Table 1_Sphingosine-1-phosphate stimulates colorectal cancer tumor microenvironment angiogenesis and induces macrophage polarization via macrophage migration inhibitory factor.xlsx

BackgroundColorectal cancer (CRC) is the most common gastrointestinal malignancy with extensive reprogramming of sphingolipid metabolism. However, the role and mechanisms of sphingosine-1-phosphate (S1P), a key bioactive molecule in sphingolipid metabolism, remain insufficiently characterized. Therefore, this study integrated multi-omics data to elucidate the characteristics and functions of S1P within the tumor microenvironment (TME) and investigated its role in angiogenesis through in vitro experiments.
MethodsWe used bulk RNA sequencing data sets (RNA-seq) to study the prognostic value and clinicopathological characteristics of the increased synthesis of S1P. In order to elucidate the contribution of S1P to the complexity of the tumor microenvironment, we employed intercellular communication analysis and functional enrichment analysis at the single-cell transcriptome (scRNA-seq) level. The expression of Sphingosine kinase 1 (SPHK1) in human tissues was verified by immunohistochemical staining (IHC). Then, we inhibited the synthesis of S1P by suppressing SPHK1 at the cellular level to explore the changes in the pro-angiogenic function of tumor cells and M2-like macrophages, as well as the direction of macrophage polarization.
ResultsS1P activity is elevated in the TME of CRC, and the increased synthesis of S1P suggests poor prognosis and early metastasis. intercellular communication analysis indicates that high S1P epithelial cells can promote angiogenesis and influence the polarization of tumor-associated macrophages (TAMs) through the macrophage migration inhibitory factor (MIF) pathway. TAMs were grouped according to gene expression patterns, in which, PCLAF+ cluster TAMs showed significantly high S1P activity, contributing to tumor growth and angiogenesis. IHC demonstrated elevated levels of SPHK1 protein expression in CRC tumor tissues. Inhibition of the synthesis of S1P in tumor cells and macrophages suppressed macrophage M2 polarization levels and reversed the pro-angiogenic phenotype by inhibiting VEGFA protein expression. Spatial transcriptomics revealed a correlation between the distribution of SPHK1 and M2-like macrophage.
ConclusionsBy integrating multi-omics data and further cellular experiments, we propose that inhibition of S1P may play an important role in inhibiting angiogenesis and reversing M2-type macrophage polarization, demonstrating its anti-tumor efficacy in CRC.

研究背景：结直肠癌（Colorectal cancer, CRC）是最常见的胃肠道恶性肿瘤，其鞘脂代谢发生广泛重编程。然而，鞘脂代谢中的关键生物活性分子——1-磷酸鞘氨醇（sphingosine-1-phosphate, S1P）的作用与机制仍未得到充分阐明。因此，本研究整合多组学数据，以阐明1-磷酸鞘氨醇在肿瘤微环境（tumor microenvironment, TME）中的特征与功能，并通过体外实验探究其在血管生成中的作用。
研究方法：本研究利用批量RNA测序（bulk RNA sequencing, RNA-seq）数据集，分析1-磷酸鞘氨醇合成增加的预后价值及其临床病理特征。为阐明1-磷酸鞘氨醇对肿瘤微环境复杂性的调控贡献，我们在单细胞转录组（single-cell transcriptome, scRNA-seq）层面开展了细胞间通信分析与功能富集分析。通过免疫组织化学染色（immunohistochemical staining, IHC）验证了鞘氨醇激酶1（sphingosine kinase 1, SPHK1）在人体组织中的表达情况。随后，我们在细胞水平上抑制鞘氨醇激酶1以阻断1-磷酸鞘氨醇的合成，探究肿瘤细胞与M2样巨噬细胞的促血管生成功能变化，以及巨噬细胞极化方向的改变。
研究结果：结直肠癌肿瘤微环境中1-磷酸鞘氨醇的活性升高，且其合成增加提示患者预后不良与早期转移。细胞间通信分析显示，高表达1-磷酸鞘氨醇的上皮细胞可通过巨噬细胞迁移抑制因子（macrophage migration inhibitory factor, MIF）通路促进血管生成，并影响肿瘤相关巨噬细胞（tumor-associated macrophages, TAMs）的极化。根据基因表达模式对肿瘤相关巨噬细胞进行聚类，其中PCLAF+聚类的肿瘤相关巨噬细胞表现出显著升高的1-磷酸鞘氨醇活性，可促进肿瘤生长与血管生成。免疫组织化学染色结果证实，结直肠癌肿瘤组织中鞘氨醇激酶1的蛋白表达水平升高。抑制肿瘤细胞与巨噬细胞中1-磷酸鞘氨醇的合成，可通过抑制血管内皮生长因子A（vascular endothelial growth factor A, VEGFA）的蛋白表达，削弱巨噬细胞M2样极化水平并逆转促血管生成表型。空间转录组分析显示，鞘氨醇激酶1的分布与M2样巨噬细胞存在相关性。
研究结论：本研究通过整合多组学数据并开展进一步细胞实验，提出抑制1-磷酸鞘氨醇通路可能在抑制血管生成与逆转M2样巨噬细胞极化中发挥重要作用，证实其在结直肠癌中的抗肿瘤功效。