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Transcriptome analysis of tissue macrophages

ABSTRACT: RNA-seq analysis of cardiac, renal, and liver macrophages. CD11b+F4/80+Ly6C-Ly6G- tissue resident macrophages were isolated from the heart, kidney, and liver of mice. Isolated RNA was subjected to RNA-seq to identify differentially expressed transcripts.

ORGANISM(S): Mus musculus

PROVIDER: GSE84586 | GEO | 2017/04/10

SECONDARY ACCESSION(S): PRJNA330530

REPOSITORIES: GEO

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Project description:Tumor-associated macrophages (TAMs) are highly heterogeneous, derived from myeloid monocyte/macrophages or tissue-resident macrophages, and play vital role in tumor progression. Here we adopted a C57BL/6 mouse model imitating the late-stage colorectal liver metastasis (CRLM) by Mc38 colorectal cancer cell injection via the portal vein. We defined the critical period at 7 to 9 days post injection (dpi) for tumor neovascularization on serial sections of CRLM biopsies. The tumor neovascularization was initiated from one of the liver blood vessels via vessel cooption and extended by vascular mimicry and growth of CD34+cells. In samples with increasing sized liver metastases of CRLM, infiltrated Ly6C+ CD11b+ F4/80 monocytes steadily gained the expression of F4/80, a Kupffer cell marker, and then transformed into Ly6C CD11bint F4/80+ cells, which, the same phenotype was also adapted by Ly6C CD11b F4/80+ Kupffer cells. F4/80+ TAMs showed proximity to neovascularization and tumor vessels. Depletion of macrophages or disturbance on macrophage polarization during the crucial period of neovascularization impeded tumor growth and vascularization and resulted in greatly reduced F4/80+ TAMs, yet increased CD11b+ cells due to inhibition of TAM differentiation. In summary, we showed dynamic F4/80+ TAM differentiation within the tumor microenvironment and strengthened its role as perivascular TAMs in CRLM. A set of aliquot samples (about 0.25 cm3) from for flow cytometry preparations (the above method and Fig. 4A) were used for bulk RNA-Sequencing, among these samples, Ht1 and Ht3 were identical samples; F4/80+cells and CD11b+ cells were selected from medium-sized CRLM (Mt2) with microbeads and MS column;A total of 20 samples were sent for analysis. These collected tissues were flash-frozen in liquid nitrogen, stored at - 80°C and shipped on dry ice to the sequencing provider (Novogene, www.novogene.com), who then extracted RNA from these samples and confirmed that all sample RNA were in good quality and adequate concentrations. The resulted RNA-Sequencing data were verified as the clean reads > 95%, Q30 > 90%, and error rate < 0.05%; Square of Pearson Correlation Coefficient (R2) between the biological replicates was > 0.89; R2 of the identical samples (Ht-1 and Ht-3) was > 0.98.

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Transcriptome analysis of tissue macrophages

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