A toll-like receptor agonist mimicking microbial signal to generate tumor-suppressive macrophages

Switching macrophages from a pro-tumor type to an anti-tumor state is a promising strategy for cancer immunotherapy. Existing agents, many derived from bacterial components, have safety or specificity concerns. Here, we postulate that the structures of the bacterial signals can be mimicked by using non-toxic biomolecules of simple design. Based on bioactivity screening, we devise a glucomannan polysaccharide with acetyl modification at a degree of 1.8 (acGM-1.8), which specifically activates toll-like receptor 2 (TLR2) signaling and consequently induces macrophages into an anti-tumor phenotype. For acGM-1.8, the degree of acetyl modification, glucomannan pattern, and acetylation-induced assembly are three crucial factors for its bioactivity. In mice, intratumoral injection of acGM-1.8 suppresses the growth of two tumor models, and this polysaccharide demonstrates higher safety than four classical TLR agonists. In summary, we report the design of a new, safe, and specific TLR2 agonist that can generate macrophages with strong anti-tumor potential in mice.

A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

Software and code
Policy information about availability of computer code Data collection 1.TEM images were captured using transmission electron microscopy JEOL 2100F (200KV.0.14nm) JEOL Ltd. 2.Flow cytometry data were acquired using BD Accuri C6. 3.Haematoxylin and eosin (H&E) staining were captured using Axio Imager A2 microscope 4.All immunostaining pictures were captured using a Leical TCS SP8 confocal microscope 5.NMR data was acquired using BZH 600MHz/54 mm ASCEND, BRUKER Ltd. 6.Quantitative real-time PCR data were collected using Agilent Technologies Stratagene Mx3005P 7.Mircoarry data was acquired by Agilent Microarray Scanner (Cat.# G2565CA, Agilent technologies, Santa Clara, CA, US) 8. Particle size was collected using Zetasizer Nano ZS, Malvern Panalytical Ltd. 9. UV data was collected using FlexStation III Multi-Mode Microplate Reader Data analysis 1.Flow cytometry data were analyzed using FlowJo X 0.7. 2.Mircoarry data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, KEGG Pathway analysis were performed in the standard enrichment computation method according to the KEGG database (https://www.genome.jp/kegg ). 3.Statistical analysis was performed using Graphpad Prism 7.01.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

April 2018
Data 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 All relevant data that are included with this study are available from corresponding author upon reasonable request. The source data underlying Figure  All studies must disclose on these points even when the disclosure is negative.

Sample size
Sample size was determined empirically for sufficient statistical power. Variations between samples were also used to determine the suitability of the sample size.
Data exclusions No data were excluded from analyses in the experiments.

Replication
All attempts at replication were successful.
Randomization In animal studies, we randomly allocated animals into groups such that as animals were added to the experiment, the numbers of animals in each group did not significantly differ.

Blinding
We needed to investigate the difference between different groups, and such difference had not been known. Thus we did not use blinding in the study.
Reporting for specific materials, systems and methods Antibody 6-10 were purchased for immunofluorescence staining.Application-specific criteria was used to pass or fail the antibodies, and all of antibodies were typically tested in multiple applications (has to show specific signal for the primary application, needs to be tested on positive and negative control samples where available and any potential cross-reaction has to be noted and supported with data from sequence alignment, internal testing, and/or external publications), please refer to the the manufacturer's description: http://www.abcam.com/primary-antibodies/how-we-validate-our-antibodies.  The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology Sample preparation
To analyze blood samples, the whole blood was collected in K2EDTA collection tubes (Terumo Medical, Somerset, NJ, USA)and treated with red blood cell lysis buffer. To analyze adherent cells (Bone marrow derived macrophages ), cells was washed with PBS and collected with scraper. All kinds of cells were adjusted to 10^5 cells/mL and blocked with 2% bovine serum albumin (BSA) and incubated with the fluorescence-conjugated antibodies in the dark for 30 min at 4 °C according to product manuals. Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.