Development of novel metastatic mouse models that recapitulate the major immune contexts of human colon cancer

本研究的目的是建立一种新型的小鼠结直肠癌(CRC)模型。 免疫反应。这些模型旨在解决当前模型中存在的局限性 CRC，以提高其在免疫治疗中的转化性研究的适宜性。免疫检查点 抑制剂使实体肿瘤的治疗发生了革命性的变化，并揭示了肿瘤的关键重要性。 免疫环境对治疗结果的影响。伴有DNA错配修复(MMR)缺陷的CRC的特征是 高负担的体细胞突变，增加的T细胞渗透，以及对检查点的良好反应 封锁。不幸的是，大多数CRC的突变负担较低，并且对这些基因耐药。 治疗。临床前小鼠模型是研究潜在反应因素的强大平台 去接受免疫治疗。然而，没有一个单一的模型能够真实地概括结肠中的原发肿瘤的发展。 微环境、转移到肝脏和肺的转移，以及潜在的主要免疫背景 人类结直肠癌免疫治疗反应的变异性。为了解决这些严重的翻译缺陷， 我们将使用一种创新的技术，使用结肠镜引导的慢病毒或肿瘤粘膜下注射 有机化合物可在结肠局灶性原位和原位肿瘤中诱发易转移的肿瘤。在目标1中， 我们将设计一种通过诱导表达一种模型来调节肿瘤免疫原性的模型 抗原。我们将剖析诱导的抗肿瘤T细胞反应的特点，并探讨其用途 通过转移抗原特异性T细胞来测试过继T细胞治疗的模型。增强过继T细胞 治疗和镜像正在进行的临床试验，我们将评估CRISPR-Cas9介导的疗效 在转移前删除T细胞中的免疫检查点。在目标2中，我们将模拟免疫反应疗法 通过靶向基本DNA MMR基因Msh2和MLH1进行CRC并使用下一代测序 描述由此产生的MMR缺陷肿瘤与熟练肿瘤的突变情况。在《目标3》中，我们将 在这些模型中进行免疫检查点阻断的临床前试验，以探索它们概括的能力 人类结直肠癌患者群体的反应。我们还将测试一种新的免疫原性组合 化疗和检查点阻断，基于免疫原性细胞死亡可能致敏的假设 突变负担低或预先存在的T细胞参与免疫攻击最少的肿瘤。这一策略是 旨在改善对大多数结直肠癌患者的治疗，这些患者的肿瘤是非免疫原性和非 对免疫疗法有反应。该研究计划的总体目标是开发和基准一组 高度可比性的CRC模型将用于解决为什么只有一小部分患者对 免疫疗法。提出的策略是创新的，因为它使用了老鼠基因研究的尖端方法 工程和癌症建模，以捕捉人类结直肠癌的关键特征。这项研究还将包括 深入描述这些模型中的免疫微环境，并与人类进行比较。

The purpose of this proposal is to develop novel mouse models of colorectal cancer (CRC) with appropriate immune responses. These models have been designed to address limitations present in current models of CRC in order to enhance their suitability for translational research in immunotherapy. Immune checkpoint inhibitors have revolutionized treatment of solid tumors, and brought to light the critical importance of tumor immune context in treatment outcome. CRC with DNA mismatch repair (MMR) deficiency is characterized by a high burden of somatic mutations, increased T cell infiltration, and a favorable response to checkpoint blockade. Unfortunately, the majority of CRC has a lower mutational burden and is refractory to these treatments. Preclinical mouse models are powerful platforms for investigating the factors underlying response to immunotherapy. However, no single model faithfully recapitulates primary tumor development in the colon microenvironment, metastatic dissemination to the liver and lung, and the major immune contexts underlying variability in immunotherapy response in human CRC. To address these significant translational deficiencies, we will use an innovative technique employing colonoscopy-guided sub-mucosal injection of lentivirus or tumor organoids to induce focal autochthonous and orthotopic tumors in the colon that readily metastasize. In Aim 1, we will engineer a model that modulates tumor immunogenicity through inducible expression of a model antigen. We will dissect the features of the induced anti-tumor T cell response and investigate the utility of this model for testing adoptive T cell therapy by transferring antigen-specific T cells. To potentiate adoptive T cell therapy and mirror ongoing clinical trials in humans, we will assess the efficacy of CRISPR-Cas9-mediated deletion of immune checkpoints in T cells prior to transfer. In Aim 2, we will model immunotherapy-responsive CRC by targeting the essential DNA MMR genes Msh2 and Mlh1 and use next-generation sequencing to characterize the mutational landscapes of resulting MMR-deficient versus proficient tumors. In Aim 3, we will perform preclinical trials of immune checkpoint blockade in these models to explore their ability to recapitulate the responses of human CRC patient populations. We will also test a novel combination of immunogenic chemotherapy and checkpoint blockade, based on the hypothesis that immunogenic cell death may sensitize tumors with low mutational burden or minimal pre-existing T cell involvement to immune attack. This strategy is aimed at improving treatment for the majority of CRC patients, whose tumors are non-immunogenic and non- responsive to immunotherapy. The overarching goal of this research plan is to develop and benchmark a set of highly comparable CRC models that will be used to address why only a fraction of patients respond to immunotherapy. The proposed strategy is innovative in that it uses cutting-edge methods in mouse genetic engineering and cancer modeling to capture critical features of human CRC. This research will also include deep characterization of the immune microenvironment in these models and a comparison to humans.