Determination of myeloid cell subpopulations during intestinal cell invasion

描述（由申请人提供）：基质细胞越来越多地被认为在组织再生和修复过程中发挥重要作用。在肠道中，丰富的结缔组织和免疫细胞支持不断更新的上皮细胞的功能和结构。上皮稳态的破坏，例如组织损伤或癌症中发生的破坏，导致先天免疫系统的骨髓细胞的募集和激活。这些在组织防御和修复中发挥着不同的作用，在某些情况下有助于疾病的发病机制。在结直肠癌中尤其如此。最近的研究表明，良性腺瘤性息肉向腺癌的转变伴随着浸润免疫细胞数量的增加。此外，结直肠癌存活率与肿瘤内的巨噬细胞和中性粒细胞密度有关。免疫细胞如何调节癌症进展还知之甚少，但有很好的证据表明，这些细胞可以促进癌细胞的生长，侵袭和转移。该提案的目标是研究免疫细胞在体内的功能，斑马鱼细胞入侵模型，共享重要的功能与目前公认的模型，人类癌细胞入侵，使用体外系统开发。 这项建议包括两个具体目标。第一个目标是确定野生型斑马鱼幼虫和myh 11-meltdown突变体肠道中的先天免疫细胞群，这些突变体响应于不受调节的平滑肌肌球蛋白功能而发展上皮细胞侵袭。免疫细胞将使用在已建立的骨髓细胞启动子控制下表达荧光报告基因的转基因系在体内定位。此外，使用荧光报告分子和特异性标记染料将用于评估极化免疫细胞中上调的活化途径。延时记录将用于研究免疫细胞和侵袭性上皮细胞之间的相互作用。遗传功能丧失研究将用于评估这些细胞在细胞侵袭表型中发挥的作用。将在驱动上皮细胞侵袭伪足形成但不支持上皮细胞的基质侵袭的转基因系中进行可比研究，从而允许在侵袭过程的特定阶段进行免疫细胞分析。第二个目标是在两种斑马鱼模型中进行先天免疫细胞的转录谱分析，并将基因表达的变化与骨髓细胞亚型相关联，特别是与癌症进展相关的基因，以及功能性测定差异调节基因对入侵过程的重要性。总的来说，这些研究将增强我们对免疫细胞如何调节肠道组织结构的理解。此外，它们可能导致识别新的标记物， 先天免疫细胞亚型在结直肠癌中具有预后意义。

DESCRIPTION (provided by applicant): Stromal cells are increasingly recognized as playing an essential role in tissue regenerative and reparative processes. In the intestine, a rich population of connective tissue and immune cells supports the function and architecture of the continuously renewing epithelium. Disruption of epithelial homeostasis, such as occurs with tissue injury or in cancer, leads to the recruitment and activation of myeloid cells of the innate immune system. These play diverse roles in tissue defense and repair and in certain instances contribute to disease pathogenesis. This is particularly true in colorectal cancer. Recent work has shown that the transition of benign adenomatous polyps to adenocarcinoma is accompanied by an increase in the number of infiltrating immune cells. Furthermore, colorectal cancer survival has been linked to both macrophage and neutrophil density within the tumors. How immune cells regulate cancer progression is poorly understood, but there is good evidence that these cells can contribute to cancer cell growth, invasion and metastasis. The goal of this proposal is to study immune cell function in an in vivo, zebrafish cell invasion model that shares important features with currently accepted models of human cancer cell invasion that were developed using in vitro systems. This proposal consists of two specific aims. The goal of the first aim is to define the innate immune cell population in the intestine of wild type zebrafish larvae and myh11-meltdown mutants that develop epithelial cell invasion in response to unregulated smooth muscle myosin function. Immune cells will be localized in vivo using transgenic lines that express fluorescent reporter genes under the control of established myeloid cell promoters. Additionally, the use of fluorescent reporters and specific labeling dyes will be used to assess the activation pathways upregulated in polarized immune cells. Time-lapse recordings will be used to study interactions between the immune cells and the invasive epithelial cells. Genetic loss-of-function studies will be used to assess what role if any these cells play in the cell invasion phenotype. Comparable studies will be performed in a transgenic line that drives formation of epithelial cell invadopodia but that does not support stromal invasion of the epithelial cells, thus allowing immune cells analyses at specific stages of the invasive process. The goal of the second aim will be to perform transcriptional profiling of innate immune cells in the two zebrafish models, and to correlate changes in gene expression with myeloid cell subtypes, particularly those linked to cancer progression as well as functionally assay the importance of differentially regulated genes to the invasion process. Collectively, these studies will enhance our understanding of how immune cells regulate tissue architecture in the intestine. Furthermore, they may lead to the identification of markers of novel innate immune cell subtypes that have prognostic significance in colorectal cancer.