An organotypic model recapitulating colon cancer microenvironment and metastasis

项目摘要 本U 01是对FOA PAR-16-105癌症组织工程协作： 用于癌症研究的仿生组织工程技术。我们将在其中解决以下方面的重大差距： 我们对转移性结直肠癌（CRC）的发生和进展的了解。这一差距主要是由于 部分原因在于目前的体外、离体甚至体内研究模型难以建立或具有 有限的适用性。因此，我们的目标是显示三种创新的转移模型的效用 （长期器官型模型和使用多器官微流体装置的模型）和新的体内模型 （免疫活性胚泡模型）。使用这些模型，我们将研究交互式细胞分泌组， 分化，迁移和侵袭的原发性和转移性成纤维细胞肿瘤微环境， 以确定CRC转移的关键因素。我们的假设是：炎症相关的成纤维细胞和 改变的代谢条件通过转录调节促进上皮转移。根据我们最近的 成功的工程新模型，我们提出了3个目标。目标1。将分泌精氨酸的基质整合到 我们的器官型模型，以研究炎症对癌症干细胞（SC）的影响， 侵袭/迁移细胞表型。目标2.利用微流控体构建肝转移模型， 芯片平台来研究转移细胞的转录组和表观遗传重编程。目标3：为了验证我们的 使用体内免疫活性鼠转移模型的体外模型。在目标1中，脱细胞人结肠 将被来自间质的正常或癌性成纤维细胞和形式为 的癌症SC和类器官，以询问这些操作如何影响该模型中的侵袭和分化。 为了补充细胞发现，我们将使用RNA-seq和ATAC鉴定转录组的改变。 seq.目的2使用创新的芯片上的身体微流控装置来测试自分泌和旁分泌增殖 以及在有利于致癌进展的条件下的分泌反应，包括缺氧和低葡萄糖。 如在目标1中，将对不同环境中的上皮细胞和成纤维细胞进行转录分析。Aim 3使用新的 在免疫活性胚泡模型中，将进行相同的转录谱分析。所有3款车型将在 使用比较和信息学方法联系起来。这种方法是创新的，因为它使用人类结肠组织， 所有的目标和询问的转录贡献和景观的可访问的染色质在每个模型。 我们的多学科团队包括一名结直肠外科医生-科学家和生物医学工程师，他们在以下方面具有专业知识： 癌症SC、信号传导、转移和器官型和器官芯片平台。其成果将 意义重大，因为它们将建立用于研究晚期CRC发病机制的改进方法， 开发源自患者的高通量细胞模型以测试治疗干预。

PROJECT SUMMARY This U01 is in response to FOA PAR-16-105 Cancer Tissue Engineering Collaborative: Enabling Biomimetic Tissue-Engineered Technologies for Cancer Research. In it we will address the substantial gap in our knowledge of the initiation and progression of metastatic colorectal cancer (CRC). This gap is due, in large part, to the fact that current in vitro, ex vivo, and even in vivo research models are hard to establish or have limited applicability. Accordingly, our objectives are to show the utility of three innovative models of metastasis (a long-term organotypic model and a model that uses multi-organ microfluidic devices) and a new in vivo model (immunocompetent blastocyst model). Using these models, we will investigate the interactive cellular secretome, differentiation, migration and invasion of the primary and metastatic fibroblast tumor microenvironment in order to identify critical contributions to CRC metastasis. Our hypothesis is: inflammation-associated fibroblasts and altered metabolic conditions promote epithelial metastasis via transcriptional regulation. Based on our recent successful engineering of novel models, we propose 3 Aims. Aim 1. To integrate cytokine-secreting stroma into our organotypic model in order to study the influence of inflammation on cancer stem cells (SCs) and on invasion/migration cell phenotypes. Aim 2. To engineer a liver metastasis model using a microfluidic body-on-a- chip platform to study transcriptome and epigenetic reprogramming of metastatic cells. Aim 3. To validate our in vitro models using in vivo immunoproficient murine metastatic models. In Aim 1, decellularized human colons will be repopulated with normal or cancerous fibroblasts from the stroma, and cancer epithelial cells in the form of cancer SCs and organoids to ask how these manipulations influence invasion and differentiation in this model. To complement the cellular findings, we will identify alterations in the transcriptome using RNA-seq and ATAC- seq. Aim 2 uses the innovative body-on-a chip microfluidic device to test autocrine and paracrine proliferative and secretory responses under conditions that favor oncogenic progression, including hypoxia and low glucose. As in Aim 1, epithelia and fibroblasts in the different milieus will be transcriptionally profiled. Aim 3 uses a new immunocompetent blastocyst model and will undergo the same transcriptional profiling. All 3 models will be linked using comparative, informatics approaches. The approach is innovative as it uses human colon tissues in all Aims and asks about the transcriptional contribution and landscape of accessible chromatin in each model. Our multidisciplinary team includes a colorectal surgeon-scientist and biomedical engineers with expertise in cancer SCs, signaling, metastases, and organotypic and organ-on-a-chip platforms. The findings will be significant, as they will establish improved approaches for studying the pathogenesis of advanced CRC, and for developing patient-derived, high-throughput cell models to test therapeutic interventions.