The Study About The Heterogeneity Of Neuroblastoma Through Single-Cell Transcriptome Sequencing

Background:Neuroblastoma(NB)is highly heterogeneous,and the prognosis is related to the age of diagnosis,tumor stage,histological type,and genetic changes.Some children with stage IVs tend spontaneous remission,while the high-risk group was difficult to cure even with multidisciplinary combined intensive therapy,and the long-term survival rate was less than 50%.Current research suggests that NB have two types of interconvertible tumor cells,including mesenchymal type(MES type)and adrenergic type(ADRN type).These two phenotypes have different degrees of differentiation and drug sensitivity.Further research on the cellular heterogeneity of NB is of great significance for guiding accurate treatment and improving the prognosis of patients.However,the existing research on cell heterogeneity relies on transcriptome sequencing technology at the cell population level,which can only reflect the characteristics of the overall transcriptome of the cells and mask the characteristics of cells with relatively low cell abundance(such as tumor stem cells).Therefore,there is an urgent need to conduct transcriptomics studies on NB at the single cell level to further clarify the regulatory mechanism of non-genetic heterogeneity and phenotypic plasticity of NB cells.The aim of this study is to assess the intra-tumoral and inter-tumoral heterogeneity of neuroblastoma by single-cell RNA sequencing,and to identify transcriptional programs that might explain patient outcome.Methods: 1.Heterogeneity analysis of primary neuroblastoma tissues: Seven primary neuroblastoma tissues with different pathological subtypes were collected,and the viable single cells(Calcein-Blue + / DRAQ5 +)was captured by flow cytometry.Single-cell c DNA library was constructed using the Smart-seq2 method.We yielded 5309 high quality single-cell transcriptomes for downstream analysis.2.Correlation between the number of circulating tumor cells and NB metastasis and progression: The peripheral blood of 20 healthy children and 25 untreated patients with NB were collected.By using CD56 immunomagnetic beads-based cell enrichment and immunofluorescence antibodies,we counted the number of CD45-/ DAPI + / GD2 + cells in 3 m L peripheral blood.The CTCs were tested again after two courses of chemotherapy in NB children and at the end of chemotherapy.The clinical data of the children were collected to assess the correlation between the number of CTCs and treatment response and prognosis.3.Heterogeneity of circulating tumor cells in high-risk NB children: Peripheral blood was collected from 2 healthy children and 3 children with untreated high-risk NB,and viable non-white single cells were captured by immunomagnetic bead negative enrichment combined with flow sorting(Calcein-Blue + / DRAQ5 + / CD45-).Single-cell c DNA library was constructed using the Smart-seq2 method.We yielded 305 high quality single-cell transcriptomes for downstream analysis.Results: 1.Heterogeneity analysis of primary neuroblastoma tissues: A total of 1429 NB cells and 3880 non-tumor cells were identified.According to the transcriptome characteristics of NB cells,1,000 ADRN-type NB cells,214 transitional NB cells,and 215 MES-type NB cells were identified.Cell state transition trajectory shows that ADRN-type NB cells have a bidirectional differentiation trajectory,that is,they can transition to transitional NB cells or MES-type NB cells.Transitional NB cells exhibit an aggressive neurodevelopmental phenotype,which is associated with poor prognosis.2.Correlation between the number of circulating tumor cells and NB metastasis and progression: The CTCs test results of 20 healthy children were negative,and the detection rate of CTCs of 25 untreated NB children was 84%.Children with tumor progression after treatment had more CTCs(P = 0.015)and higher serum NSE levels(P = 0.010)before treatment.At the same time,the positive rate of CTCs in these children was higher after two courses of chemotherapy(P = 0.028).Patients with more than 11 CTCs per 3 m L blood had inferior progression-free survival(19.4 months vs.32.5 months,P = 0.007).3.Heterogeneity of circulating tumor cells in high-risk NB children: 274 CTCs and 31 non-tumor cells were captured from the peripheral blood of 2 healthy children and 3 untreated children with high-risk NB.After data reduction and clustering,3 CTCs cell clusters and 1 Non-tumor cell cluster were identified.Analysis of copy number variations showed that CTCs had characteristic NB copy number changes(17q + / 1p-/ 11q-).Compared with non-tumor cells,CTCs have up-regulated 141 genes,which are mainly involved in oxidative phosphorylation and NADH dehydrogenase complex.Most CTCs are ADRN-type NB cells,and a small number of CTCs are active mitotic and poor differentiated MES-type NB cells,which are associated with inferior prognosis in children with NB.Conclusions: 1.NB has extensive cellular heterogeneity,including intra-tumoral heterogeneity and inter-tumoral heterogeneity.2.There are transitional NB cells in the primary neuroblastoma tissues,which have an aggressive neurodevelopmental phenotype,which are related to the poor prognosis of patients,MYCN gene amplification and tumor metastasis.3.The number of CTCs at the initial diagnosis is related to neuroblastoma progression and metastasis.4.CTCs have phenotypic heterogeneity,and mesenchymal CTCs with active cell division and poor differentiation are associated with poor prognosis in children with NB.