Next Generation Sequencing Facilitates Quantitative Analysis of Splenic Macrophage Subpopulations identified by Ly6c and F4/80 Transcriptomes in mouse T cell acute lymphoblastic leukemia
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE101621 | GEO | 2020/07/19
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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Project description:The experiment was designed to obtain a broader unbiased view of the changes in islet macrophages following low dose STZ challenge. Mice were purchased from Jackson Laboratory (Bar Harbor, ME). 16-20-week-old C57BL/6J males were given 30 mg/kg STZ or acetate buffer (control) i.p. (intraperitoneal injection) for 5 consecutive days. Following the first STZ or buffer injection mice were sacrificed on day 14 and islets were isolated by collagenase digestion. Freshly isolated islets were dispersed in 0.02% Trypsin-EDTA for 3 minutes followed by up to 1 minute of pipetting under a stereomicroscope to obtain a single cell solution. Islet media was added to stop the reaction. Islets from 10 mice were pooled per sample (N). Dispersed islets were washed with FACS buffer (1% heat inactivated FBS, 1 mM EDTA, 11 mM glucose in PBS). Cells were kept on ice and pre-incubated with Fc Block (1:100) for 5 minutes, followed by 30 min incubation with CD45-eFluor 450 (1:250; clone 30-F11), Ly-6C-APC (1:1,200; clone HK1.4), CD11b-PE (1:1,200; clone M1/700, F4/80-FITC (1:150; clone BM8), CD11c-PECy7 (1:150; clone N418), and the viability dye 7AAD (1:2,000). Unstained, single stains, and fluorescence minus one controls were used for setting gates and compensation. Viable, single CD45+Ly6c-Cd11b+Cd11c+F4/80+ cells were sorted using a BD FACS Aria IIu directly into lysis buffer, and the RNeasy Plus Micro Kit from Qiagen was used to isolate total RNA. Total RNA quality control quantification was performed using an Agilent 2100 Bioanalyzer. All RNA samples had an RNA integrity number (RIN) ≥9.1. The NeoPrep Library Prep System from Ilumina was used for library preparation followed by sequencing using standard Illumina methods and Ilumina NextSeq500.
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Project description:Loss-of-function mutations in the lysosomal nucleoside transporter SLC29A3 lead to the accumulation of nucleosides in lysosomes and result in histiocytosis, characterized by the excessive accumulation of phagocytes in multiple organs. However, the underlying mechanism by which lysosomal nucleoside storage drives histiocytosis remains poorly understood. Herein, we provide evidence that histiocytosis in Slc29a3–/– mice is dependent on Toll-like receptor 7 (TLR7), which senses a combination of guanosines and oligoribonucleotides. TLR7 increased phagocyte populations by driving the proliferation of immature Ly6C high splenic macrophages and their subsequent maturation into Ly6C low phagocytes in Slc29a3–/– mice. Interestingly, TLR7 activation in nucleoside-laden splenic macrophages failed to induce inflammatory responses. Our data demonstrate that TLR7 responses to lysosomal nucleoside stress drive unique immune responses distinct from inflammation in SLC29A3-related disorders.
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Project description:Background & aimsColorectal cancer is a leading cause of cancer death, and a major risk factor is chronic inflammation. Despite the link between colitis and cancer, the mechanism by which inflammation leads to colorectal cancer is not well understood.MethodsTo investigate whether different forms of inflammation pose the same risk of cancer, we compared several murine models of colitis (dextran sodium sulfate [DSS], 2,4,6-trinitrobenzene sulfonic acid, 4-ethoxylmethylene-2-phenyloxazol-5-one, Citrobacter rodentium, Fusobacterium nucleatum, and doxorubicin) with respect to their ability to lead to colonic tumorigenesis. We attempted to correlate the severity of colitis and inflammatory profile with the risk of tumorigenesis in both azoxymethane-dependent and Dclk1/APCfl/fl murine models of colitis-associated cancer.ResultsDSS colitis reproducibly led to colonic tumors in both mouse models of colitis-associated cancer. In contrast, all other forms of colitis did not lead to cancer. When compared with the colitis not associated with tumorigenesis, DSS colitis was characterized by significantly increased CD11b+F4/80+Ly6Chigh macrophages and CD11b+Ly6G+ neutrophils. Interestingly, depletion of the CD11b+F4/80+Ly6Chigh macrophages inhibited tumorigenesis, whereas depletion of CD11b+Ly6G+ neutrophils had no effect on tumorigenesis. Furthermore, the macrophage-derived cytokines interleukin-1β, tumor necrosis factor-α, and interleukin-6 were significantly increased in DSS colitis and promoted stemness of Dclk1+ tuft cells that serve as the cellular origin of cancer.ConclusionsWe have identified CD11b+F4/80+Ly6Chigh macrophages as key mediators of cancer initiation in colitis-associated cancer. Development of new therapies that target these cells may provide an effective preventative strategy for colitis-associated cancer.
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Project description:Background & aimsImmunotherapies that induce T-cell responses have shown efficacy against some solid malignancies in patients and mice, but these have little effect on pancreatic ductal adenocarcinoma (PDAC). We investigated whether the ability of PDAC to evade T-cell responses induced by immunotherapies results from the low level of immunogenicity of tumor cells, the tumor's immunosuppressive mechanisms, or both.MethodsKras(G12D/+);Trp53(R172H/+);Pdx-1-Cre (KPC) mice, which develop spontaneous PDAC, or their littermates (controls) were given subcutaneous injections of a syngeneic KPC-derived PDAC cell line. Mice were then given gemcitabine and an agonist of CD40 to induce tumor-specific immunity mediated by T cells. Some mice were also given clodronate-encapsulated liposomes to deplete macrophages. Tumor growth was monitored. Tumor and spleen tissues were collected and analyzed by histology, flow cytometry, and immunohistochemistry.ResultsGemcitabine in combination with a CD40 agonist induced T-cell-dependent regression of subcutaneous PDAC in KPC and control mice. In KPC mice given gemcitabine and a CD40 agonist, CD4(+) and CD8(+) T cells infiltrated subcutaneous tumors, but only CD4(+) T cells infiltrated spontaneous pancreatic tumors (not CD8(+) T cells). In mice depleted of Ly6C(low) F4/80(+) extratumoral macrophages, the combination of gemcitabine and a CD40 agonist stimulated infiltration of spontaneous tumors by CD8(+) T cells and induced tumor regression, mediated by CD8(+) T cells.ConclusionsLy6C(low) F4/80(+) macrophages that reside outside of the tumor microenvironment regulate infiltration of T cells into PDAC and establish a site of immune privilege. Strategies to reverse the immune privilege of PDAC, which is regulated by extratumoral macrophages, might increase the efficacy of T-cell immunotherapy for patients with PDAC.
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