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10x Single-cell Sequencing for Smad3 wild-type and knockout neutrophils

ABSTRACT: The role of neutrophils in tumor is still controversial and largely unknown. We previously identified an anticancer tumor microenvironment from mice lacking Smad3, but the underlying mechanism is still largely unclear. Interestingly, depletion of neutrophils largely suppressed the anticancer phenotype of Smad3-KO mice against syngeneic murine Lewis lung carcinoma (LLC). Here, we discovered that Smad3 is essential for promoting N1/N2 polarization of the tumor_x0002_associated neutrophils (TANs) in lung carcinoma. We found that Smad3 deficiency dramatically increased N1 TANs associated with an anticancer phenotype in mice and detected a negative correlation between Smad3 activation and anticancer N1 associated with a higher mortality of NSCLC. By conducting a neutrophil-specific single-cell RNA-sequencing, we uncovered that Smad3 is essential for completing the N1/N2 polarization of TANs, whereas N1 will be the dominated phenotype in the Smad3-KO TME by analyzing the developmental pathways of TANs with pseudo-time at transcriptome level. Mechanistically, direct binding of Smad3 on the developmental genes in neutrophils under cancer condition was evidenced by ChIP-sequencing at genomic level. Importantly, both neutrophil-specific and pharmaceutical inhibition of Smad3 effectively enhanced the anticancer activity of human and murine TANs, thereby suppressing the progression of lung carcinoma in vivo. Thus, Smad3 may represent a precision therapeutic target for enhancing the anticancer activity of TANs via blocking N1/N2 polarization in NSCLC. in C57BL6 (wild-type) and Smad3-deficient (Smad3-/- ) mice and submitted for cell encapsulation and library construction by Chromium controller with 5’ expression kit (10x genomics)

ORGANISM(S): Mus musculus

PROVIDER: GSE186530 | GEO | 2023/03/16

REPOSITORIES: GEO

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Project description:The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naive neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC). In the current study, a transcriptomics approach was used in mice to compare these cell types. Our data show that the three populations of neutrophils are significantly different in their mRNA profiles with NN and G-MDSC being more closely related to each other than to TAN. Structural genes and genes related to cell-cytotoxicity (i.e. respiratory burst) were significantly down-regulated in TAN. In contrast, many immune-related genes and pathways, including genes related to the antigen presenting complex (e.g. all six MHC-II complex genes), and cytokines (e.g. TNF-a, IL-1-a/b), were up-regulated in G-MDSC, and further up-regulated in TAN. Thirteen of the 25 chemokines tested were markedly up-regulated in TAN compared to NN, including striking up-regulation of chemoattractants for T/B-cells, neutrophils and macrophages. This study characterizes different populations of neutrophils related to cancer, pointing out the major differences between TAN and the other neutrophil populations. Various types of myeloid cells have been shown to promote tumor progression by direct immune suppression and by production of angiogenic factors, matrix-degrading enzymes, or growth factors. In untreated tumors, neutrophils have been reported to produce angiogenic factors and matrix-degrading enzymes, support the acquisition of a metastatic phenotype, and suppress the anti-tumor immune response. Neutrophils, like all other leukocytes, move into tissues from the blood under the influence of specific chemokines (e.g. KC/CXCL-1, MIP-2a/CXCL-2 and GCP-2/CXCL-6), cytokines (e.g. TNFa and IFN-x), and cell adhesion molecules located on their own surface (e.g. CD11b) and on the surface of endothelial cells (e.g. selectins, ICAM-1 and PECAM-1). When they traffic into tumors, they are referred to as TAN. In mice, TAN can be defined by the specific surface markers CD11b and Ly6G with low expression of macrophage markers such as F4/80. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immune suppressive cells that are produced excessively in cancer. They comprise at least two subsets -granulocytic (Ly6G+, G-MDSC) and monocytic cells (Ly6C+, M-MDSC), potentially with different immunosuppressive properties. It has been previously shown that MDSC can enter tumors and differentiate to mature macrophages (TAM) or neutrophils (TAN). However, since no definitive markers have been established, it is unknown whether intratumoral N2 neutrophils (N2 TAN) are granulocytic MDSC from spleen that are attracted to the tumor or if they are typical blood-derived neutrophils that are then converted to an N2 phenotype by the tumor microenvironment, specifically by the high local concentrations of TGF-b. The purpose of this study was to use a transcriptomics approach to gain further information about TANs by comparing the RNA profile of these cells to naive bone-marrow neutrophils (NN) and to the granulocytic fraction of myeloid derived suppressor cells (G-MDSC). We examined which pathways and gene-groups varied amongst these 3 populations of neutrophils and performed a detailed analysis on pathways related to the main functions of neutrophils, such as respiratory burst, granule proteins, phagocytosis, apoptosis, structural genes, antigen presentation and specific immune effects. Our data defines TAN as a unique population of neutrophils, quite distinct from both NN and G-MDSC.

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10x Single-cell Sequencing for Smad3 wild-type and knockout neutrophils

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