Macrophage-based cell therapy is promising in solid tumors, but the efficient acquisition of macrophages remains a challenge. Induced pluripotent stem cell (iPSC)-induced macrophages are a valuable source, but time-consuming and costly. The application of reprogramming technologies allows for the generation of macrophages from somatic cells, thereby facilitating the advancement of cell-based therapies for numerous malignant diseases. The composition of CD45+ myeloid-like cell complex (MCC) and induced macrophage (iMac) were analyzed by flow cytometry and single-cell RNA sequencing. The engraftment capacity of CD45+ MCC was evaluated by two transplantation assays. Regulation of c-Myc on MafB was evaluated by ChIP-qPCR and promoter reporter and dual luciferase assays. The phenotype and phagocytosis of iMac were explored by flow cytometry and immunofluorescence. Leukemia, breast cancer, and patient-derived tumor xenograft models were used to explore the anti-tumor function of iMac. Here we report on the establishment of a novel methodology allowing for reprogramming fibroblasts into functional macrophages with phagocytic activity by c-Myc overexpression. Fibroblasts with ectopic expression of c-Myc in iPSC medium rapidly generated CD45+ MCC intermediates with engraftment capacity as well as the repopulation of distinct hematopoietic compartments. MCC intermediates were stably maintained in iPSC medium and continuously generated functional and highly pure iMac just by M-CSF cytokine stimulation. Single-cell transcriptomic analysis of MCC intermediates revealed that c-Myc up-regulated the expression of MafB, a major regulator of macrophage differentiation, to promote macrophage differentiation. Characterization of the iMac activity showed NF-κB signaling activation and a pro-inflammatory phenotype. iMac cells displayed significantly increased in vivo persistence and inhibition of tumor progression in leukemia, breast cancer, and patient-derived tumor xenograft models. Our findings demonstrate that c-Myc alone is enough to reprogram fibroblasts into functional macrophages, supporting that c-Myc reprogramming strategy of fibroblasts can help circumvent long-standing obstacles to gaining “off-the-shelf” macrophages for anti-cancer immunotherapy.

中文翻译：

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单独的 c-Myc 足以将成纤维细胞重编程为功能性巨噬细胞


基于巨噬细胞的细胞疗法在实体瘤中很有前景，但巨噬细胞的有效获取仍然是一个挑战。诱导多能干细胞（iPSC）诱导的巨噬细胞是一个有价值的来源，但耗时且昂贵。重编程技术的应用允许从体细胞产生巨噬细胞，从而促进多种恶性疾病的细胞疗法的进步。通过流式细胞术和单细胞 RNA 测序分析 CD45+ 骨髓样细胞复合物 (MCC) 和诱导巨噬细胞 (iMac) 的组成。通过两种移植测定评估CD45+MCC的植入能力。通过 ChIP-qPCR、启动子报告基因和双荧光素酶测定评估 c-Myc 对 MafB 的调节。通过流式细胞术和免疫荧光研究了 iMac 的表型和吞噬作用。使用白血病、乳腺癌和患者来源的肿瘤异种移植模型来探索 iMac 的抗肿瘤功能。在这里，我们报告了一种新方法的建立，该方法允许通过 c-Myc 过表达将成纤维细胞重编程为具有吞噬活性的功能性巨噬细胞。在 iPSC 培养基中异位表达 c-Myc 的成纤维细胞快速生成具有植入能力以及不同造血区室重新增殖的 CD45+ MCC 中间体。 MCC中间体稳定地维持在iPSC培养基中，仅通过M-CSF细胞因子刺激即可连续产生功能性和高纯度的iMac。 MCC中间体的单细胞转录组分析表明，c-Myc上调MafB（巨噬细胞分化的主要调节因子）的表达，从而促进巨噬细胞分化。 iMac 活性特征显示 NF-κB 信号激活和促炎表型。 iMac 细胞在白血病、乳腺癌和患者来源的肿瘤异种移植模型中显示出显着增加的体内持久性和对肿瘤进展的抑制。我们的研究结果表明，仅使用 c-Myc 就足以将成纤维细胞重编程为功能性巨噬细胞，这支持了成纤维细胞的 c-Myc 重编程策略可以帮助克服获得“现成”巨噬细胞用于抗癌免疫治疗的长期障碍。