Fig. 3

RNA LLPS, TGFβ1 downregulation, and antitumor immune activation in 4T1 tumor models after intratumoral CP administration. Mice bearing 4T1 tumors were intratumorally injected with the CPs (3 mg/kg body weight; dextran was used as a control reagent) 14 days before the mice were sacrificed, and the tissues were analyzed accordingly. a Representative confocal images of RNA droplets in tumor sections stained with SYTO RNAselect Green (scale bar, 5 μm). b Tumor cells and different leukocytes isolated from 4T1 tumor tissues were analyzed for RNA droplets marked by SYTO RNAselect Green (scale bar, 5 μm). c Western blot analysis of TGFβ1 in the tumor tissues after CP treatment. d ELISA analysis of TGF-β1 in tumor tissues after CP treatment. e Immunofluorescent staining of TGFβ1 in tumor tissue sections (scale bar, 50 μm). f Mean mouse tumor weights 14 days after the first CP treatment. g ELISAs of TNFα, IL10, and IL12; h the frequency of CD3⁺ T cells, CD4⁺ T cells, and CD8⁺ T cells; and i the frequency of Th1 cells, Th2 cells, Th17 cells, and Treg cells in the tumor tissues harvested from the same experiment as (f). j Evaluation of the antitumor activity of the CPs in the TGFβ1 knockout 4T1 cell animal model. Tumor size was normalized based on untreated 4T1^TGFβ1+/+ cell-bearing mice, n = 10 per group. Data are expressed as the mean ± SEM, and the differences between experimental groups were analyzed by two-way ANOVA with Sidak’s multiple comparisons test (i) and one-way ANOVA with Dunnett’s test (d, f–h and j). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001