Analysis of the Signaling and Mechanical Cues Promoting Invasion in Melanoma

描述(申请人提供)：转移性黑色素瘤是皮肤癌中最致命的形式，也是最具侵袭性的癌症类型之一，能够迅速扩散到原发部位。这一建议旨在分析黑色素瘤细胞遗传和信号状态的变化以及细胞微环境对细胞侵袭程度和假定转移潜力的综合影响。我们分析的主要假设是，黑色素瘤细胞的侵袭性是由不同因素的组合决定的，细胞微环境的状态是主要因素，包括细胞外基质的结构和可溶性化学因素的存在。细胞微环境的改变，如细胞外基质纤维的排列，可以使原本不转移的细胞变得更具侵袭性，而表面上转移的细胞的侵袭性可以通过环境变化和对信号级联调节的有针对性的干预来缓和。我们还假设，细胞不仅可以通过分泌或降解细胞外基质成分，还可以通过主动施加力，从而使细胞外基质纤维沿着最近的锚定点(包括血管系统)方向排列，从而主动与微环境相互作用。这反过来可以产生更多的各向异性力量，允许细胞更多地变形ECM，最终利用重新排列的ECM结构向血管迁移。我们将与ICBP@MIT密切合作测试这些假设，首先利用使用shRNA文库干扰遗传靶标的新方法(目标1)以及广泛的环境扰动，然后使用icBP@MIT提供的最先进的统计分析工具来降低结果数据集的复杂性，将多种环境和遗传扰动与攻击性细胞行为(目标2)联系起来，我们将在此过程中使用几种新颖的分析方法，增加数据采集和处理的可管理性，以及增强实验的仿生性质。最后，我们将通过一系列测试，从细胞培养到组织构建，再到模型组织水平(目标3)，测试目标2中开发的模型所产生的预测。我们预计，拟议的研究将揭示环境和遗传因素之间在控制黑色素瘤转移事件方面的新的、以前没有预料到的相互作用，并可能提出与这种致命疾病作斗争的新方法。这一结果也可能与其他转移性癌症相关。 公共卫生相关性：早期检测因缺乏基于生物标记物的检测测试而进一步受阻。造成这种情况的关键原因之一是缺乏了解 黑色素瘤转移开关的潜在机制，以及允许转移细胞成功和快速地通过与转移扩散相关的所有障碍的机制，包括有效地迁移到血管和进入血管。这 该项目旨在填补这一关键空白。

DESCRIPTION (provided by applicant): Metastatic melanoma is the deadliest form of skin cancer and one of the most aggressive cancer types, capable of rapid spreading out of the primary site. This proposal is aimed at analyzing the combined effect of changes in the genetic and signaling states of melanoma cells and the cell micro-environment on the degree of cell aggressiveness and putative metastatic potential. The main hypothesis underlying our analysis is that aggressiveness of melanoma cells is determined by a combination of different factors, with a major contribution from the state of cell microenvironment, including the structure of the ECM and the presence of soluble chemical factors. Alteration in the cell micro-environment, such as alignment of ECM fibers, can permit an otherwise non-metastatic cell to become more aggressive, whereas the aggressiveness of ostensibly metastatic cell can be moderated by environmental changes and targeted intervention in regulation of signaling cascades. We also hypothesize that cells can actively interact with the micro-environment not only by secreting or degrading ECM components, but by actively exercising forces on and thus aligning ECM fibers in the direction of a nearest anchor point, including the vasculature. This can in turn generate more anisotropic forces allowing cells to deform ECM even more and ultimately take advantage of the re-aligned ECM structure to migrate towards blood vessels. We will test these hypotheses in close collaboration with ICBP@MIT, first taking advantage of the new methodologies using shRNA libraries to perturb genetic targets (Aim 1) along with a broad range of environmental perturbations and then use the state of the art statistical analysis tools available at ICBP@MIT to reduce complexity of resulting datasets, linking multiple environmental and genetic perturbation to aggressive cell behavior (Aim 2), We will use several novel assays in the process, increasing manageability of data acquisition and processing, as well as enhancing the biomimetic nature of experimentation. Finally, we will test the predictions arising from the models developed in Aim 2 through a series of tests, ranging from cell culture to tissue construct to model tissue levels (Aim 3). We anticipate that the proposed research will reveal new, previously unanticipated interplay between environmental and genetic factors in control of metastatic events in melanoma and potentially suggest new ways of battling this deadly disease. The results might also have relevance to other metastatic cancers. PUBLIC HEALTH RELEVANCE: Early detection is further hampered by the lack of biomarker-based detection tests. One of the critical reasons for this situation is the lack of understanding of the mechanisms underlying the metastatic switch in melanoma and the mechanisms allowing metastatic cells to successfully and quickly navigate all the hurdles associated with the metastatic spread, including effective migration to and intravasation into the blood vessels. This project is aimed at filling this critical gap.