Fig 2: M-LECP recruited to ZR-75 human tumors retain myeloid-lymphatic identity but downregulate T-cell and erythroid markers.Human ZR-75 breast tumor cells were orthotopically implanted into SCID mice (N = 5). When tumors reached ~300mm3, mice were infused i.v. with 1.5x106 of GFP+ M-LECP differentiated in vitro using standard CSF-1/LPS protocol. Four weeks after transfer, tumors were excised and triple-stained for GFP, Lyve-1, and (A) CD11b, (B) Ter-119 or (C) CD3e. Nuclei (blue) shown in merged images were visualized by Hoechst stain. Yellow arrowheads indicate GFP+/Lyve-1+ cells that lack markers of other lineages. Cells with rare co-expression of erythroid or lymphoid markers in GFP+/Lyve-1+ cells are indicted by yellow arrows. All images were acquired at 400X magnification. (D) The mean percentage of GFP+/Lyve-1+ cells expressing CD11b, Ter-119 or CD3e was determined on 3–4 tumor sections each derived from individual mice. A minimum of 100 GFP+/Lyve-1+ cells identified in multiple sections were analyzed to calculate the percentage cells positive for each marker. (E) Identical analysis was applied to native Lyve-1+ cells lacking GFP expression. Results are presented as the means ± S.D. “Exogenous” and “Endogenous” labels refer to adoptively transferred (GFP+) or tumor-induced host M-LECP (GFP-), respectively. Statistically significant differences between Lyve-1+ cells with myeloid and other markers were determined by Student’s t-test with P-values of <0.001 indicated by triple stars.

Fig 3: IL-10 pathway regulates transcription of specific markers of multiple lineages in BM myeloid precursors.Marker expression levels in CSF-1/LPS differentiated BM cells from WT mice in the presence of anti-IL-10R antibodies and from IL-10R KO mice were compared with the levels in WT cells treated with a control antibody. Cells harvested on the sixth day of differentiation were analyzed by RT-qPCR for mRNA expression of markers of (A) lymphatic-specific, (B) endothelial, (C) myeloid, (D) erythroid, (E) T-cell, and (F) B-cell lineages. All analyses were performed in triplicate for each target, normalized to β-actin, and independently reproduced twice. Data for each target are reported as the mean fold-change in treated cells compared with the WT cells treated with control rat IgG ± S.D. Statistical significance of expression changes compared with control group was determined by Student’s t-test with P-values indicated by *<0.05, **<0.01, and ***<0.001.

Fig 4: ACh reduces cytokine release from cultured resident macrophages. (A) GFP-expressing macrophages were isolated by flow cytometry from the colonic muscularis propria of Cx3cr1GFP;CCR2RFP mice. (B) Cx3cr1GFP macrophages were cultured in vitro and formed colonies in the presence of CSF-1 (dashed box enlarged in inset). (C) Transcript levels of Tnf in the presence of LPS (100 ng/mL) were reduced significantly by the addition of ACh in a dose-dependent manner (n = 3 per group). Channelrhodopsin-expressing cholinergic neurons (ChATChR2-tdT) and muscularis macrophages (Cx3cr1tdT) were co-cultured in vitro. (D) After addition of LPS, IL1β can be seen expressed by tdT+/CD11b+ macrophages (white arrows), and not by tdT+/CD11b-negative cholinergic neurons (yellow arrows). (E) Quantification of IL1β immunofluorescence (MFI, mean fluorescence intensity) is shown in control conditions, in the presence of LPS, and with LPS after BLS to activate ChAT-expressing neurons (n = 8 per group). Scale bars: 200 μm (B), 50 μm (D). Data are shown as means ± SEM. ∗∗P < .01, ∗P < .05.

Fig 5: MSU-42011 polarizes bone marrow-derived macrophages (BMDMs) towards an anti-tumor phenotype. BMDMs were isolated from C57BL/6 mice and differentiated with MCSF. On day 5, BMDMs were treated with conditioned media (CM) from E18-14C-27 cells, either alone or with 300 nM RXR agonist. (A) BMDMs were harvested for flow cytometry to detect percentage of live single cells that were F4/80 + CD206+. All data normalized to conditioned media-treated cells and presented as mean of three experimental runs. Error bars represent standard error. (B–E) RNA was extracted, and gene expression was detected by qPCR. Error bars represent standard error of three biological replicates. * p < 0.05.