Gene expression profiles of interactions between colorectal cancer-associated fibroblasts and myeloid cells in 2D co-cultures and in novel next generation triple cultures containing colorectal cancer patient-derived organoids

In this study colorectal cancer (CRC) patient-derived cancer-associated fibroblasts (CAFs) and primary monocytes or macrophages were co-cultured in 2D set-up and single-cell transcriptomics was employed to analyze the phenotypic and functional changes of the myeloid cells and CAFs upon their interaction. The results showed that CAFs instruct monocytes to gain a tumor-associated macropages (TAM)-like cells phenotype. In the next step, novel next generation tumor organoid cultures of myelid cells, CAFs and patient-derived CRC organoids (PDOs) were utilized to mimic the tumor microenvironment and study the phenotypic changes of cells under chemotherapy and oncolytic virus treatments. The sequencing data revealed that both treatments triggered distinct phenotypic changes and identified immunomodulatory effects of chemotherapy in TAM-like cells. Oncolytic virus treatment led to strong upregulation of Type I and II interferon signaling, while both therapies activated multiple cell-cell communication pathways, highlighting key molecular interactions relevant to the tumor microenvironment. This co-culture model provided insights into TAM-like responses, aiding precision cancer treatment evaluations. Co-cultures of CAFs and myeloid cells and single cultures (controls) were performed for 24h and followed by scRNA-seq. Triple cultures were established for 3 days, followed by the 24h treatment with chemotherapy (20 𝝁M oxaliplatin + 0.5 mM 5-fluorouracil), oncolytic virus (H1N1-NS1116) or mock (control). Single cells were stained with DAPI and live cells were sort purified and used for scRNA-seq. *************************************************************** Submitter states that missing raw files are due to patient privacy concerns. ***************************************************************

本研究将结直肠癌（colorectal cancer, CRC）患者来源的癌相关成纤维细胞（cancer-associated fibroblasts, CAFs）与原代单核细胞或巨噬细胞进行二维共培养，并采用单细胞转录组测序（single-cell transcriptomics）分析二者相互作用后髓系细胞与癌相关成纤维细胞的表型及功能变化。研究结果显示，癌相关成纤维细胞可诱导单核细胞获得肿瘤相关巨噬细胞（tumor-associated macrophages, TAMs）样表型。
后续研究构建了包含髓系细胞、癌相关成纤维细胞及患者来源结直肠癌类器官（patient-derived CRC organoids, PDOs）的新型下一代肿瘤类器官共培养体系，以模拟肿瘤微环境，探究细胞在化疗与溶瘤病毒处理后的表型变化。测序数据表明，两种处理方式均引发了显著的表型改变，并揭示了化疗对肿瘤相关巨噬细胞样细胞的免疫调节作用。溶瘤病毒处理可强烈上调I型与II型干扰素信号通路，而两种疗法均激活了多条细胞间通信通路，凸显了与肿瘤微环境相关的关键分子互作机制。
该共培养模型为肿瘤相关巨噬细胞样细胞应答提供了研究视角，有助于精准癌症治疗的评估。
将癌相关成纤维细胞与髓系细胞的共培养体系以及单独培养的对照组均培养24小时，随后进行单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq）。构建三细胞共培养体系并培养3天，随后分别用化疗药物（20 μM奥沙利铂+0.5 mM 5-氟尿嘧啶）、溶瘤病毒（H1N1-NS1116）或空白对照（mock）处理24小时。使用DAPI对单细胞进行染色，分选纯化活细胞后用于单细胞RNA测序。
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提交者声明，原始数据文件缺失系因患者隐私保护相关问题。
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