Malignant Cell Engagement in Immune Circuits of Human Microsatellite-stable and Microsatellite-instable Colorectal Cancer

项目摘要 癌症免疫学中的一个重要问题是恶性细胞和T细胞如何沟通和影响 抗肿瘤免疫的功效。为了生成这些细胞如何相互作用的假设，我们应用了Single 细胞RNA测序以分析700,000个恶性、免疫和间质细胞的转录 队列：(1)65例原发未经治疗的结直肠癌患者(31例MSS和34例MSI)，分别为 特点是免疫治疗应答率差异很大；(2)治疗前后的样本 20例BRAF突变的转移性结直肠癌患者接受BRAFi/Meki/抗PD1治疗。这些数据让我们得出了 这一提议的中心假设是：恶性细胞能强烈表达干扰素刺激基因(ISGs) 促进抗肿瘤免疫，而肿瘤细胞慢性干扰素反应损害抗肿瘤免疫 和治疗反应。这项拟议的工作有望揭示对人类肿瘤的基本见解。 为设计稳健的生物标记物和新的治疗方法提供了一个新的视角 战略。我们将解决三个主要问题。首先，与MSI/响应者相关的ISG计划是否在 恶性肿瘤细胞是驱动抗肿瘤免疫的肿瘤内前馈环路的一部分吗？这将是 通过空间映射ISG+恶性细胞和 未经处理的原发结直肠癌标本中的T细胞，以及经治疗的患者的治疗后标本中的T细胞 BRAFi/Meki/anti-PD-1，这将揭示该空间网络与肿瘤消退的关联。第二, 什么是人类结直肠癌恶性ISGs的驱动因素，干扰素是否会影响免疫反应和 通过对人类CRC细胞进行表观遗传学重新编程对靶向治疗的反应？为了解决这个问题，我们将 使用干扰素和天然免疫刺激诱导结直肠癌机构体中的ISGs，绘制转录和 在新分离的CRC细胞中发现的表观遗传特征，并命名为ISG诱导 上游监管机构。干扰素刺激和随后休息的有机化合物将被评估为表观遗传学 记忆和调节的二次免疫和药物反应。第三，预先存在的ISG签名如何 在恶性肿瘤细胞中影响CTL介导的抗肿瘤反应？利用多肽同源T细胞系，我们将测试 负载ISG+和ISG-肽的CRC有机化合物的体外杀伤作用。我们将进一步测试ISG如何 通过暂时控制可移植瘤中恶性ISG的表达来调控体内肿瘤杀伤 模特们。综上所述，我们使用scRNAseq来预测恶性细胞与T细胞的相互作用，然后测试这些 利用空间图谱、单细胞表观基因组学和人类基因组学等尖端技术的假设 器官类T细胞共培养。这一结果应该能阐明对干扰素有反应的恶性细胞如何影响。 抗肿瘤免疫和耐药性，为R01提案生成基础结果和模型系统， 并为独立的职业生涯做好准备，研究驱动人类肿瘤的细胞网络 豁免权。

Project Summary An important question in cancer immunology is how malignant cells and T cells communicate and impact the efficacy of anti-tumor immunity. To generate hypotheses for how these cells interact, we have applied single cell RNA sequencing to profile the transcriptomes of >700,000 malignant, immune, and stromal cells in 2 cohorts: (1) 65 patients with two types of primary untreated CRC (31 MSS and 34 MSI tumors) which are characterized by vastly different immunotherapy response rates; (2) pre- and post-treatment specimens from 20 BRAF-mutant metastatic CRC patients treated with BRAFi/MEKi/anti-PD1. These data led us to formulate the central hypothesis for this proposal: malignant cells that express interferon-stimulated genes (ISGs) acutely promote anti-tumor immunity, while chronic interferon responses in malignant cells impair anti-tumor immunity and therapeutic responses. The proposed work is anticipated to reveal fundamental insights into human tumor immunology and provide a novel perspective for the design of robust biomarkers and new therapeutic strategies. We will address three major questions. First, is the MSI/responder-associated ISG program in malignant cells part of an intra-tumoral feed-forward loop that is driving anti-tumor immunity? This will be answered by spatially mapping the intratumoral cellular interaction network between ISG+ malignant cells and T cells in untreated primary CRC specimens, and in post-treatment specimens of patients treated with BRAFi/MEKi/anti-PD-1, which will reveal association of this spatial network with tumor regression. Second, what are the drivers of malignant ISGs in human CRC, and can interferons impact immune responses and responses to targeted therapy by epigenetically reprogramming human CRC cells? To address this, we will use interferons and innate immune stimuli to induce ISGs in CRC organoids, map the transcriptional and epigenetic signatures to the signatures found in freshly isolated CRC cells, and nominate ISG-inducing upstream regulators. IFN-stimulated and subsequently rested organoids will be assessed for epigenetic memory and for modulated secondary immune and drug responses. Third, how do pre-existing ISG signatures in malignant cells impact CTL-mediated anti-tumor responses? Using peptide-cognate T cell lines, we will test the in vitro killing of ISG+ and ISG- peptide-loaded CRC organoids. We will furthermore test how ISGs modulate in vivo tumor killing by temporally controlling malignant ISG expression in transplantable tumor models. In summary, we use scRNAseq to predict malignant cell – T cell interactions, and then test these hypotheses with cutting-edge technologies such as spatial profiling, single cell epigenomics, and human organoid T cell co-cultures. The results should elucidate how malignant cells that respond to interferons impact anti-tumor immunity and resistance, generate foundational results and model systems for an R01 proposal, and prepare the candidate for an independent career to study the cellular networks that drive human tumor immunity.