Pediatric Translational Medicine Institute, Department of Hematology & Oncology, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, China.
State Key Laboratory of Systems Medicine for Cancer, Renji-Med X Clinical Stem Cell Research Center, Ren Ji Hospital, Shanghai Cancer Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, China.
J Hematol Oncol. 2024 Sep 12;17(1):83. doi: 10.1186/s13045-024-01605-x.
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.
基于巨噬细胞的细胞疗法在实体瘤中很有前景，但高效获取巨噬细胞仍然是一个挑战。诱导多能干细胞（iPSC）诱导的巨噬细胞是一种有价值的来源，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，从而促进了许多恶性疾病的基于细胞的治疗的进展。
通过流式细胞术和单细胞 RNA 测序分析 CD45 髓样样细胞复合物（MCC）和诱导巨噬细胞（iMac）的组成。通过两种移植实验评估 CD45 MCC 的植入能力。通过 ChIP-qPCR、启动子报告和双荧光素酶测定评估 c-Myc 对 MafB 的调节作用。通过流式细胞术和免疫荧光探索 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。
本研究报告了一种新的方法学，通过过表达 c-Myc 可将成纤维细胞快速重编程为具有吞噬活性的功能性巨噬细胞。在 iPSC 培养基中表达 c-Myc 的成纤维细胞迅速产生具有植入能力的 CD45 MCC 中间产物，并重新填充不同的造血区室。MCC 中间产物在 iPSC 培养基中稳定维持，并仅通过 M-CSF 细胞因子刺激即可连续产生功能性且高度纯的 iMac。MCC 中间产物的单细胞转录组分析表明，c-Myc 上调了巨噬细胞分化的主要调节因子 MafB 的表达，以促进巨噬细胞分化。iMac 活性的表征显示 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著增加的体内持久性和肿瘤进展抑制作用。
我们的研究结果表明，c-Myc 单独足以将成纤维细胞重编程为功能性巨噬细胞，这表明 c-Myc 重编程成纤维细胞的策略可以帮助克服获得用于癌症免疫治疗的“现成”巨噬细胞的长期障碍。
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