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

关于细菌组织的时空动力学，仍然存在一个关键的知识缺口 传播，以及不同种类的细菌如何广泛和不同地影响表型 被称为癌症的标志。作为一个特别说明性的例子，核梭杆菌是 口腔革兰氏阴性厌氧菌一种常驻的、不活动的、革兰氏阴性的口腔厌氧菌，具有传播和引起疾病的能力 脑、肺和肝的致命感染，常见于结直肠癌(CRC) 组织。已知，这种细菌进入宿主细胞是激活前体细胞的关键成分。 致癌途径，然而，组织传播机制过去最初的细胞进入 人们对口腔仍知之甚少。最近的研究表明，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，以携带到远处的转移部位，如 作为肝脏。我们最近证明了这些细菌入侵CRC有机化合物，并且 耐人寻味地定位到在CRC进展中至关重要的隐蔽利基。这些观察结果表明 细菌在结直肠癌中的潜在作用，为进一步了解核杆菌是如何获得 接触到通常无菌的组织。我们相信，组织工程学最近的突破将使 能够揭示细菌对肿瘤微环境的广泛影响的研究 (TME)，包括但不限于i)通过血管和肿瘤内的传播动力学 血管周围生态位，以及II)细菌通过调节上皮细胞到血管内皮细胞对结直肠癌进展的影响。 间充质转化(EMT)已被认为与结肠干细胞的生态位有关。在这个高度 探索性R21项目我们将克服此类研究的主要限制，包括过度 肿瘤和细菌细胞在体外的增殖，以及体内有限的时空分辨率。我们会 利用组织工程结合微流控分析和细菌基因技术 能够在模拟如下的体外生物系统中对肿瘤-微生物相互作用进行高分辨率研究 人类的TME。我们将致力于以下目标：1)开发一种基于微流控球体的组织 模型能够模拟核盘藻的趋向性，从循环出来进入结肠TME，和2) 无偏转录组和表观组谱分析F。 在完全定义的CRC TME条件下的核线虫感染。更广泛地说，这个项目可能会导致 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，帮助确定肿瘤-微生物的新角色 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靶点 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.