A Tissue Engineering Approach to Analyzing Host-Microbe Interactions in Cancer

关于细菌组织的时空动力学， 传播，以及各种细菌如何广泛和差异地影响表型 被称为癌症的标志。作为一个特别说明性的实例，具核梭杆菌是 一种口腔中的常驻、非运动、革兰氏阴性厌氧菌，具有传播和引起 脑、肺和肝的致命感染，并且通常在结肠直肠癌（CRC）中大量发现 组织.已知该细菌进入宿主细胞是激活促细胞因子的关键组分。 致癌途径，但F.有核质通过初始细胞进入 对口腔的了解仍然很少。最近研究表明，这种细菌能够 在受感染的宿主肿瘤细胞内存活足够长的时间以被携带沿着到远处转移部位， 就像肝脏。我们最近已经证明这些细菌侵入CRC类器官， 有趣地定位于在CRC进展中至关重要的隐窝小生境。这些观察表明， 细菌在CRC中的潜在作用，并提供了进一步了解F.核增益 接触正常无菌组织我们相信最近在组织工程方面的突破将使 能够揭示肿瘤微环境中细菌的广泛影响的研究 (TME)包括但不限于i）通过血管和肿瘤内的扩散动力学 血管周围生态位，和ii）细菌对CRC进展的影响，通过调节上皮细胞对 间充质转化（EMT），已牵连在结肠干细胞龛。在这个高度 探索性R21项目，我们将克服这些研究的主要局限性，其中包括过度- 体外肿瘤和细菌细胞的增殖，以及体内有限的时空分辨率。我们将 利用组织工程结合微流体分析和细菌遗传技术， 能够在体外生物系统中进行肿瘤-微生物相互作用的高分辨率研究， 人类的TME我们将继续进行以下目标：1）开发微流体球体为基础的组织 能够模仿F.有核细胞趋向性离开循环并进入结肠TME，以及2） 对由F. 在完全定义的CRC TME条件下的核感染。更广泛地说，该项目可能会导致 与竞争对手相比，具有更高吞吐量、准确性和成本效益的适应性技术 分析宿主-病原体动态相互作用的方法，有助于确定肿瘤-微生物的新作用 在复杂的癌症过程中的相互作用，并有助于识别和筛选未来的目标， 癌症疫苗或疗法。

There remains a critical knowledge gap regarding the spatiotemporal dynamics of bacterial tissue dissemination, as well as how a diverse range of bacteria broadly and differentially influence phenotypes referred to as the hallmarks of cancer. As one particularly illustrative example, Fusobacterium nucleatum is a resident, non-motile, Gram-negative anaerobe of the oral cavity with the ability to disseminate and cause lethal infections of the brain, lungs, and liver, and is often found in abundance in colorectal cancer (CRC) tissue. It is known that entry of this bacterium into host cells is a critical component in the activation of pro- oncogenic pathways, yet the tissue dissemination mechanisms for F. nucleatum past initial cellular entry in the oral cavity remains poorly understood. Recently it was demonstrated that this bacterium is able to survive within infected host tumor cells for long enough to be carried along to distant metastatic sites such as the liver. And we have more recently demonstrated that these bacteria invade CRC organoids, and intriguingly localize to crypt niches of critical importance in CRC progression. These observations suggest a potential role for bacteria in CRC and provide a rationale to further understand how F. nucleatum gains access to normally sterile tissues. We believe that recent breakthroughs in tissue engineering will enable studies that are capable of revealing the broad-ranging effects of bacteria in the tumor microenvironment (TME), including but not limited to i) the dissemination dynamics through blood vessels and within the tumor perivascular niche, and ii) the impacts of bacteria on CRC progression via regulation of epithelial-to- mesenchymal transition (EMT) that has been implicated in the colon stem cell niche. In this highly exploratory R21 project we will overcome the major limitations to such studies, which include the over- proliferation of tumor and bacterial cells in vitro, and the limited spatiotemporal resolution in vivo. We will leverage tissue engineering combined with microfluidic analysis and bacterial genetic technologies to enable high-resolution studies of tumor-microbe interactions in an in vitro biological system modeled after the human TME. We will proceed with the following aims: 1) Develop a microfluidic spheroid-based tissue model capable of mimicking F. nucleatum tropism out of the circulation and into the colon TME, and 2) Perform unbiased transcriptome and epigenome profiling of epithelial pathway activation resulting from F. nucleatum infection under fully-defined CRC TME conditions. More broadly, this project could result in an adaptable technology with higher throughput, accuracy, and cost-effectiveness compared with competing methods to analyze dynamic host-pathogen interactions, help to define new roles for tumor-microbe interactions in complex cancer processes, and aid in the identification and screening of future targets for cancer vaccines or therapies.