Intratumoral Heterogeneity Of Cervical Squamous Cell Carcinoma And Concurrent Chemoradiotherapy-induced Modulation Of Immune Microenvironment And Systematic Immunity

Objective:We used single-cell RNA sequencing（sc RNA-seq）to investigate the heterogeneity of epithelial cells and immune cells in cervical squamous cell carcinoma and concurrent chemoradiotherapy（CCRT）-induced modulation of immune microenvironment.Meanwhile,we explore the impact of CCRT on systemic immunity in patients with locally advanced cervical squamous cell carcinoma.Materials and Methods:1.Single-cell transcriptomic data of 52289 cells was obtained from cervical squamous cell carcinoma,cervical intraepithelial neoplasia（CIN）and adjacent normal cervical tissues,including 22253 cells from 4 cervical squamous cell carcinoma tissues,20892 cells from 3 CIN tissues and 9144 cells from 2 normal cervical tissues.Bioinformatics analysis was used to qualify the raw data,and then identify cell types,analyze differential genes,gene copy number variation（CNV）and gene set variation（GSVA）for epithelial cells and immune cells.2.Single-cell transcriptomic data of 21835 cells was obtained from cervical squamous cell carcinoma biopsies at times of pre-CCRT and mid-CCRT,including9912 cells from 3 pre-CCRT tissues and 11923 cells from 3 paired mid-CCRT tissues.Bioinformatics analysis was used to qualify the raw data,and then identify cell types,analyze differential genes,and the gene ontology of immune cells.Then,we further analyzed the effect of CCRT on the proportion and function of immune cells in microenvironment.3.A total of 58 patients with locally advanced cervical squamous cell carcinoma who admitted to our hospital and treated with CCRT from April 2018 to July 2019 were enrolled.The peripheral blood was collected at times of pre-,mid-and post-CCRT.Luminex x MAP was used to detect levels of 16 immune checkpoint molecules in patients’plasma.According to Response Evaluation Criteria in Solid Tumors（RECIST 1.1）,tumor response was evaluated 1 month after CCRT,including complete response（CR）,partial response（PR）,stable disease（SD）and progressive disease（PD）.The changes of levels of 16 soluble immune checkpoints induced by CCRT and the differences of levels of 16 soluble immune checkpoints between patients with CR and PR/SD were analyzed.Results:1.A total of 12 groups of epithelial cells were found,including 3 groups of normal cervical epithelial cells,3 groups of metaplastic squamous epithelial cells and6 groups of malignant squamous epithelial cells.There were differences in CNV among epithelial cell populations.The CNV of 6 groups of malignant squamous epithelial cells was significantly higher than that of 3 groups of normal epithelial cells and 3 groups of metaplastic squamous epithelial cells.GSVA analysis of 6groups of malignant squamous epithelial cells showed that there were significant differences in enriched signal pathway,immune response,stress state and metabolic pathway between them.There were 4 groups of CD8⁺T cells(CD8⁺T_cy,CD8⁺T_pre-ex,CD8⁺T_ex1,CD8⁺T_ex2),4 groups of CD4⁺T cells(CD4⁺T_naive1,CD4⁺T_naive2,CD4⁺T_reg1,CD4⁺T_reg2),2 groups of NK cells（NK1,NK2）,1 group of ILC3s,3 groups of DC cells（c DC1,c DC2,p DC）,3 groups of myeloid-derived suppressor cell（MDSC）-like macrophages（M?1-M?3）,and 4 groups of tumor-associated macrophages（M?4-M?7）.2.Three groups of CD4⁺T(CD4⁺T_naive1,CD4⁺T_naive2,CD4⁺T_reg),4 groups of CD8⁺T cells(CD8⁺T_cy,CD8⁺T_pre-ex,CD8⁺T_ex1,CD8⁺T_ex2),2 groups of NK cells（NK1,NK2）,2 groups of DC（c DC,p DC）and 8 groups of macrophages（M?1-M?8）were found in cervical squamous cell carcinoma pre-CCRT and mid-CCRT.We found that CCRT significantly increased the proportion of CD4⁺T_naive1cells（P=0.035）and decreased the proportion of CD4⁺T_regcells（P=0.043）.The proportion of CD8⁺T_pre-excells were increased with a trend（P=0.083）,while the proportion of CD8⁺T_ex1and CD8⁺T_ex2were decreased with a trend（P=0.08,0.18）;NK1 cells were significantly decreased（P=0.043）,while the proportion of NK2 cells were significantly increased（P=0.043）.However,there was no significant change in myeloid cells（P>0.05）.CCRT decreased the cytotoxic score and exhaustive score of cytotoxic CD8⁺T_cycells,CD8⁺T_ex1cells and CD8⁺T_ex2cells,while increased the cytotoxic score of NK cells and decreased the exhaustion score of CD4⁺T_regcells.3.Compared with pre-CCRT,soluble BTLA,CD28,TIM-3,CD40,LAG-3,TLR-2,GITRL,PD-1,CTLA-4,B7-1,B7-2,PD-L1 and ICOS in the peripheral blood were significantly increased post-CCRT（all P<0.05）,and most of them were gradually increased along with CCRT.We also found that CCRT increased the soluble immune checkpoint molecules in both CR and PR/SD groups.Comparing the levels of soluble immune checkpoint molecules between CR group and PR/SD group at times of pre-CCRT,mid-CCRT and post-CCRT,we found that the level of CD40 in PR/SD group was higher than that in CR group（P<0.05）at the time of mid-CCRT;the level of LAG-3 in PR/SD group was higher than that in CR group（P<0.01）at the time of post-CCRT.Conclusions:In this study,we used sc RNA-seq to investigate the heterogeneity of cervical squamous cell carcinoma,and found that there were multiple cell subsets in epithelial cells and immune cells.We further investigated CCRT-induced modulation of immune microenvironment and systematic immunity and found that CCRT changed the proportion and function of immune cell subsets in the microenvironment and the levels of immune checkpoint molecules in peripheral blood,which provide insights for the combination of CCRT and immunotherapy.