Fig 2: Runx2 was expressed in heart-infiltrating myeloid cells. (A) Paraffin-embedded sections of infarcted myocardium were prepared from the heart 7 days after MI. The sections were stained with anti-Runx2 antibody. Representative images are shown. (bar = 25 µm). (B) Representative flow cytometry plot of leukocytes enriched from MI7d hearts. (C) Frozen sections were prepared from the hearts 7 days after MI. The sections were co-stained with anti-Runx2 and anti-CD68 antibodies. Representative images are shown. Arrows, Runx2+ CD68+ cells: arrow heads, Runx2+ CD68- cells. (bar = 50 µm). (D and E) Flow cytometry analysis of Runx2 expression on F4/80+ CD86+ M1 macrophages (upper) and F4/80+ CD206+ M2 macrophages (lower). CD11b + myeloid cells were enriched from MI4d (D) or MI7d (E) hearts. Isotype control antibodies were used for positive/negative gate setting.

Fig 3: oHSV2-aPD1 Treatment Facilitates Amplification of Immune Effector Cells in the SpleenC57BL/6J-Pdcd1 mice were subcutaneously inoculated with 2 × 105 B16R cancer cells and treated with oHSV2 or oHSV2-aPD1 as described in the Materials and Methods. Single cells were isolated from spleens collected 5 days after the last treatment. (A–L) These cells were incubated with CD16/32 antibody and stained with antibodies to determine quantities of T cells (A–C), NKs (defined as CD3-NK1.1+) (D), memory T cells (defined as CD4+CD44+ or CD8+CD44+) (E and F), DCs (defined as CD45+CD11b+CD11c+Ly6G-) (G), and CD8+ T cell activation (H–L). Data are presented as individual replicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Fig 4: PS3D1@DMXAA amplifies the immunostimulation activity of DMXAA and promotes the formation of a T cell–inflamed TME.Mice bearing B16.F10 tumors were intravenously treated with PBS (1), free DMXAA (2), PS3D1 (3), a physical mixture of empty PS3D1 and DMXAA (4), or PS3D1@DMXAA (5) for five times, 2 days apart. (A) Selected gene expression levels in the tumors were analyzed by qPCR on day 14 (n = 5, two-way ANOVA with Tukey’s test). (B and C) IFN-ß, IFN-?, tumor necrosis factor–a (TNF-a) (B, n = 4), and selected chemokines (C, n = 5) within the B16.F10 TME were measured by bead-based multiplex LEGENDplex (two-tailed Student’s t test). (D and E) Secretion of IFN-? (D) and TNF-a (E) in sera of mice on day 7 (n = 5). (F and G) Representative flow cytometric analysis of DC maturation (CD80+CD86+ DCs of CD45+CD11c+ DCs) (F) and quantification (G) in the tdLNs on day 14 (n = 5). (H to J) Representative flow cytometric plots (H) and the corresponding quantification of tumor-infiltrating CD8+ (I) and CD4+ (J) T cells on day 14 (n = 4). (K) Ratio of CD8+ to CD4+ T cells in the TME. (L) Quantification of tumor-infiltrating IFN-?+CD8+ in CD8+ T cells (n = 4). (M) Flow cytometric quantification of the counts of macrophages (MF; CD11b+F4/80+), monocytic (m-MDSC; CD11b+Ly6c+Ly6g-) and granulocytic MDSC (g-MDSC; CD11b+Ly6c+Ly6g+SSChi), neutrophils (CD11b+Ly6c+Ly6g+SSClo), natural killer cells (NK1.1+), and DCs (CD11c+MHC-II+) in the TME on day 14 (n = 4, two-way ANOVA with Tukey’s test). (N and O) Representative flow cytometric plots (N) and the corresponding quantification (O) of M2 macrophages (CD206+) in F4/80+CD11b+CD45+ cells (n = 4). Data are means ± SD, and statistical significance was calculated by one-way ANOVA with Tukey’s test unless otherwise indicated. ***P < 0.001, **P < 0.01, and *P < 0.05.

Fig 5: In situ clearance of EMT6-Luc cells in EMT6 tumor-primed mice. Primary tumor-bearing (EMT6 or 4T1) or the naive BALB/c mice were injected with EMT6-Luc cells (100K) via the tail vein 3-weeks post implantation. a, b Live animal and lung ex vivo bioluminescent images show entrapped EMT6-Luc cells after 2 h of tail vein injection in naive, EMT6, and 4T1 tumor-bearing mice, respectively. c, d Live imaging of mice and ex vivo imaging of lungs at 3 days post injection of indicated animals. The reduced signal intensity of EMT6-Luc cells in the lungs of EMT6 tumor-bearing mice compared with naive and 4T1 tumor-bearing mice is associated with less number of tumor cells. e, f Pan-keratin staining of tumor cells and TUNEL-positive apoptotic cells in the lungs of the indicated animals at day 3. g Flow-cytometry analyses of CD8+ T cells in the lungs of naive, EMT6, and 4T1 tumor-bearing mice. h, i Flow-cytometry analysis shows an elevated CD11b+Ly6Cint cell population (gMDSC) in the lungs of 4T1 tumor-bearing mice compared with naive and EMT6 tumor-bearing mice. j The CD11b-Ly6C+ population is significantly increased in only EMT6 tumor-bearing mice at day 3 post i.v. injection. Results are presented as mean ± SD (n = 3). Scale bar: 50 µm. *P < 0.05, **P < 0.005, ***P < 0.0005, one-way analysis of variance test