Project-2:Defining the role of compartmentalized neuro-lymphatic networks on CRC and metastatic progression

结直肠癌（CRC）是美国第二大致命癌症，影响超过14万人 在美国，每年大约有5万人死于这种疾病，主要是由于转移性进展。肠道是 人体最大的免疫细胞集合，保持在接近外来抗原的饮食，丰富 在近端区域，以及微生物群，其积累在远端区域。广阔而高度联系的 肠淋巴管形成了主要的细胞和抗原转运途径，到达引流淋巴结，负责 获得性免疫的启动，在调节结直肠癌转移中发挥四种作用：免疫 细胞可以（i）通过免疫细胞杀伤预防CRC进展和早期传播或转移，（ii）促进 通过释放炎性细胞因子的CRC进展和转移，（iii）防止转移性定植 在肝脏中，或（iv）促进肝脏中的转移性定植。我们提供的数据表明， 肠淋巴引流到功能不同的淋巴结有助于同时诱导 肠道中的免疫抑制和炎症免疫反应;然而，肠道炎症的相关性 与结直肠癌和转移的关系尚不清楚。除了分隔的淋巴网络，肠道 也有大量的肠神经元在功能上与它们所占据的每个区域相协调。逆行 从不同的肠道区域和特定的细胞分选独立转录组学追踪，我们发现了新的 神经元回路和肠神经元在感知肠组织中的扰动中的作用;无论肠 神经元感知和调节CRC转移进展仍然是未知的。总体而言，项目2的重点是 了解肠相关神经元如何感知和提供调节CRC进展的信号， 原发性肿瘤和转移性肿瘤的肝转移和肠淋巴引流如何区室化 肝脏部位调节抗肿瘤反应和早期传播。在目标1中，我们假设原发性CRC， 以及肝转移，被肠相关神经元群体特异性地感知，这反过来， 影响肿瘤和转移进展。这个问题将使用工具来解决可视化和电路- 图，单细胞和主动翻译转录组学，以及针对特定神经元的化学遗传学方法 子集在目标2中，我们假设肠淋巴系统与原发性CRC和早期CRC之间存在沟通。 转移性接种到局部和全身免疫系统，调节抗肿瘤反应。这将是 解决了结合现代清除和活成像技术，单细胞转录组学和新的免疫 细胞相互作用方法。通过结合这些方法，Mucida实验室的专业知识，与Sohail Tavazoie实验室的 在癌症和转移生物学的专业知识（项目1），Birsoy实验室的体外筛选和细胞的专业知识， 代谢（项目3），以及Cao和Saeed Tavazoie实验室在单细胞和生物信息学方面的专长 通过分析，我们试图确定神经淋巴网络在CRC进展和转移中的作用， 阵

Colorectal cancer (CRC) is the second most deadly cancer in the United States, affecting over 140,000 people each year, killing approximately 50,000 in the US, largely by metastatic progression. The intestine hosts the body’s largest collection of immune cells, maintained in close proximity to foreign antigens from the diet, enriched in the proximal regions, and microbiota, which accumulate in the distal regions. The vast and highly connected gut lymphatic vessels form a major cell- and antigen transport route to the draining lymph nodes, responsible for the initiation of adaptive immunity, which could play four roles in regulating colorectal cancer metastasis: immune cells could (i) prevent CRC progression and early dissemination or metastasis by immune-cell killing (ii) promote CRC progression and metastasis through release of inflammatory cytokines, (iii) prevent metastatic colonization in the liver, or (iv) promote metastatic colonization in the liver. We provide data that compartmentalization of intestinal lymphatic drainage to functionally distinct lymph nodes facilitates the simultaneous induction of immune-suppressive and inflammatory immune responses in the gut; however, the relevance of gut lymphatics to CRC and metastasis remains underexplored. In addition to compartmentalized lymphatic networks, the gut also hosts a large number of enteric neurons functionally tuned to each region they occupy. Using retrograde tracing from distinct intestinal regions and specific cell-sorting independent transcriptomics, we uncovered novel neuronal circuits and a role for enteric neurons in sensing perturbations in the intestinal tissue; whether enteric neurons sense and modulate CRC metastatic progression remains unknown. Overall, Project 2 is focused on understanding how enteric-associated neurons sense and provide signals that regulate CRC progression and liver dissemination and how compartmentalized intestinal lymphatic drainage of primary tumor and metastatic liver sites regulate anti-tumor responses and early dissemination. In Aim 1, we hypothesize that primary CRC, as well as liver metastasis, are specifically sensed by populations of enteric-associated neurons, which in turn, influence tumor and metastatic progression. This question will be addressed using tools to visualize and circuit- map, single cell and active translating transcriptomics, and chemogenetic approaches targeting specific neuronal subsets. In Aim 2, we hypothesize that the intestinal lymphatic system communicates primary CRC and early metastatic seeding to the local and systemic immune system, modulating anti-tumor responses. This will be addressed combining modern clearing and live imaging techniques, single cell transcriptomics and novel immune cell-interaction approaches. By combining these approaches, expertise of Mucida lab, with Sohail Tavazoie lab’s expertise in cancer and metastasis biology (Project 1), the Birsoy lab’s expertise in in vitro screenings and cell metabolism (Project 3), and the Cao and Saeed Tavazoie labs expertise in single-cell and bioinformatics analyses, we seek to determine the role of neuro-lymphatic networks in CRC progression and metastatic formation.