Re-defining the sphingosine rheostat as a stromal-immune program associated with immune suppression in metastatic colorectal cancer

Background Thirty years ago, the “sphingosine rheostat” model proposed a pro-survival/pro-apoptotic role for sphingosine-1-phosphate/ceramide in tumor cells. In cancer, ceramide-derived sphingosine-1-phosphate increases, raising the sphingosine-1-phosphate/ceramide ratio, whereas in normal tissue ceramide synthesis predominates. We hypothesize that the rheostat presents more relevance in the context of immune and stromal components rather than tumor epithelial cells and may govern both inflammation and immunosuppression. Methods Quantitative liquid chromatography–tandem mass spectrometry analysis of the sphingosine-1-phosphate/ceramide pathway was performed in resected primary ( n = 480) and in metastatic colorectal cancer tumors ( n = 94). An integrative multi-omics approach combined high-throughput quantitative real time PCR, targeted lipidomics, a large gene-expression dataset ( n = 2,373), clinical data, standard as well as high-definition spatial transcriptomics, and public single cell RNA-sequencing. Results Metastatic colorectal cancer specimens showed enrichment in sphingosine-1-phosphate and sphinganine-1-phosphate, whereas ceramide (24:1) was decreased. Gene-expression analysis and quantitative real time PCR confirmed increased sphingosine-1-phosphate and sphinganine-1-phosphate synthesizing enzymes (SPHK1) and receptors S1PR3-5 in metastatic colorectal cancer vs. primary colorectal cancer. Cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) and public single cell RNAseq data analysis showed SPHK1/S1PR3 expression was predominant in stromal, and immune tumor microenvironment cells, whereas SPHK2 predominated in epithelial cells. A three-gene sphingosine-1-phosphate signature was associated with stromal enrichment, myeloid derived suppressor cells, M2 macrophages, advanced stage, poor survival, nodal infiltration and weak to negative correlations with validated checkpoint inhibitor response scores. Conclusions Metastatic colorectal cancer lesions display a sphingosine-1-phosphate /ceramide imbalance with negative associations to patient prognosis and checkpoint inhibitor response gene-signatures. These findings reposition the rheostat as primarily active in the immunosuppressive tumor microenvironment rather than in tumor cells. Our findings warrant the exploration of new therapeutic strategies potentially rebalancing the sphingosine-1-phosphate/ceramide ratio, reprograming the immunosuppressive tumor microenvironment and enhancing immunotherapy responses, thereby addressing an unmet need in colorectal cancer treatment.