Engineer Biomimetic Microfluidic Models to Investigate and Reprogram Tumor Associated Neutrophils for Cancer Therapy

项目摘要 中性粒细胞在肿瘤发展的不同阶段发挥关键作用。在小鼠中， 中性粒细胞导致胶质母细胞瘤（GBM）和肺癌中肿瘤生长减少，但促进肿瘤生长 在转移前肺和其他实体瘤中，表明它们在转移前肺和其他实体瘤中的阶段特异性和组织依赖性功能。 肿瘤进展。嗜中性粒细胞还可以促进癌细胞对化疗、放疗和化疗的抵抗。 通过释放各种细胞因子在不同肿瘤中进行免疫治疗。尽管这些临床前和动物研究 关于肿瘤相关中性粒细胞（TAN），我们对肿瘤相关中性粒细胞（TAN）的机制理解仍然存在知识差距 人嗜中性粒细胞调节GBM中的癌症进展和治疗抗性，这是由于短寿命和 对中性粒细胞基因编辑的抗性以及分离阶段特异性TAN的技术障碍。解决 针对这一空白，我们提出利用微流体和人类诱导多能干细胞（hiPSC）的力量 探讨中性粒细胞在人类GBM发育中的多样性和可塑性。阐明基本的 该机制还将使急需的开发和评估以嗜中性粒细胞为靶点的癌症治疗成为可能。 我们的中心假设是，微流体模型将重演人类肿瘤的不同阶段 进展，提供了一个平台，表型和机制的理解，中性粒细胞的作用， GBM发展。为了验证这一假设，我们将实施一种新的间质性肿瘤微环境， 芯片（iT-MOC），并在不同条件下询问嗜酸性粒细胞介导的肿瘤进展和治疗抗性。 目标1中的GBM生长阶段。然后在目标2中，我们将确定形态，极化，寿命和 GBM浸润中性粒细胞的抗肿瘤细胞毒性。在目标3中，我们将重新编程肿瘤相关中性粒细胞， - 通过具有嵌合抗原受体（汽车）的hiPSC的基因工程化而朝向抗肿瘤效应细胞， microRNAs（miRNAs）。这是一种新的方法，因为人类中性粒细胞不能被遗传修饰。成功 这些目标的完成将提供一个创新的平台来研究TAN的多样性和可塑性，并提供 深入了解将它们重新编程为抗肿瘤效应细胞和CAR-中性粒细胞的概念验证， 靶向癌症治疗。

Project Summary Neutrophils play critical roles during different stages of tumor development. In mice, systemic depletion of neutrophils results in decreased tumor growth in glioblastoma (GBM) and lung cancer, but promote tumor growth in pre-metastatic lung and other solid tumors, indicating their stage-specific and tissue-dependent functions in tumor progression. Neutrophils could also facilitate cancer cell resistance to chemotherapy, radiotherapy, and immunotherapy in different tumors by releasing various cytokines. Despite these preclinical and animal studies on tumor-associated neutrophils (TANs), a knowledge gap remains in our mechanistic understanding of how human neutrophils regulate cancer progression and therapeutic resistance in GBM, due to the short life and resistance to gene editing of neutrophils as well as technical hurdles in isolating stage-specific TANs. To address this gap, we propose to harness the power of microfluidics and human induced pluripotent stem cells (hiPSCs) to interrogate the diversity and plasticity of neutrophils in human GBM development. Elucidating the underlying mechanism will also enable the much-needed development and evaluation of neutrophil-targeted cancer therapy. Our central hypothesis here is that the microfluidic model will recapitulate the different stages of human tumor progression, providing a platform for phenotypic and mechanistic understanding of the roles of neutrophils in GBM development. To test this hypothesis, we will implement a novel interstitial tumor-microenvironment-on- chip (iT-MOC), and interrogate neutrophil-mediated tumor progression and therapeutic resistance at different GBM growth stages in Aim 1. Then in Aim 2, we will determine the morphology, polarization, life-span and antitumor cytotoxicity of GBM-infiltrating neutrophils. In Aim 3, we will reprogram tumor-associated neutrophils towards antitumor effector cells via genetic engineering of hiPSCs with chimeric antigen receptors (CARs) and microRNAs (miRNAs). This is a novel approach as human neutrophils cannot be genetically modified. Successful completion of these aims will offer an innovative platform to study the diversity and plasticity of TANs, and provide insights into reprograming them towards antitumor effector cells and the proof-of-concept for CAR-neutrophils in targeted cancer therapy.