Fig 2: Optimization of cell seeding density. (a) Photograph of a microdevice and a syringe pump. (b) Live/dead images of OP9 cells after 4-day culture in the microdevice. Channel depth is 0.2, 0.5, or 1.0 mm. (c) The number of blood cells produced in the channel. Flk-1+ cells were seeded at a density ranging from 2 × 103 cells/cm2 to 10 × 103 cells/cm2. Mean ± SD, n = 3. (d) Phase-contrast and immunofluorescence images of the blood and endothelial cells. Arrowheads indicate blood cells. Immunofluorescence staining was performed for the hematopoietic marker CD41, which is expressed on all hematopoietic stem and progenitor cells in the early embryo and the endothelial cell marker CD31.

Fig 3: SDF-1a induces expression of active form of E-selectin ligands in EC.(A) Increased expression of E-selectin ligand, CD44, in luminal EC of ischemic hindlimb wounds injected with ?mSDF-1a compared to PBS injected non-wounded hindlimb in NOD mice (n = 8 mice/group). (A) right: Co-expression (yellow) of CD44 (green) and CD31 (red) in vessels was detected by immunostaining. Representative images are shown (images of isotype-matched non-specific control Ab are not shown). Left: Quantification of CD44 expression in vessels. Data are presented as mean ± SD of ratio of CD44:CD31 signals from 5 random selected sections of high power field (LPF, X 20) of each wound sample. CD31 signal was established as “1” in each section and relative amount of CD44 signal was normalized accordingly. (B) Immunoblotting analysis of SDF-1a-induced expression of three E-selectin ligands in HMVEC at various time points (?hSDF-1a: 100 ng/ml). ß-actin is used as loading control. Experiments were repeated three times and similar results were obtained. (C) Binding of FITC-conjugated HECA452 to HMVEC stimulated with ?hSDF-1a or BSA. Fluorescent signals were quantified (top). Representative fluorescent images were exhibited (bottom). (D) Binding of FITC-conjugated HECA452 to human EPC stimulated with ?hSDF-1a or BSA (100 ng/ml). Fluorescent signals were quantified (top). Representative fluorescent images were exhibited (bottom). Data are analyzed by 2-tailed Student’s t-test and presented as mean ± SEM of fluorescent signals based on triplicate wells in each condition and totally three independent experiments in (C,D).

Fig 4: Single-cell RNA sequencing reveals immune and epithelial cell heterogeneity in paired normal-cancer samples(A) Schematic describing experimental set-up for 10x genomic single-cell RNaseq of matched tumor-normal prostate samples from n = 10 patients.(B) UMAP of 15,492 cells post-QC from all prostate samples.(C) UMAP of embedding density of source of samples (normal—green to blue gradient, top; tumor—orange to red gradient, bottom).(D) Violin plot of canonical marker genes for each cell types found in prostate. Gene expression values per cell are standardized to a range from 0 to 10.(E) Patient demographics displayed as a color-coded heatmap and stacked bar charts of single-cell cell type proportions.(F) Quantification of absolute cells counts by flow cytometry per 0.5mg of normal and malignant human prostatic tissue for the indicated immune subsets. Data are a combination of n = 6 donors with each dot representing an individual donor.(G) Quantification of absolute cells counts by flow cytometry per murine prostate lobe for the indicated immune subsets. Each dot represents an individual mouse (n = 3 biological replicates). Abbreviations: MNP—mononuclear phagocyte; DC—dendritic cell; ILC—innate-like lymphoid cell; PMN—polymorphonuclear; NK natural killer (H) Confocal imaging of CD3, CD4 and CD8 in human normal and tumor prostate. Scale bars, 50 µm. (I) Confocal imaging of CD3, CD8, F4/80 and CD31 in normal murine prostate. Scale bars, 100 µm. See also Figure S1 and Table S1.

Fig 5: Immune landscape of the prostate includes a prostate-specific macrophage subset enriched in metallothionein transcripts(A) UMAP of 793 cells in myeloid compartment after integration of myeloid/MNP cells from n = 10 patients with Henry et al. myeloid/MNP cells.(B) Mean expression dot plot of top five significant marker genes for each myeloid cluster. Marker genes were identified using Wilcoxon rank sum test and p adj < 0.05 was considered statistically significant. Size of circles indicate percentage of cells expressing the gene and increasing color gradient from white to red corresponds to increasing expression value.(C) UMAP plot of predicted MNP clusters in prostate cancer single cell data from (Karthaus et al., 2020, Chen et al., 2021, Crowley et al., 2020).(D) (top) Representative RNAscope images of probes targeting MT1 family genes (magenta) and CD68 (yellow). ‘L’ indicates lumen. Arrows point to single cells that are marked by both probes in sub-panels i and ii. Scale bar, 20 µm. (bottom) Representative immunofluorescence microscopy images of a human prostate section labeled for metallothionein (a-MT)/isotype control (yellow), HLA-DR (cyan), CD206 (purple) and DAPI (blue). White arrows point to structure displaying colocalization of a-MT with HLA-DR and/or CD206 labeling. Scale bars, 50 µm.(E) Mean expression dot plot of Zinc transporter genes for each myeloid cluster. Size of circles indicate percentage of cells expressing the gene and increasing color gradient from white to red corresponds to increasing expression value.(F) Heatmap of mean AUCell enrichment of F4/80hi/lo gene sets, corresponding to yolk sac (YS) versus hematopoetic stem-cell (HSC) lineage. Row enrichment value is scaled from 0 to 1 and presented as an increasing gradient from black, gray, yellow to orange which corresponds to increasing enrichment score.(G) Representative immunofluorescence microscopy images of cross sections of mouse prostate labeled for F4/80 (green), MHCII (red), CD11b (blue), CD31 (yellow) and phalloidin. Scale bars, 120 µm.(H) Cell counts per gram of prostate for rat IgG2a isotype or anti-Csf1r antibody (Ab) treated male mice. N = 5 per group. ****p < 0.0001; n.s denotes not significant (p > 0.05) (Two-way ANOVA with Tukey’s multiple correction).(I) Zinc concentration of anterior prostate lobe, liver lobe, and kidney from male mice treated with either rat IgG2a isotype control or anti-Csf1r Ab. N = 6 per group. (shown is representative quantification from one of two independent experiments). *p < 0.05; n.s. not significant (Mann-Whitney test). See also Figures S4–S5.