Exploring The Clinical Value And Mechanisms Of SPP1~+ Macrophages In Colorectal Cancer Based On Multi-Omics

Background:Colorectal Cancer(CRC)is a malignancy with high incidence and mortality rates globally.Macrophages in the CRC tumor microenvironment play a significant role in tumor development and metastasis.With the advancement of single-cell sequencing technologies in recent years,researchers have gained a deeper understanding of the heterogeneity of immune cell subpopulations,particularly macrophages,within the tumor microenvironment.Among them,SPP1~+macrophages have been increasingly recognized as key participants.Despite the acknowledged importance of SPP1~+macrophages,their specific functions,regulatory mechanisms,and roles in the development of CRC are not fully understood.ObjectiveThis study aims to systematically analyze the characteristics,clinical value,and functional mechanisms of SPP1~+macrophages in CRC to provide new biomarkers and potential therapeutic targets for the diagnosis and treatment of CRC.MethodsThis research employed a comprehensive multi-omics approach to systematically elucidate the role of SPP1~+macrophages in CRC.By integrating analysis of seven public CRC single-cell RNA sequencing datasets,we constructed a cellular atlas of macrophages in CRC.Using single-cell analysis,spatial transcriptomics,quantitative PCR,and immunofluorescence,among other multi-omics techniques,we systematically studied the tissue expression distribution and spatial distribution of SPP1~+macrophages.Subsequently,we assessed the relationship between SPP1~+macrophages and clinical characteristics,disease progression,and prognosis of CRC patients by combining clinical information.Finally,through functional enrichment analysis,intercellular communication analysis,Western Blot,chromatin immunoprecipitation,cellular function experiments,and animal models,we conducted an in-depth study of the biological functions of SPP1~+macrophages in CRC and their specific mechanisms in promoting tumor progression.ResultsIn terms of basic features,we identified four subgroups of macrophages in CRC tissues,including FCN1~+macrophages,C1QC~+macrophages,SPP1~+macrophages,and MKI67~+macrophages.Single-cell sequencing results show that SPP1 is highly expressed only in certain myeloid cells(i.e.,SPP1~+macrophages),and is almost not expressed in other myeloid cells,other immune cells,or non-immune cells.Multi-omics results reveal that SPP1~+macrophages are specifically enriched in both primary and liver metastatic lesions of CRC.Spatial transcriptomics and immunofluorescence results further discover that the spatial distribution of SPP1~+macrophages tends to be enriched in the core areas of the tumor.Pseudotime analysis supports the following hypothesis regarding the origin of SPP1~+macrophages:monocytes from peripheral blood are recruited to colon tissue and first differentiate into FCN1~+macrophage-like monocytes,then polarize into C1QC~+macrophages and SPP1~+macrophages,respectively.In terms of clinical value,compared to normal tissues,the proportion of SPP1~+macrophages in CRC samples is significantly increased and shows an increasing trend during the development,progression,and metastasis of the tumor,and is closely related to the poor prognosis of CRC patients.Single-cell sequencing results show that the expression of immune checkpoints PD-L1 and HLA-G in SPP1~+macrophages is significantly higher than in other macrophage subgroups.Patients with CRC with high infiltration of SPP1~+macrophages have characteristics of high genomic instability and mutations.The Sub Map algorithm also predicts that patients with CRC with high SPP1~+macrophage infiltration are more likely to benefit from immune checkpoint blockade therapy.In addition,single-cell and spatial transcriptomics results show that the expression of CSF1R in C1QC~+macrophages is significantly higher than in SPP1~+macrophages.Regarding the functional mechanism,spatial transcriptomics results show the enrichment of glycolysis and hypoxia characteristics in CRC tumor regions.Cell and animal experiments show that SPP1~+macrophages can promote CRC cell proliferation,invasion,and migration.Cell communication analysis suggests that the SPP1 protein secreted by SPP1~+macrophages may mediate interactions with other cells through the SPP1-CD44,SPP1-PTGER4,and SPP1-α4β1 axes.Finally,experimental results suggest that the upregulation of the upstream transcription factor SOX2 can promote the expression of SPP1,and the SPP1 protein can promote the expression of the downstream hypoxia-inducible factor HIF-1αin CRC cells and the glycolysis process.Conclusion1.Four macrophage subgroups can be identified in CRC tissues,including FCN1~+macrophages,C1QC~+macrophages,SPP1~+macrophages,and MKI67~+macrophages.Inflammation,phagocytosis,malignancy,and proliferation are the most significant characteristics of these four macrophage subgroups,respectively.2.In terms of tissue distribution,SPP1~+macrophages are specifically enriched in both primary and liver metastatic lesions of CRC;in terms of spatial distribution,SPP1~+macrophages tend to be enriched in the core areas of the tumor;pseudotime analysis supports the following hypothesis regarding the origin of SPP1+macrophages:monocytes from peripheral blood are recruited to colon tissue and first differentiate into FCN1~+macrophage-like monocytes,then polarize into C1QC~+macrophages and SPP1~+macrophages,respectively.3.SPP1~+macrophages can serve as a malignant biomarker for CRC diagnosis,disease assessment,and prognosis.4.SPP1~+macrophages may be involved in the formation of hypoxia and glycolysis characteristics in the CRC tumor microenvironment;SPP1~+macrophages can promote CRC cell proliferation,invasion,and migration;the increase in upstream transcription factor SOX2 can promote the expression of SPP1,and the SPP1 protein can promote the expression of HIF-1αin downstream CRC cells and the glycolysis process.5.CRC patients with high levels of SPP1~+macrophages have characteristics such as higher genomic instability and mutations and are more likely to benefit from immunotherapy;the expression of CSF1R in C1QC~+macrophages is significantly higher than in SPP1~+macrophages,suggesting that current therapies targeting CSF1R might preferentially deplete the protective C1QC~+macrophage subgroup rather than the malignant SPP1~+macrophages,which may be one of the reasons for the limited efficacy of anti-CSF1R monotherapy in cancer patients.