Relationship between cytomegalovirus infection and glioblastoma immune microenvironment
Objective To analyze the transcriptome maps of cytomegalovirus(CMV)-infected glioblastoma tissue using single-cell transcriptome sequencing technology,and to explore the relationship between CMV infection and glioblastoma immune microenvironment.Methods Twenty patients with glioblastoma were diagnosed and treated in the Second Affiliated Hospital of Xinjiang Medical University from December 2022 to August 2023.The glioblastoma tissue was collected and the presence of CMV particles and genetic materials in glioblastoma tissue was identified by colloidal gold immunoelectron microscopy and droplet digital PCR.The viral copy number was compared between CMV-positive and CMV-negative glioblastoma tissues.Single-cell transcriptome sequencing technology was used to analyze the transcriptome maps of 6 CMV-positive and 3 CMV-negative glioblastoma patients,and to identify the immune cell subset co-expressing macrophage marker and tumor cell marker.Immunofluorescence staining and flow cytometry were used to validate this immune cell subset.The differentially expressed genes and involved signaling pathways were analyzed.Results The results of colloidal gold immunoelectron microscopy showed that there were few gold-labeled electron-dense granules in CMV-positive tissue,which were consistent with the morphology of CMV particles.The results of droplet digital PCR showed that there were 12 CMV-positive and 8 CMV-negative patients.The viral copy number of CMV-positive tissue[(150±30)copies/mL]was higher than that of CMV-negative tissue[(10±5)copies/mL](t=7.200,P＜0.001).Single-cell transcriptome sequencing identified 6 major cell types in both CMV-positive and CMV-negative tissues,including tumor cell,macrophage,oligodendrocyte,endothelial cell,stromal cell and T lymphocyte,as well as a novel kind of immune cell subset,namely double-positive tumor-associated macrophage(TAM)co-expressing macrophage marker CD68 and tumor cell marker SOX2(CD68+SOX2+TAM).The results of immunofluorescence staining showed that there was a co-expression of CD68 and SOX2 in CMV-positive tissue,while the co-expression of CD68 and SOX2 was not obvious in CMV-negative tissue.The results of flow cytometry showed that CD68+SOX2+TAM was enriched in CMV-positive tissue.The percentage of CD68+SOX2+TAM in CMV-positive tissue[(4.86±0.25)％]was higher than that in CMV-negative tissue[(0.94±0.41)％](t=8.160,P＜0.001).The results of function analysis showed that the differentially expressed genes of CD68+SOX2+TAM in CMV-positive and CMV-negative tissues were mainly associated with CMV infection,involving signaling pathways of the interaction of viral proteins with cytokines and their receptors.Conclusion CMV infection may exist in patients with glioblastoma,and it can induce the production of double-positive TAM co-expressing macrophage marker and tumor cell marker(CD68+SOX2+TAM),remodeling the glioblastoma immune microenvironment.
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