Department of Orthopaedic Surgery, Shanghai Key Laboratory of Orthopaedic Implants, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200011, China.
Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC, USA.
Theranostics. 2023 Sep 4;13(14):4905-4918. doi: 10.7150/thno.86921. eCollection 2023.
The liver metastasis accompanied with the loss of liver function is one of the most common complications in patients with triple-negative breast cancers (TNBC). Lineage reprogramming, as a technique direct inducing the functional cell types from one lineage to another lineage without passing through an intermediate pluripotent stage, is promising in changing cell fates and overcoming the limitations of primary cells. However, most reprogramming techniques are derived from human fibroblasts, and whether cancer cells can be reversed into hepatocytes remains elusive. Herein, we simplify preparation of reprogramming reagents by expressing six transcriptional factors (HNF4A, FOXA2, FOXA3, ATF5, PROX1, and HNF1) from two lentiviral vectors, each expressing three factors. Then the virus was transduced into MDA-MB-231 cells to generated human induced hepatocyte-like cells (hiHeps) and single-cell sequencing was used to analyze the fate for the cells after reprogramming. Furthermore, we constructed a Liver-on-a-chip (LOC) model by bioprinting the Gelatin Methacryloyl hydrogel loaded with hepatocyte extracellular vesicles (GelMA-EV) bioink onto the microfluidic chip to assess the metastasis behavior of the reprogrammed TNBC cells under the 3D liver microenvironment . The combination of the genes , , , , and could reprogram MDA-MB-231 tumor cells into human-induced hepatocytes (hiHeps), limiting metastasis of these cells. Single-cell sequencing analysis showed that the oncogenes were significantly inhibited while the liver-specific genes were activated after lineage reprogramming. Finally, the constructed LOC model showed that the hepatic phenotypes of the reprogrammed cells could be observed, and the metastasis of embedded cancer cells could be inhibited under the liver microenvironment. Our findings demonstrate that reprogramming could be a promising method to produce hepatocytes and treat TNBC liver metastasis. And the LOC model could intimate the 3D liver microenvironment and assess the behavior of the reprogrammed TNBC cells.
三阴性乳腺癌（TNBC）患者最常见的并发症之一是肝转移伴肝功能丧失。谱系重编程是一种在不经过中间多能阶段的情况下直接将一种谱系的功能性细胞类型诱导为另一种谱系的技术，它在改变细胞命运和克服原代细胞的局限性方面具有很大的潜力。然而，大多数重编程技术都是从人成纤维细胞中衍生出来的，癌细胞是否可以逆转成肝细胞仍然难以捉摸。在这里，我们通过表达两个慢病毒载体中的六个转录因子（HNF4A、FOXA2、FOXA3、ATF5、PROX1 和 HNF1）来简化重编程试剂的制备，每个载体表达三个因子。然后将病毒转导到 MDA-MB-231 细胞中，生成人诱导的肝细胞样细胞（hiHeps），并使用单细胞测序分析细胞重编程后的命运。此外，我们通过将载有人肝细胞外囊泡（GelMA-EV）生物墨水的明胶甲基丙烯酰水凝胶生物墨水打印到微流控芯片上，构建了一个肝脏芯片（LOC）模型，以评估重编程的 TNBC 细胞在 3D 肝脏微环境下的转移行为。基因、、、、和的组合可以将 MDA-MB-231 肿瘤细胞重编程为人类诱导的肝细胞（hiHeps），限制这些细胞的转移。单细胞测序分析表明，谱系重编程后，癌基因明显受到抑制，而肝脏特异性基因被激活。最后，构建的 LOC 模型表明，在肝脏微环境下，可以观察到重编程细胞的肝表型，并抑制嵌入癌细胞的转移。我们的研究结果表明，重编程可能是一种产生肝细胞和治疗 TNBC 肝转移的有前途的方法。LOC 模型可以模拟 3D 肝脏微环境，并评估重编程的 TNBC 细胞的行为。
