The Mechanism Of Clustering Invasion And Metastasis In Breast IMPC Revealed By Single-cell RNA Sequencing

Bacground and ObjectiveInvasive micropapillary carcinoma(IMPC)is a special type of breast cancer that can occur in multiple organs throughout the body and grow in groups,with the biological characteristics of higher lymphatic infiltration and easier lymph node metastasis.The IMPC was initially reported in breast cancers by the WHO classification.It has the morphological characteristics of a mulberry-like tumor cell mass surrounded by obvious interstitial spaces,lacking a central bundle of vessels.Our team’s previous research results showed that:IMPC tumor cell clusters were showed reversed tumor cell clusters in the vasculature,metastasis,or in vivo and in vitro.Under the electron microscope,IMPC tumor cell clusters are rich in stem cell characteristics.The surface of the IMPC tumor cell cluster facing the interstitial side is rich in microvilli,and the corresponding cytoplasm is rich in a large number of motor fibers,mitochondria,Golgi,etc.The clinical characteristics of IMPC are high vascular invasion,easy recurrence and metastasis.Based on this,the research hypothesis of cell clustering invasion and metastasis of IMPC tumor cell group was proposed,and a series of research verifications were carried out.In this study,the isolated and purified human breast cancer primary IMPC cells were analyzed using Bulk RNA-seq technology,and invasive ductal carcinoma-non-special(IDC-NOS)primary cells were used as controls.And using single-cell transcriptome sequencing technology to sequence mixed IMPC,qualitative and localized research and analysis of the lineage relationship and function between each cell subpopulation within the IMPC tumor cell group.In this study,the cell map of human breast IMPC was successfully drawn,and the results showed that the IMPC tumor cell mass was derived from IDC-NOS.And verify and analyze the mechanism of IMPC tumor cell cluster polarity reversal,group invasion and metastasis.It provides theoretical and experimental basis for the research of IMPC’s precise diagnosis and treatment.Methods1.Fresh human breast IMPC and IDC-NOS tumor tissues were digested and separated,and the obtained primary IMPC tumor cell clusters and primary IDC-NOS tumor cells were separately cultured to observe their biological characteristics;2.The 3-D culture technique and immunofluorescence staining of primary cells were used to define and verify the characteristics of the polarity reversal of primary IMPC suspended tumor cell mass;3.The primary IMPC suspended tumor cell clusters and primary IDC-NOS adherent tumor cells were isolated and purified,and the Bulk transcriptome sequencing was performed with their normal tissues as controls.Then we can analyze the differences between their transcriptomes,and explore the molecular and functional differences between IMPC and IDC primary tumor cells;4.Through single-cell transcriptome sequencing analysis,we draw a single-cell map of mixed IMPC(IMPC with IDC)reveal the heterogeneity of its tumor cells,explore the lineage level between its cell subtypes,and find the fate-determining genes at its trajectory differentiation.Results1.We successfully cultured primary IMPC tumor cells of breast,and verified the biological characteristics of its cluster growth pattern and polarity inversion on primary cells;2.We successfully cultured primary IDC tumor cells of breast with high success rate,which can be stably passed to 8-10 generations;3.Through the separation and purification method,we obtained primary IMPC suspended tumor cell mass and primary IDC adherent tumor cells.We analyzed the differences between their transcriptomes by Bulk transcriptome sequencing.The result shows that IMPC does have a different gene expression profile than IDC.And the transcriptome results suggest that,compared with IDC,IMPC primary cells have a more vigorous ability to take in nutrients from the surroundings and lysosomal metabolism,and IMPC primary cells are in the cell skeleton regulation and its fatty acid metabolism are also more active;4.Using differential genes between IMPC primary cells and IDC primary cells,using big data to predict breast cancer molecular typing,suggesting that IMPC is more inclined to Lumina type;5.We collected 3 mixed IMPCs(IMPC with IDC)for single cell sequencing.CNV copy number variation speculated that most of the primary breast cells tested were tumor cells;6.Based on the genes specifically expressed by clusters,we mapped the single-cell map of human breast mixed IMPC,which contains 9 cell types,including IMPC inside cells,IMPC outside cells(interstitial side),and IDC-NOS cells,Basal cells,villous cells,ciliary cells,interstitial-like IDC cells,IDC-NOS mesenchymal stem cells and immune cells.We compared with Bulk RND-seq results to verify the reliability of cell group;7.We have predicted the molecular typing of the measured single cells,which suggests that the molecular characteristics of IMPC tend to be Lumina type;8.Through functional enrichment analysis of each subpopulation,we found that the IMPC cell population is associated with peroxidase,insulin and insulin-like growth factor signaling pathways,fatty acid metabolism,glycolysis,oxidative phosphorylation,and reactive oxygen species pathways;9.Pseudo-time analysis suggests that in mixed IMPC primary cells,IDC cell subsets may be differentiated into IMPC subpopulations,and the differentiation node genes among them are obtained by analysis;10.Pseudo-time analysis reveals the evolutionary relationship between the inner cell subgroup of the IMPC and the outer cell group of the IMPC,and the differentiation node genes among them are obtained by analysis;11.Using the differential genes between IMPC cell subsets and IDC cell subsets,referring to breast cancer big data,the correlation between IMPC-specific expression genes and lymph node metastasis in breast cancer patients was analyzed,and it was found that IMPC-specific expression genes("S100P","SERHL2","TFF1","IFI27","C19orf33","NEAT1","GDF15","CYP1B1","FTH1","NQO1")were correlated with breast cancer lymph node metastasis and it has significance for the prognosis of breast cancer.Conclusions1.Primary IMPC tumor cells can maintain the biological characteristics in tissues of IMPC cells in vitro;2.Bulk RNA-seq analysis of the transcriptome information of the isolated and purified IMPC and IDC-NOS revealed that IMPC and IDC-NOS have very different gene expression profiles;3.The human breast mixed IMPC single cell map contains 9 cell types.There is an evolutionary relationship between cells outside the IMPC clusters and cells inside the cell clusters,suggesting that external cells may gradually evolve from internal cells;4.Pseudo-time analysis suggests that IDC cells may differentiate into IMPC cells;5.Differential genes between IMPC cell subsets and IDC cell subsets are associated with lymph node metastasis and have prognostic significance.