Characteristics Of Tumor Microenvironment In Small Cell Lung Cancer And Its Role In Function And Mechanism Of Chemotherapy Resistance

PurposeSmall cell lung cancer(SCLC),a highly invasive neuroendocrine tumor,accounts for about 15% of all lung cancers(LC).It is characterized by rapid growth,low differentiation,and occurrence with local or distant metastasis at early stage.Additionally,SCLC is sensitive to initial chemotherapy,but it is easy to develop chemotherapy drug resistance.When SCLC is resistant to different drugs,it will lead to the phenomenon of no drug available,contributing to poor prognosis of SCLC patients.In view of the clinical characteristics of SCLC,this project will explore the characteristics of SCLC tumor tissue microenvironment and its role in function and mechanism of chemotherapy resistance from multiple perspectives.According to the World Health Organization(WHO)classification of LC,SCLC can be divided into two subtypes: combined SCLC(CSCLC)and pure SCLC(PSCLC).CSCLC mainly consists of SCLC and non-small cell lung cancer(NSCLC)components,while NSCLC components may include one or more different tumors.In order to comprehensively analyze the microenvironment characteristics of SCLC tumor tissues,this study will first explore the genomic characteristics of CSCLC and PSCLC tumor tissues,hoping to analyze the characteristics of gene mutations in CSCLC and PSCLC tumor tissues and the characteristics of gene mutations in different components of CSCLC.This study may provide a new perspective for exploring the development and occurrence of SCLC.To further analyze the influence of microenvironment characteristics of SCLC tumor tissue on chemotherapy resistance,single-cell transcriptome sequencing will be used in this project to explore the effect of abnormal gene expression and its activation on chemotherapy resistance at the single-cell level.This study is expected to explain the new potential mechanism of chemotherapy resistance in SCLC.The microenvironment characteristics of SCLC tumor tissues are closely related to the chemotherapy resistance.However,the impact of tumor immune microenvironment on immunotherapy is still unclear.In comparison with the tumor immune microenvironment in NSCLC,this study will further reveal the characteristics of tumor immune microenvironment in SCLC,and provide potential targets for the exploration of SCLC treatment.MethodCurrent study can be divided into threeparts: In the first part,CSCLC and PSCLC samples were screened from our hospital,and tissue separation was performed by hematoxylin-eosin staining,immunohistochemical staining and microdissection.Then,targeted DNA sequencing and bioinformatics were used to analyze the mutation characteristics of CSCLC and PSCLC genes as well as the gene mutation characteristics between different components of CSCLC.In the second part,SCLC samples were collected first,and single-cell suspension preparation and single-cell transcriptomic sequencing were then performed.Subsequently,analysis between drug-resistant patients and non-drug-resistant patients was done by bioinformatics.Moreover,gene transfection,q RT-PCR,western blot and CCK8 experiments were used to explore the mechanism of SCLC chemotherapy resistance.In the third part,single-cell transcriptome sequencing data of NSCLC were collected,meanwhile whole exon sequencing(WES)was performed on SCLC samples.Analysis of single-cell transcriptome sequencing data of NSCLC and SCLC mentioned above was explored via bioinformatics.Additionally,multi-target immunofluorescence staining and WES results were applied to explore the characteristics of tumor immune microenvironment in SCLC and the differences between SCLC and NSCLC.Result1.For SCLC patients who underwent surgical treatment,the prognosis of patients with PSCLC is better than that of patients with CSCLC;TP53 and RB1 mutations were common in both CSCLC and PSCLC,but the common mutations were few and the mutant genes were different.In CSCLC,the proportion of common mutations in NSCLC and SCLC components was more than 50%,among which TP53 and RB1 were the most common mutations.There were specific genetic changes in different NSCLC components of CSCLC,such as TERT,EGFR,NKX2-1 and SDHA genetic mutation in adenocarcinoma(AD),and SOX2,CDKN2 A and FAM135 B genetic mutation in squamous cell carcinoma(SCC).These results suggested that there were widespread gene mutations in the microenvironment of SCLC tumor tissue,which was responsible for the occurrence and development of SCLC.2.Single-cell transcriptome sequencing results showed that chemotherapyresistant SCLC tissues were enriched in WNT,EMT and TGF-β pathways,and FZD8,EDIL3,FOS and other genes were significantly differential expressed.Via preliminary screening,it was found that down-regulated expression of FZD8 could improve the chemotherapy sensitivity of SCLC,indicating that chemotherapy resistance of SCLC was closely related to WNT signaling pathway.It is further demonstrated that the expression and activation of abnormal genes in the tumor microenvironment of SCLC may lead to the formation of chemotherapy resistance in SCLC.3.Single-cell transcriptome sequencing,WES and public database results indicated that there were few mutations of MHC-Ⅰ related genes in SCLC tumors,and the level of MHC-Ⅰ related genes in SCLC cell lines was significantly lower than that in NSCLC cell lines.Compared with NSCLC,cell cycle pathways were more active in SCLC,but immune-related pathways were less active in SCLC.Compared with the tumor immune microenvironment in NSCLC,exhausted CD8+T cells were less enriched in SCLC,yet there were more TGFBR2+NK cells with impaired cytotoxicity in SCLC.Above results suggested that SCLC had immunosuppressive tumor microenvironment.ConclusionIn the microenvironment of SCLC tumor tissue,there were widespread gene mutations.Moreover,it was suggested that NSCLC and SCLC components in CSCLC may originate from the same pluripotent single clone.Additionally,abnormal expression and activation of FZD8 in WNT signaling pathway was closely related to chemotherapy resistance in SCLC tumor tissue microenvironment.Furthermore,compared with NSCLC,the microenvironment of SCLC tumor tissue had more active cell cycle pathways,and lower active immune-related pathways.Moreover,SCLC owned immunosuppressive tumor microenvironment.