PO.TB10.18 · 肿瘤生物学

代表巨噬细胞状态及免疫逃逸潜能机制决定因素的主调控蛋白的阐明与药理学靶向

Elucidation and pharmacologic targeting of master regulator proteins representing mechanistic determinants of macrophage state and immunoevasive potential

编号 4938 展板 26 时间 4/21 09:00–12:00 区域 Section 30 主讲 Gaetano Viscido, MS;PhD

分会场 Novel Experimental Platforms and Causal Inference

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作者与单位 Authors & Affiliations

Gaetano Viscido¹, Mikko Turunen¹, Zhouzerui Liu², Justyn Chang², Meghna S. Raman², Leo B. Dupire¹, Hanrui Zhang³, Tim Olsen², Jeremy Worley⁴, Aleksandar Obradovic⁵, Andrea Califano⁶

¹Systems Biology Department, Columbia University, New York, NY,²Chan Zuckerberg Biohub, New York, NY,³Cardiovascular Genomics Program, Department of Medicine, Columbia University Irving Medical Center, New York, NY,⁴Columbia University and Chan Zuckerberg Biohub, New York, NY,⁵Columbia University, New York, NY,⁶Dept. of Systems Biology and Dept. of Medicine and Dept. of Biomedical Informatics and Dept. of Biochemistry & Molecular Biophysics, and HICCC, Columbia University Irving Medical Center; Chan Zuckerberg Biohub, New York, NY

摘要 Abstract

中文摘要

目的：巨噬细胞（mΦ）在肿瘤微环境（TME）内表现出广泛的转录可塑性。M1巨噬细胞促进抗肿瘤免疫，而调节性M2巨噬细胞则驱动肿瘤进展和对免疫检查点治疗的耐药。在近期一项研究中，Obradovic等人（Cell 2021）使用VIPER——一种从转录组数据推断蛋白活性的基于网络的算法——在透明细胞肾癌（ccRCC）中鉴定出一个高度免疫抑制的TAM亚群，其特征为TREM2⁺/C1Q⁺/APOE⁺（TCA⁺）表达。TCA⁺巨噬细胞与不良预后、转移和免疫逃逸相关。我们旨在鉴定从机制上控制TCA⁺程序的主调控（MR）蛋白，这些蛋白可能作为可操作的靶点，用于选择性清除这些细胞或将其重编程为中性、抗肿瘤或促炎状态。方法：我们通过Perturb-seq进行了汇集的单细胞CRISPR干扰（CRISPRi），靶向由VIPER从TCA⁺与M0/M1巨噬细胞差异表达基因中鉴定出的50个候选MR，随后进行时间分辨的scRNA-seq。将THP-1单核细胞分化并使用IL-4和IL-13极化至M2状态48小时，然后在跨越M2极化的七个时间点（0、6、12、24、48、96、192小时）进行分析。每个时间点分析约100,000个细胞，同时以约10,000个未扰动的THP-1来源M0、M1和M2巨噬细胞作为参照。我们旨在使用PLATE-seq生成TCA⁺巨噬细胞在超过350种药物作用下的扰动RNA-seq谱，以鉴定能够靶向单个MR（OncoTarget）或逆转全局MR活性特征（OncoTreat）的化合物，从而表型模拟经验证的遗传扰动。未发表数据：我们生成了一个全面的数据集，包含约700,000个时间分辨（每个时间点约100,000个细胞）的遗传扰动巨噬细胞，能够在M2/TCA+极化的整个过程中对MR特异性转录反应进行高分辨率表征。该数据集捕获了由候选MR的靶向抑制所诱导的早期、中期和晚期转录变化，为TCA+状态的获得和维持所依赖的调控架构提供了动态视图。我们将其与超过350个药物扰动谱整合，建立一个匹配的药理学资源，用于下游鉴定能够调节MR活性或全局性改变TCA+转录程序的化合物。结论：本研究提供了免疫抑制性TCA⁺巨噬细胞中调控程序的大规模、时间分辨图谱，旨在鉴定并靶向主调控因子以逆转其免疫抑制表型。该框架为中和巨噬细胞介导的免疫抑制、改善对免疫检查点治疗的反应提供了一条途径。

查看英文原文 English abstract

PURPOSE: Macrophages (mΦ) exhibit extensive transcriptional plasticity within the tumor microenvironment (TME). While M1 mΦ promote anti-tumor immunity, regulatory M2 mΦ drive tumor progression and resistance to immune checkpoint therapy. In a recent study, Obradovic et al. (Cell 2021) used VIPER, a network-based algorithm that infers protein activity from transcriptomic data, to identify a highly immunosuppressive TAM subset in clear cell renal carcinoma (ccRCC) characterized by TREM2⁺/C1Q⁺/APOE⁺ (TCA⁺) expression. TCA⁺ mΦ were associated with poor prognosis, metastasis, and immune evasion. We aim to identify Master Regulator (MR) proteins that mechanistically control the TCA⁺ program and may serve as actionable targets for selective depletion or reprogramming of these cells toward neutral, antitumor or pro-inflammatory states. METHODS: We performed pooled single-cell CRISPR interference (CRISPRi) via Perturb-seq targeting 50 candidate MRs identified by VIPER from genes differentially expressed in TCA⁺ versus M0/M1 mΦs, followed by time-resolved scRNA-seq. THP-1 monocytes were differentiated and polarized to the M2 state using IL-4 and IL-13 for 48 hours, then profiled across seven time points spanning M2 polarization (0, 6, 12, 24, 48, 96, 192 hr). Approximately 100,000 cells per time point were analyzed, along with ~10,000 unperturbed THP-1-derived M0, M1, and M2 mΦs as references. We aim to generate perturbational RNA-seq profiles of TCA⁺ mΦs with >350 drugs using PLATE-seq to identify compounds that either target individual MRs (OncoTarget) or invert the global MR-activity signature (OncoTreat), thus phenocopying validated genetic perturbations. UNPUBLISHED DATA: We generated a comprehensive dataset comprising ~700,000 time-resolved (~100,000 cells per time point), genetically perturbed mΦs, enabling high-resolution characterization of MR-specific transcriptional responses across the full course of M2/TCA + polarization. The dataset captures early, intermediate, and late transcriptional changes induced by targeted repression of candidate MRs, providing a dynamic view of the regulatory architecture underlying the acquisition and maintenance of the TCA + state. We will integrate with >350 drug-perturbation profiles to establish a matched pharmacologic resource for downstream identification of compounds capable of modulating MR activity or globally shifting the TCA + transcriptional program. CONCLUSION: This study provides a large-scale, time-resolved map of regulatory programs in immunosuppressive TCA⁺ mΦs, with the aim of identifying and targeting Master Regulators to invert their immunosuppressive phenotype. This framework offers a path to neutralize mΦ-mediated immunosuppression and improve responses to immune checkpoint therapy.

利益披露 Disclosure

G. Viscido, None.. M. Turunen, None.. Z. Liu, None.. J. Chang, None.. M. S. Raman, None.. L. B. Dupire, None.. H. Zhang, None.. T. Olsen, None.. J. Worley, None.. A. Obradovic, None. A. Califano, DarwinHealth Independent Contractor, Stock.

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