Bioengineering approaches to map mechanotransduction in the living cell

 描述（由申请人提供）：越来越多的证据表明，实体瘤中的癌细胞具有高度异质性，但由于表面标记物方法已被证明不可靠，且其有效性存在争议，因此鉴定和分离这些极高致瘤性的肿瘤再增殖细胞具有挑战性。在上一个资助周期中，我们开发了一种新的机械方法，使用软纤维蛋白基质从黑色素瘤细胞中选择和生长癌细胞亚群，并证明它们在野生型同源甚至非同源小鼠中具有高度致瘤性。在这种竞争性的更新，我们建议阐明力如何调节基因表达，这是至关重要的，在阐明力诱导分化的高度致瘤性的肿瘤重建细胞的潜在机制。我们的初步研究结果表明，H3 K9甲基化水平的表观遗传变化是肿瘤再增殖细胞中力和Sox 2表达之间的联系。我们进一步表明，粘着斑激酶（FAK）活性在自我更新的肿瘤重建细胞中很高，但在软3D基质中侵入的肿瘤重建细胞中很低，细胞质中的FAK活性和细胞核中的H3 K9甲基化呈负相关，并且沉默FAK导致H3 K9甲基化和肿瘤重建细胞自我更新的抑制。重要的是，通过细胞表面上的整合素的生理量级的局部力被证明能够展开染色质片段并诱导基因表达，这表明活细胞中基因表达的直接力传播途径。基于这些初步结果，我们提出了3个具体目标来阐明活细胞中的机械转导机制：目标1：阐明表面力如何调节肿瘤再增殖细胞中H3 K9的甲基化和自我更新基因表达;目标2：阐明TRC生长中FAK活性和H3 K9甲基化如何调节;目标3：以确定表面力如何改变细胞核中的基因表达。我们的初步结果支持所有3个目标的可行性。 我们将采用几种生物工程方法对活细胞施加力，并结合联合收割机FRET技术，3D成像，BAC（细菌人工染色体）转基因技术来可视化，定量和映射活细胞的细胞质和细胞核中的机械转导过程。长期目标是了解3D矩阵中的力和机械微环境如何调节自我更新基因和分化基因的表达，这对于阐明高度致瘤性肿瘤重建细胞如何在软低力微环境中维持自我更新至关重要。

 DESCRIPTION (provided by applicant): Increasing evidence demonstrates that cancer cells from solid tumors are highly heterogeneous, but it has been challenging to identify and to isolate those extremely tumorigenic tumor-repopulating cells as surface marker methods have been proven to be unreliable and their effectiveness has been controversial. During the last funding cycle, we developed a novel mechanical method of selecting and growing a subpopulation of cancer cells from melanoma cells using a soft fibrin matrix and demonstrated that they are highly tumorigenic in wild-type syngeneic and even nonsyngeneic mice. In this competitive renewal, we propose to elucidate the underlying mechanisms of how forces regulate gene expression, which is crucial in elucidating force-induced differentiation of highly tumorigenic tumor-repopulating cells. Our preliminary results suggest that epigenetic changes of H3K9 methylation levels are a link between force and Sox2 expression in the tumor-repopulating cells. We further show that focal adhesion kinase (FAK) activity is high in self-renewing tumor-repopulating cells but low in invading tumor-repopulating cells in the soft 3D matrix, that FAK activity in the cytoplasm and H3K9 methylation in the nucleus is inversely associated, and that silencing FAK leads to H3K9 methylation and inhibition of self-renewal of tumor-repopulating cells. Importantly, a local force of physiologic magnitudes via integrins on the cell surface is shown to be able to unfold chromatin segments and induce gene expression, suggesting a direct force-propagating pathway for gene expression in a live cell. Built on these preliminary results, we propose 3 specific aims to elucidate mechanotransduction mechanisms in the living cell: Aim 1: To elucidate how a surface force regulates methylation of H3K9 and self-renewal gene expression in tumor- repopulating cells; Aim 2: To elucidate how FAK activity and H3K9 methylation are regulated in TRC growth; Aim 3: To determine how a surface force alters gene expression in the nucleus of a cell. Our preliminary results support the feasibility of all 3 aims. We will employ several bioengineering approaches to apply forces to the living cell, and combine FRET technology, 3D imaging, BAC (bacterial artificial chromosome) transgene techniques to visualize, to quantitate, and to map mechanotransduction processes in the cytoplasm and in the nucleus of the living cell. The long term goal is to understand how forces and mechanical microenvironment in the 3D matrix regulate expression of self-renewing genes and differentiation genes, fundamental in elucidating how highly tumorigenic tumor-repopulating cells sustain self-renewal in soft low-force microenvironment.