饰剂 从表观基因组数据中识别，以确定驱动刚度诱导的染色质的分子机制 重塑成功完成这项提案将首次揭示机械化的程度， 癌症背景下的记忆，以及3D培养中细胞的表观基因组景观。

Tumor stiffness has been implicated as a driving force in tumor progression and metastasis. Matrix stiffness has been shown to alter the phenotype of breast epithelial cells from growth-arrested in soft conditions to malignant and invasive in stiff conditions. However, it is unknown whether invading cells retain `memory' of stiff environments after dissemination from the primary tumor, and if so, by what mechanism. This proposal aims to determine the extent and basis of mechanical memory of stiff microenvironments in mammary epithelial cells. Using a novel 3D model in which matrix stiffness can be tuned independently of ligand density, polarized, growth arrested mammary acini can be generated in soft gels while stiff gels give rise to malignant, invasive phenotypes. After culture in either soft or stiff conditions, the gel stiffness will be altered to determine the reversibility of the phenotypes initially established. These experiments will demonstrate whether mechanical memory exists for breast epithelial cells or in cancer contexts in general. Next chromatin accessibility and transcription factor occupancy will be assessed genome-wide using a transposition-based assay (ATAC-seq) suited for low cells numbers required for 3D culture. Epigenetic modifiers will be screened based on candidates identified from epigenomic data to identify the molecular mechanism driving stiffness-induced chromatin remodeling. Successful completion of this proposal will reveal, for the first time, the extent of mechanical memory in a cancer context, and the epigenomic landscape of cells in 3D culture.