Effects of Microenvironmental Stiffness on Epigenetic Regulation

肿瘤僵硬已被认为是肿瘤进展和转移的驱动力。矩阵刚度 已被证明可以改变乳腺上皮细胞的表型，从软性条件下的生长停滞到 在僵硬的条件下恶性和侵袭性的。然而，目前尚不清楚入侵细胞是否保留了僵硬的“记忆” 从原发肿瘤扩散后的环境，如果是，通过什么机制。这项建议旨在 确定乳腺上皮细胞僵硬微环境机械记忆的程度和基础。 使用一种新的3D模型，其中基质刚性可以独立于配体密度、极化、 生长受阻的乳腺腺泡可在软凝胶中产生，而硬凝胶可导致恶性的、侵袭性的 表型。在柔软或僵硬的条件下培养后，凝胶硬度将发生变化，以确定 最初建立的表型的可逆性。这些实验将证明机械 记忆存在于乳腺上皮细胞或一般的癌症环境中。下一步染色质可及性和 转录因子的占有率将使用基于转座的分析方法(atac-seq)在全基因组范围内进行评估。 适用于3D培养所需的低细胞数。表观遗传修饰物将根据候选者进行筛选 从表观基因组数据确定僵硬诱导染色质的分子机制 改建。这项提案的成功完成将第一次揭示出机械化的程度 癌症背景下的记忆，以及3D培养中细胞的表观基因组图景。

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.