重塑肿瘤微环境（TME）以使癌细胞受益对于肿瘤进展至关重要。虽然弥漫型胃癌（DGC）优先与TME相互作用，但是这种复杂网络的精确机制仍不清楚。本研究旨在调查DGC进展的相互激活机制。进行了共培养巨噬细胞、非癌纤维细胞（NF）和DGC细胞的大规模细胞术分析。RNA测序用于检查纤维细胞的基因表达。DGC细胞经细胞因子处理，以检查其对特征变化的影响。 TCGA和熊本大学队列用于评估体外发现的临床相关性。队列分析揭示了DGC患者预后不良。纤维细胞和巨噬细胞与DGC细胞相互作用，在DGC组织的浸润前形成细胞簇。原始的三维共培养系统确定了非恶性细胞对DGC侵袭性生长的促进作用。我们显著地确认了一种混合极化的巨噬细胞细胞类型，该类型带有M1 / M2细胞表面标记的三维共培养系统。混合极化的巨噬细胞中的IL-1β促使NF转化为类癌相似的（CAF-like）细胞，通过诱导IL-6、IL-24和白血病抑制因子（LIF）的分泌促进了DGC细胞的恶性表型。此外，IL-6和促红细胞生成素2（GM-CSF）共同维持了混合极化巨噬细胞的稳定状态。最后，我们发现混合极化巨噬细胞在DGC组织中经常被检测到。这些发现表明，混合极化的巨噬细胞通过DGC细胞和非恶性细胞之间的相互作用存在一种新型亚型。 ©2022.作者（们）独有许可国际胃癌联合会和日本胃癌联合会使用。

Remodeling the tumor microenvironment (TME) to benefit cancer cells is crucial for tumor progression. Although diffuse-type gastric cancer (DGC) preferentially interacts with the TME, the precise mechanism of the complicated network remains unknown. This study aimed to investigate the mutual activation mechanism underlying DGC progression.Mass cytometry analysis of co-cultured macrophages, noncancerous fibroblasts (NFs), and DGC cells was performed. RNA sequencing was applied to examine gene expression in fibroblasts. DGC cells were treated with cytokines to examine their effect on characteristic changes. The TCGA and Kumamoto University cohorts were used to evaluate the clinical relevance of the in vitro findings.Cohort analysis revealed that DGC patients had a poor prognosis. The fibroblasts and macrophages interacted with DGC cells to form a cell cluster in the invasive front of DGC tissue. The original 3D triple co-culture system determined the promotional effects of nonmalignant cells on DGC invasive growth. We notably identified a mixed-polarized macrophage cell type with M1/M2 cell surface markers in a triple co-culture system. IL-1β from mixed-polarized macrophages induced the conversion of NFs to cancer-associated fibroblast-like (CAF-like) cells, promoting the malignant phenotype of DGC cells by inducing the secretion of IL-6, IL-24, and leukemia inhibitory factor (LIF). Moreover, IL-6 and colony stimulating factor 2 (GM-CSF) cooperated to maintain the stable state of mixed-polarized macrophages. Finally, we found that mixed-polarized macrophages were frequently detected in DGC tissues.These findings demonstrated that mixed-polarized macrophages exist as a novel subtype through the reciprocal interaction between DGC cells and nonmalignant cells.© 2022. The Author(s) under exclusive licence to The International Gastric Cancer Association and The Japanese Gastric Cancer Association.