Single-Cell and Spatial Transcriptomics Revealing the Role of IDO1 in HPV+ Cervical Cancer Tumor Immune Microenvironment and Its Implications in Radiotherapy and Immunotherapy

Purpose/Objective(s)Persistent infection of human papillomavirus (HPV) is one major etiology of cervical cancer (CC). By now, anti-PD-1 immunotherapy is approved for advanced CC patients, but the response rate was just about 10-20%, tumor immune microenvironment (TIME) might be one factor that affect the efficacy. The indoleamine 2,3-dioxygenase (IDO), a metabolic immune checkpoint, is recently shown to have a correlation-ship with HPV carcinogenesis in CC, with unknown mechanism. This study, using the single cell transcriptomic single-cell sequencing and spatial transcription sequencing analysis/immunologic technology, aimed to exam the role of IDO1 expression in HPV+ CC TIME and explore the changes after radiotherapy.Materials/MethodsNewly diagnosed advanced HPV- CC and HPV+ CC patients were tested for the tumor and tumor immune microenvironment (TIME) heterogeneity and their changes after fractionated radiation therapy. Tumor tissues were collected, single cell suspension was made for Single-cell RNA sequencing (SCRNAseq) using the 10 × Genomics, while frozen tissue was embedded for spatial transcriptome sequencing (STRNAseq). Seurat 4.0 was used to cluster and annotate cell clusters and map SCRNAseq data to the STRNAseq data. The specific characters of cell clusters were computed by Gene Set Enrichment Analysis (GSEA). SPOTLight and CellChat were used to analyze cell location and interaction respectively.ResultsA total of 28631 cells were clustered into 31 cell subsets in HPV- CC and HPV+ CC tissues, including baseline (Pre HPV- CC and Pre HPV+ CC) and 3-week after radiotherapy (Post 3w HPV- CC and Post 3w HPV+CC). There were 10431 epithelial cells (Epi) in all these 4 tumor tissues, with heterogenous IDO1 expression, including IDO1-high Epi, IDO1-low Epi, and IDO1-neg Epi. Interestingly, more than 99% of Epi in Pre HPV- CC tissues were IDO1-neg cells, while more than 99% in Pre HPV+ CC tissue were IDO1-high. Furthermore, the proportion of IDO1-high Epi in Pre HPV+ CC patient dropped to 16.7% after radiotherapy, while the proportion of IDO1-low Epi rase to 63.3%. Using GSEA, the characters of IDO1-high Epi group was shown to have positive regulation of leukocyte chemotaxis and negative regulation of cell adhesion and differentiation. IDO1-high Epi cells also had the hallmark of interferon gamma response. These cells could mainly receive regulative information of interferon gamma pathway from exhausted CD8 T cells, which could affect the apoptosis of tumor cells.ConclusionThis study comprehensively analyzed the immune suppressive role of IDO1-high Epi cells in HPV+ CC TIME at the single-cell transcriptional scale and explored their functional characters in CC radiotherapy. This would be able to provide more evidence to combine with radiotherapy and immunotherapy to improve patients’ prognosis. Persistent infection of human papillomavirus (HPV) is one major etiology of cervical cancer (CC). By now, anti-PD-1 immunotherapy is approved for advanced CC patients, but the response rate was just about 10-20%, tumor immune microenvironment (TIME) might be one factor that affect the efficacy. The indoleamine 2,3-dioxygenase (IDO), a metabolic immune checkpoint, is recently shown to have a correlation-ship with HPV carcinogenesis in CC, with unknown mechanism. This study, using the single cell transcriptomic single-cell sequencing and spatial transcription sequencing analysis/immunologic technology, aimed to exam the role of IDO1 expression in HPV+ CC TIME and explore the changes after radiotherapy. Newly diagnosed advanced HPV- CC and HPV+ CC patients were tested for the tumor and tumor immune microenvironment (TIME) heterogeneity and their changes after fractionated radiation therapy. Tumor tissues were collected, single cell suspension was made for Single-cell RNA sequencing (SCRNAseq) using the 10 × Genomics, while frozen tissue was embedded for spatial transcriptome sequencing (STRNAseq). Seurat 4.0 was used to cluster and annotate cell clusters and map SCRNAseq data to the STRNAseq data. The specific characters of cell clusters were computed by Gene Set Enrichment Analysis (GSEA). SPOTLight and CellChat were used to analyze cell location and interaction respectively. A total of 28631 cells were clustered into 31 cell subsets in HPV- CC and HPV+ CC tissues, including baseline (Pre HPV- CC and Pre HPV+ CC) and 3-week after radiotherapy (Post 3w HPV- CC and Post 3w HPV+CC). There were 10431 epithelial cells (Epi) in all these 4 tumor tissues, with heterogenous IDO1 expression, including IDO1-high Epi, IDO1-low Epi, and IDO1-neg Epi. Interestingly, more than 99% of Epi in Pre HPV- CC tissues were IDO1-neg cells, while more than 99% in Pre HPV+ CC tissue were IDO1-high. Furthermore, the proportion of IDO1-high Epi in Pre HPV+ CC patient dropped to 16.7% after radiotherapy, while the proportion of IDO1-low Epi rase to 63.3%. Using GSEA, the characters of IDO1-high Epi group was shown to have positive regulation of leukocyte chemotaxis and negative regulation of cell adhesion and differentiation. IDO1-high Epi cells also had the hallmark of interferon gamma response. These cells could mainly receive regulative information of interferon gamma pathway from exhausted CD8 T cells, which could affect the apoptosis of tumor cells. This study comprehensively analyzed the immune suppressive role of IDO1-high Epi cells in HPV+ CC TIME at the single-cell transcriptional scale and explored their functional characters in CC radiotherapy. This would be able to provide more evidence to combine with radiotherapy and immunotherapy to improve patients’ prognosis.