Cervical cancer (CC) is the fourth leading cause of deaths in gynecological malignancies. Although the etiology of CC has been extensively investigated, the exact pathogenesis of CC remains incomplete. Recently, single-cell technologies demonstrated advantages in exploring intra-tumoral diversification among various tumor cells. However, single-cell transcriptome analysis (single-cell RNA sequencing [scRNA-seq]) of CC cells and microenvironment has not been conducted. In this study, a total of 20,938 cells from CC and adjacent normal tissues were examined by scRNA-seq. We identified four tumor cell subpopulations in tumor cells, which had specific signature genes with different biological functions and presented different prognoses. Among them, we identified a subset of cancer stem cells (CSCs) that was related to the developmental hierarchy of tumor progression. Then, we compared the expressive differences between tumor-derived endothelial cells (TECs) and normal ECs (NECs) and revealed higher expression of several metabolism-related genes in TECs. Then, we explored the potential biological function of ECs in vascularization and found several marker genes, which played a prior role in connections between cancer cells and ECs. Our findings provide valuable resources for deciphering the intra-tumoral heterogeneity of CC and uncover the developmental procedure of ECs, which paves the way for CC therapy.

Immune cells are essential for defending the body's balance and have increasingly been implicated in controlling tumor growth. In cervical cancer (CC), the immune landscape is extensively connected with human papillomavirus (HPV) status. Recent insights from studies have revealed that as a result of infection with HPV, immune cell populations such as lymphocytes or monocytes change during carcinogenesis. Immune therapy, in particular checkpoint inhibitors, those targeting PD-1 or PD-L1, has shown promising efficacy. This article reviews the immune landscape and immunotherapy of CC.

No abstract

Cervical cancer (CC) is one of the most common malignancy in women worldwide. It is characterized by a natural continuous phenomenon, that is, it is in the initial stage of HPV infection, progresses to intraepithelial neoplasia, and then develops into invasion and metastasis. Determining the complexity of tumor microenvironment (TME) can deepen our understanding of lesion progression and provide novel therapeutic strategies for CC. We performed the single-cell RNA sequencing on the normal cervix, intraepithelial neoplasia, primary tumor and metastatic lymph node tissues to describe the composition, lineage, and functional status of immune cells and mesenchymal cells at different stages of CC progression. A total of 59913 single cells were obtained and divided into 9 cellular clusters, including immune cells (T/NK cells, macrophages, B cells, plasma cells, mast cells and neutrophils) and mesenchymal cells (endothelial cells, smooth muscle cells and fibroblasts). Our results showed that there were distinct cell subpopulations in different stages of CC. High-stage intraepithelial neoplasia (HSIL) tissue exhibited a low, recently activated TME, and it was characterized by high infiltration of tissue-resident CD8 T cell, effector NK cells, Treg, DC1, pDC, and M1-like macrophages. Tumor tissue displayed high enrichment of exhausted CD8 T cells, resident NK cells and M2-like macrophages, suggesting immunosuppressive TME. Metastatic lymph node consisted of naive T cell, central memory T cell, circling NK cells, cytotoxic CD8+ T cells and effector memory CD8 T cells, suggesting an early activated phase of immune response. This study is the first to delineate the transcriptome profile of immune cells during CC progression using single-cell RNA sequencing. Our results indicated that HSIL exhibited a low, recently activated TME, tumor displayed immunosuppressive statue, and metastatic lymph node showed early activated phase of immune response. Our study enhanced the understanding of dynamic change of TME during CC progression and has implications for the development of novel treatments to inhibit the initiation and progression of CC.

BackgroundNovel therapies are needed to treat recurrent and advanced cervical cancer (CC), as their prognosis remains very poor. Although therapies targeting the programmed cell death protein 1 (PD-1) pathway have been approved for CC, a large subset of patients exhibit innate resistance. Using checkpoint inhibitors in combination could enhance their efficacy.MethodsBlood samples, tumor specimens, and peritumorous (PT) tissues were obtained from patients with CC. The inhibitory receptor expression and phenotypical analysis of CD8+ T cells in CC specimens were analyzed by flow cytometry. The ligands of CD96 expressed by tumor cells were measured by immunohistochemistry and immunofluorescence. Sensitivity to pembrolizumab was evaluated by an ex vivo treatment assay based on the single-cell culture of CC specimens. The efficacies of PD-1 and/or CD96 blockades were explored using an ex vivo treatment assay and an human papillomavirus-positive TC-1 xenograft mouse model in vivo.ResultsWe found that CD96 expression was elevated on CD8+ tumor-infiltrating lymphocytes (TILs) from patients with CC who were insensitive to the PD-1 blockade. These CD96-expressing CD8+ TILs often coexpressed PD-1. The ratio of the CD96+CD8+/CD96−CD8+ T-cell gene signature from the scRNA-seq data was significantly associated with the poor survival of patients with cervical squamous cell carcinoma and endocervical adenocarcinoma. The costimulatory receptor CD226, which competes with CD96, was downregulated in tumors compared with blood and PT tissue. CD96 and T-cell immunoreceptor with Ig and ITIM domains (TIGIT) were upregulated on intratumoral CD8+ T cells. The CD226/CD96/TIGIT signaling ligands were widely expressed in CC tumor tissues. Phenotypical profiling showed that PD-1+CD96+CD8+ TILs exhibited a terminally exhausted effector phenotype with high levels of T-cell immunoglobulin mucin receptor 3 (TIM-3) and granzyme B (GZMB) and extremely low levels of proinflammatory cytokines and cytotoxic molecules. PD-1+CD96 cells exhibited a precursor exhausted phenotype with TCF-1 positivity. CD96 was further upregulated by CD8+ TILs on PD-1 blockade. Treatment with the CD96 blockade significantly enhanced the PD-1 blockade to blunt tumor growth and improve the function of CD8+ TILs in both mouse and CC specimen models.ConclusionsOur findings showed that CD96 and PD-1 cooperatively and negatively regulate the function of CD8+ TILs, and CD96 blockade has promise for use in combination with PD-1 blockade for the treatment of CC.