Fig 2: Impaired embryo development and decidual integrity in Ezh2 Amhr2-Cre cKO mice. (A) Embryo development in control and Ezh2 Amhr2-Cre cKO mice at E7.5. (B–E) Immunostaining of DES using uteri from control and Ezh2 Amhr2-Cre cKO mice at E6.5. At least three animals for each genotype were examined. Panels (D,E) are higher power images for panels (B,C), respectively. Scale bar = 20 µm (D,E) and 200 µm (B,C). (F) qRT-PCR analysis of Prl8a2, Klrg1, Il11ra1, Spp1, and Prf1 mRNA expression using implantation sites from control and Ezh2 Amhr2-Cre cKO mice at E7.5. n = 6. Data are mean ± s.e.m, ***p < 0.001. (G–L) Immunostaining of PRF1 using implantation sites from control and Ezh2 Amhr2-Cre cKO mice at E7.5. Panels (I–L) represent two implantation sites from different Ezh2 Amhr2-Cre cKO mice. Panels (H,J,L) are higher power images for panels (G,I,K), respectively. At least three animals for each genotype were examined. MS, mesometrial side. AMS, antimesometrial side. Dashed blue arrows indicate embryonic-uterine orientation. Scale bar = 200 µm (G,I,K) and 20 µm (H,J,L). (M) Qualification of the number of PRF1-positive cells in control and Ezh2 Amhr2-Cre cKO mice at E7.5. n = 6. Data are mean ± s.e.m. * p < 0.05. (N) Subfertility in Ezh2 Amhr2-Cre cKO mice during a 3-month breeding trial. n = 5–6. Data are mean ± s.e.m. ** p < 0.01 and *** p < 0.001.

Fig 3: T cell immune effect following PDT treatment in mice bearing hPD-1/PD-L1 MC38 tumors. (A) IHC of CD3, CD4, CD8, Gzmb, and Prf1 was performed in tumor tissues at the end of the experiment (image size represents ×400). (B) Quantitation of CD3, CD4, CD8, Gzmb, and Prf1 expression in tumor tissues. Relative protein expression was quantitated using the ImageJ software. Dots represents individual values. (C–E) Gzmb (C), IFN-γ (D), and IL-2 (E) levels in mouse sera isolated on day 20 post-PDT treatment. The assays were performed in triplicate at each concentration. Error bars represent the means ± SDs. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Fig 4: The infiltration of CD103+CD8+ T cells was increased in the omentum of mice treated by inetetamab along with atezolizumab(A and B) Representative images of IHC (GZMB and PRF1) staining of the omentum from mice (left, magnification 400×, scale bar: 20 μm). Data are represented as mean ± SD. (C) Representative images of mIHC staining of the omentum from mice (left, magnification 400×, scale bar: 25 μm). p values were calculated using the one-way analysis of variance. ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig 5: Role of CD36 in RNAi-lipid conjugate-mediated delivery to tumor-associated immune cellsTDLNs were collected from Pan02 tumor-bearing C57BL/6 WT mice (300–400 mm3), and LNs from non-tumor-bearing C57BL/6 WT mice to measure and compare Cd36 mRNA levels (A). TDLNs were also collected from Pan02 tumor-bearing CD36 KO mice and Cd36 and Arg1 mRNA levels were determined and compared with the levels in TDLNs of tumor-bearing WT mice (B). In a separate study, established Pan02 tumors in WT and KO mice were treated with two doses (3 days apart) of RNAi-PDL1 (25 mg/kg, subcutaneously [s.c.]) or PBS, and TDLNs collected 7 days post administration of the last dose for Cd274 (C), Cd36 (D), and Arg1 (E) mRNA analysis. In the same study, tumors from WT mice and KO mice were collected at the same time point and Cd8 (F) and Gzmb (G) mRNA levels measured. The rest of the tumor samples were used for immunohistochemistry staining for CD8 and PRF1 (H) and quantitative image analysis (I) (Table S7). To assess anti-tumor efficacy, Pan02 tumor-bearing WT and KO mice were treated with four doses of RNAi-Placebo or RNAi-PDL1 (25 mg/kg, subcutaneously [s.c.]) as outlined in the figures, and tumor growth monitored (J–L). To see if Cd36 levels correlate with tumor immunophenotypes, mouse background, and Arg1 levels, Cd36 mRNA levels in TDLNs of resistant tumor phenotype were compared with the levels in TDLNs of partially inflamed MC-38 tumor-bearing mice. Cd36 mRNA levels in TDLN of tumor-bearing immunocompetent mice were compared with tumor-bearing immunocompromised mice (M), and Arg1 levels in the TDLN of the resistant phenotype were compared with the levels in partially sensitive tumor TDLNs (N). n = 3–4 for qPCR experiments, n = 5 for IHC and efficacy analysis; error bars represent the SEM; ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001 by unpaired t test. Representative images were presented in the paper, but all the tumor samples were used for image quantification analysis.