AbstractChimeric antigen receptor (CAR) T-cell therapies have shown remarkable results in patients with hematological malignancies. However, their success in treating solid tumors has been limited. As an alternative candidate for the CAR therapy, CAR-macrophages (CAR-M) have demonstrated activation and phagocytosis directed by tumor-associated antigen (TAA), showing promise in the treatment of solid tumors.

摘要嵌合抗原受体（CAR）T细胞疗法在血液系统恶性肿瘤患者中显示出显着的效果。然而，他们在治疗实体瘤方面的成功是有限的。作为CAR治疗的替代候选者，CAR巨噬细胞（CAR-M）已经证明由肿瘤相关抗原（TAA）指导的活化和吞噬作用，在实体瘤的治疗中显示出前景。

Nevertheless, the mechanisms by which CARs direct tumor chemotaxis and invasion of CAR-M remain poorly understood. In this study, we aim to investigate the role of CARs in CAR-M attachment and infiltration using 3D tumor spheroids, which were created by utilizing a novel self-assembling nucleic acid nanostructure decorated living cells (NAC).

然而，CAR指导肿瘤趋化性和CAR-M侵袭的机制仍然知之甚少。在这项研究中，我们旨在研究CAR在使用3D肿瘤球体的CAR-M附着和浸润中的作用，3D肿瘤球体是通过利用新型自组装核酸纳米结构修饰的活细胞（NAC）产生的。

Our results demonstrated that CAR-M exhibited higher invasion and killing capacity in 2D model and 3D tumor spheroids. In summary, the 3D NAC assembled tumor spheroid model provides a suitable platform for target screening and pharmacodynamic evaluation of CAR-M..

我们的研究结果表明，CAR-M在2D模型和3D肿瘤球体中表现出更高的侵袭和杀伤能力。总之，3D NAC组装的肿瘤球体模型为CAR-M的靶标筛选和药效学评估提供了合适的平台。。

IntroductionIn recent years, immunotherapy has revolutionized cancer treatment. CAR-T, as the first personalized cellular therapeutic strategy, has successfully been employed in treating refractory hematologic malignancies, particularly targeting CD191,2. However, significant challenges persist in treating solid tumors, such as low infiltration and high heterogeneity in the tumor microenvironments (TME)3.

引言近年来，免疫疗法彻底改变了癌症治疗。CAR-T作为第一种个性化的细胞治疗策略，已成功用于治疗难治性血液系统恶性肿瘤，特别是靶向CD191,2。然而，治疗实体瘤仍然存在重大挑战，例如肿瘤微环境（TME）中的低浸润和高异质性3。

To address this, CAR-NK (Natural Killer) cell therapy has emerged as a promising alternative strategy, demonstrating efficacy in targeting solid tumor antigens in clinical trials4,5,6. Another alternative strategy is CAR-M (CAR-macrophage) therapy, which offers advantages in immune cell trafficking and infiltration7,8.

为了解决这个问题，CAR-NK（自然杀伤）细胞疗法已经成为一种有前途的替代策略，在临床试验中证明了靶向实体瘤抗原的功效4,5,6。另一种替代策略是CAR-M（CAR巨噬细胞）疗法，它在免疫细胞运输和浸润方面具有优势7,8。

For instance, Michael Klichinsky and colleagues delivered CAR structures into human primary macrophages using adenovirus transduction, resulting in a distinctive phagocytic effect, enhanced expression of proinflammatory factors, and cross-presentation of tumor antigens to bystander T cells in vivo9.Other studies have focused on enhancing phagocytosis to improve the anti-tumor activity of CAR-macrophages10,11,12.

例如，Michael Klichinsky及其同事使用腺病毒转导将CAR结构传递到人原代巨噬细胞中，产生独特的吞噬作用，增强促炎因子的表达，并在体内将肿瘤抗原交叉呈递给旁观者T细胞9。其他研究集中在增强吞噬作用以改善CAR-巨噬细胞的抗肿瘤活性10,11,12。

This includes combining anti-CD47 antibodies to disrupt CD47-Sirpα axis interaction and enhance phagocytosis11, optimizing intracellular PI3K-signaling/Megf7 domain11 and MerTK12, and using exogenous chemicals to induce a proinflammatory M1 phenotype in CAR-M, thereby enhancing phagocytosis, tumor killing capability, and cytokine release on target cells13.

这包括结合抗CD47抗体以破坏CD47-Sirpα轴相互作用并增强吞噬作用11，优化细胞内PI3K信号传导/Megf7结构域11和MerTK12，以及使用外源性化学物质诱导CAR-M中的促炎M1表型，从而增强吞噬作用，肿瘤杀伤能力和靶细胞上的细胞因子释放13。

In summary, CAR-macrophages show promise as a potential treatment for solid tumors.Animal tumor models are commonly utilized for evaluating the efficacy and safety of cell therapies in a preclinical setting. However, these models are time-consuming, expensive, and raise ethical concerns. As an .

总之，CAR巨噬细胞有望成为实体瘤的潜在治疗方法。动物肿瘤模型通常用于评估临床前环境中细胞疗法的功效和安全性。然而，这些模型耗时，昂贵，并引起道德问题。作为一个。

Data availability

数据可用性

All data are available in the main text and supplementary file. All the source data is provided in Supplementary Information.

所有数据均可在正文和补充文件中找到。所有源数据均在补充信息中提供。

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20240207010CX).Author informationAuthor notesThese authors contributed equally: Qinyao Zhu, Xiaofang He, Junhua Liu, Heming Wang.Authors and AffiliationsRocRock Biotechnology Co. Ltd, Suzhou, ChinaQinyao Zhu, Junhua Liu, Xiaojiao Shan & Xiushan YinJoint Laboratory of Biomaterials and Translational Medicine, Puheng Technology Co., Ltd, Suzhou, ChinaXiaofang He, Heming Wang & Guangqi SongResearch Center of Bioengineering, Medical Innovation Research Division of Chinese PLA General Hospital, Beijing, ChinaLuo ZhangChina-Japan Union Hospital of Jilin University, Changchun, ChinaYicheng ZhaoState Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun, ChinaYicheng ZhaoNHC Key Laboratory of Biotechnology Drugs (Shandong Academy of Medical Sciences), Biomedical Sciences College, Shandong First Medical University, Jinan, ChinaXiushan YinAuthorsQinyao ZhuView author publicationsYou can also search for this author in.

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PubMed Google ScholarContributionsX.Y., Y.Z., G.S., and L.Z. conceived and designed the experiments. Q.Z., X.H., J.L., H.W., and X.S performed the experiments. Q.Z., X.H., and J.L. analyzed the data. Q.Z., X.H., and H.W. wrote the manuscript. X.Y. and Y.Z. supervised the research project.Corresponding authorsCorrespondence to.

PubMed谷歌学术贡献x。Y、 ，Y.Z.，G.S。和L.Z.构思并设计了实验。Q、 Z.，X.H.，J.L.，H.W。和X.S进行了实验。Q、 Z.，X.H。和J.L.分析了数据。Q、 Z.，X.H。和H.W.写了手稿。十、 Y.和Y.Z.监督了这项研究项目。通讯作者通讯。

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Reprints and permissionsAbout this articleCite this articleZhu, Q., He, X., Liu, J. et al. DNA origami assembled spheroid for evaluating cytotoxicity and infiltration of chimeric antigen receptor macrophage (CAR-M).

转载和许可本文引用本文Zhu，Q.，He，X.，Liu，J。等人。DNA折纸组装球体用于评估嵌合抗原受体巨噬细胞（CAR-M）的细胞毒性和浸润。

Commun Biol 7, 1302 (2024). https://doi.org/10.1038/s42003-024-07009-4Download citationReceived: 16 January 2024Accepted: 03 October 2024Published: 11 October 2024DOI: https://doi.org/10.1038/s42003-024-07009-4Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

Commun Biol 71302（2024）。https://doi.org/10.1038/s42003-024-07009-4Download引文接收日期：2024年1月16日接受日期：2024年10月3日发布日期：2024年10月11日OI：https://doi.org/10.1038/s42003-024-07009-4Share本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。复制到剪贴板。

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