Bioengineering approaches to map mechanotransduction in the living cell

 描述(申请人提供)：越来越多的证据表明，实体肿瘤中的癌细胞是高度异质性的，但识别和分离这些高度致瘤性的肿瘤再繁殖细胞一直是一项挑战，因为表面标记方法已被证明是不可靠的，其有效性一直存在争议。在上一个资金周期中，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培养癌细胞亚群，并证明它们在野生型同基因小鼠甚至非同基因小鼠中具有高度的致瘤性。在这一竞争性更新中，我们建议阐明力量如何调节基因表达的潜在机制，这对于阐明力量诱导高致瘤性肿瘤再生细胞的分化至关重要。我们的初步结果表明，H3K9甲基化水平的表观遗传学变化是肿瘤再生细胞中FORCE和SOX2表达之间的联系。我们进一步表明，粘着斑激酶(FAK)在自我更新的肿瘤再生细胞中活性高，但在软3D基质中侵袭性肿瘤再生细胞中活性低，细胞质中的FAK活性与细胞核中的H3K9甲基化呈负相关，沉默FAK导致H3K9甲基化和抑制肿瘤再生细胞的自我更新。重要的是，通过细胞表面整合素产生的生理强度的局部作用力被证明能够展开染色质片段并诱导基因表达，这表明在活细胞中基因表达是一种直接的力传播途径。在这些初步结果的基础上，我们提出了3个特定的目标来阐明活细胞中的机械转导机制：目的1：阐明表面力如何调节肿瘤再填充细胞中H3K9的甲基化和自我更新基因的表达；目的2：阐明FAK活性和H3K9甲基化如何在TRC生长中调节；目标3：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三个目标的可行性。 我们将使用几种生物工程方法对活细胞施加力，并结合FRET技术、3D成像、BAC(细菌人工染色体)转基因技术来可视化、量化和绘制活细胞胞浆和细胞核中的机械转导过程。长期目标是了解3D基质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，这是阐明高致瘤性肿瘤再繁殖细胞如何在柔软的低力微环境中维持自我更新的基础。

 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.