间充质基质细胞 (MSC) 对肿瘤的趋向性使其可用作抗肿瘤因子的载体，而体外转录 mRNA (IVT mRNA) 是一种有前景的有效瞬时表达工具，无插入突变风险。粒细胞巨噬细胞集落刺激因子（GM-CSF）是一种通过刺激特异性免疫反应而具有抗肿瘤特性的细胞因子。这项工作的目的是通过 IVT mRNA 转染生成修饰的 MSC，以过表达 GM-CSF，并确定其单独或与阿霉素 (Dox) 联合在肝细胞癌 (HCC) 小鼠模型中的治疗效果。DsRed 或 GM-CSF IVT mRNA 是从用特异性引物设计的 cDNA 模板生成的，然后进行逆转录。使用 Lipofectamine 用 DsRed (MSC/DsRed) 或 GM-CSF IVT mRNA (MSC/GM-CSF) 转染 MSC。通过流式细胞术测定基因表达和细胞表面标记。通过ELISA测定GM-CSF分泌。在体外实验中，使用来自小鼠的 J774 巨噬细胞系和骨髓单核细胞来测试 GM-CSF 功能。通过将 Hepa129 细胞皮下接种 (s.c.) 到 C3H/HeN 小鼠中来建立 HCC 模型。 s.c.之后评估了注射 MSC/GM-CSF、Dox 或其组合、肿瘤大小和小鼠存活率。收集肿瘤样本进行 mRNA 分析和流式细胞术。 IVT mRNA 转染后 2 小时至 15 天观察 MSC 的 DsRed 表达。在 HCC 小鼠模型中施用表达 DsRed 的 MSC 后，肿瘤生长保持不变，并且表达 GM-CSF 的 MSC 保持其表型特征和迁移能力。改良的 MSC 分泌的 GM-CSF 诱导小鼠单核细胞分化为树突状细胞，并促进 J774 巨噬细胞系的促炎表型。在体内，与单独治疗相比，MSC/GM-CSF 与 Dox 组合可显着降低 C3H/HeN 小鼠的 HCC 肿瘤生长，并延长小鼠的生存期。此外，MSC/GM-CSF  Dox治疗组的肿瘤表现出促炎基因表达升高，CD8  T细胞和巨噬细胞浸润增加。我们的结果表明，IVT mRNA转染是获得用于治疗目的的修饰MSC的合适策略。 MSC/GM-CSF 与低剂量的 Dox 相结合，通过增加促炎性肿瘤微环境，增强 HCC 的抗肿瘤反应，产生协同效应。© 2024。作者。

Mesenchymal stromal cells (MSCs) tropism for tumours allows their use as carriers of antitumoural factors and in vitro transcribed mRNA (IVT mRNA) is a promising tool for effective transient expression without insertional mutagenesis risk. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with antitumor properties by stimulating the specific immune response. The aim of this work was to generate modified MSCs by IVT mRNA transfection to overexpress GM-CSF and determine their therapeutic effect alone or in combination with doxorubicin (Dox) in a murine model of hepatocellular carcinoma (HCC).DsRed or GM-CSF IVT mRNAs were generated from a cDNA template designed with specific primers followed by reverse transcription. Lipofectamine was used to transfect MSCs with DsRed (MSC/DsRed) or GM-CSF IVT mRNA (MSC/GM-CSF). Gene expression and cell surface markers were determined by flow cytometry. GM-CSF secretion was determined by ELISA. For in vitro experiments, the J774 macrophage line and bone marrow monocytes from mice were used to test GM-CSF function. An HCC model was developed by subcutaneous inoculation (s.c.) of Hepa129 cells into C3H/HeN mice. After s.c. injection of MSC/GM-CSF, Dox, or their combination, tumour size and mouse survival were evaluated. Tumour samples were collected for mRNA analysis and flow cytometry.DsRed expression by MSCs was observed from 2 h to 15 days after IVT mRNA transfection. Tumour growth remained unaltered after the administration of DsRed-expressing MSCs in a murine model of HCC and MSCs expressing GM-CSF maintained their phenotypic characteristic and migration capability. GM-CSF secreted by modified MSCs induced the differentiation of murine monocytes to dendritic cells and promoted a proinflammatory phenotype in the J774 macrophage cell line. In vivo, MSC/GM-CSF in combination with Dox strongly reduced HCC tumour growth in C3H/HeN mice and extended mouse survival in comparison with individual treatments. In addition, the tumours in the MSC/GM-CSF + Dox treated group exhibited elevated expression of proinflammatory genes and increased infiltration of CD8 + T cells and macrophages.Our results showed that IVT mRNA transfection is a suitable strategy for obtaining modified MSCs for therapeutic purposes. MSC/GM-CSF in combination with low doses of Dox led to a synergistic effect by increasing the proinflammatory tumour microenvironment, enhancing the antitumoural response in HCC.© 2024. The Author(s).