RNA m6A methylation orchestrates cancer growth and metastasis via macrophage reprogramming

N6-methyladenosine (m6A) is a reversible mRNA modification that has been shown to play important roles in various biological processes. However, the roles of m6A modification in macrophages are still unknown. Here, we discover that ablation of Mettl3 in myeloid cells promotes tumour growth and metastasis in vivo. In contrast to wild-type mice, Mettl3-deficient mice show increased M1/M2-like tumour-associated macrophage and regulatory T cell infiltration into tumours. m6A sequencing reveals that loss of METTL3 impairs the YTHDF1-mediated translation of SPRED2, which enhances the activation of NF-kB and STAT3 through the ERK pathway, leading to increased tumour growth and metastasis. Furthermore, the therapeutic efficacy of PD-1 checkpoint blockade is attenuated in Mettl3-deficient mice, identifying METTL3 as a potential therapeutic target for tumour immunotherapy.


Statistics
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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Angang Yang Rui Zhang Jan 22, 2021 The amplification signal of qPCR data was acquired by Bio-Rad CFX Manager 3.1. m6A sequencing was performed (CloudSeq Biotech, Shanghai, China).
Microsoft excel 2016 was used to calculate mean, standard deviation and P value. Survival curves were calculated using the the Gehan-Breslow-Wilcoxon Test. The flow cytometry data were analyzed by Flowjo VX. Bioluminescence imaging and data processing were performed using the IVIS Spectrum imager equipped with the Living Image® 4.3.1 software.

Research sample
All experiments were performed in technical triplicate with at least 3 independent biological replicates.
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For IHC experiment, blinded staining and blinded analysis were performed. Investigators were blinded to group allocation during data collection and/or analysis. For majority of the other experiments, blind was not applicable since they were in vivo studies in which the treat groups needed to be clear when performing the experiments.
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nature research | reporting summary
B16 and LLC cells were purchased from the American Type Culture Collection (ATCC).
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nature research | reporting summary
October 2018

Animals and other organisms
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Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
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Recruitment
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Study protocol

Data collection
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Methodology
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All experimental mice were bred and maintained under specific pathogen-free conditions, fed standard laboratory chow, and kept on a 12-h light/dark cycle and temperature and humidity were kept at 22±1°C, 55%±5%. All mice were on the C57BL/6 genetic background, maintained in individual cages and used between 6 and 12 weeks of age. Co-housed Cre-negative littermate mice were used as control animals in all experiments. And the male and female mice (same-sex pairs) was used in experiments.
The study did not involve wild animals.
The study did not involve field-collected samples.
All animal procedures used in this experiment were performed in accordance with protocols approved by the Animal Experiment Administration Committee of Fourth Military Medical University.
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Describe the experimental replicates, specifying number, type and replicate agreement.
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For flow cytometric analysis and cell sorting, issues or tumors were excised from the host mice, minced, and digested with 10 U/ mL collagenase I (Gibco) and 30 U/mL DNase I in RPMI medium for 60 minutes at 37°C, and filtered through a 40-m nylon filter (BD Biosciences) to obtain single-cell suspensions. After the red blood cells were lysed, the remaining cells were washed twice with complete RPMI medium and stained with the antibodies.
Cells were either analyzed on a BD Fortessa (BD) or sorted by AriaIIIu (BD).
Analysis of flow cytometry data was performed using Flowjo VX.
The purity of sorted cells was detected with purity >90% were used.
Cell populations were determined by FSC-H/FSC-A gate. The boundaries were determined by the clear cell subpopulations and isotype controls.
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Specify in Tesla
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