Fig 1: RNase1 ectopic expression inhibits tumor progression in an immunocompetent mouse model. (A) The schedule of treatment in BALB/c mice orthotopically injected with the indicated 4T1 cells into the mammary fat pad (MFP) followed by treatment with vehicle or compound 1 (Cpd1). The arrows indicate the time of treatment. n = 9 mice per group. (B-D) Average tumor volume (B), tumor growth curve (C), and waterfall plot analysis (D) of 4T1 cells in BALB/c mice treated with vehicle or Cpd1 as indicated. Tumor volume was measured at the indicated time points, and tumors were dissected at the end point. The gray box indicates the duration of treatment. The waterfall plot generated from the tumor growth curve indicates tumor progression in each mouse under individual treatment. The number of mice that experienced tumor progression in each group is shown in parentheses. (E-G) Relative fold changes of tumor volume (E), tumor weight (F), and representative images (G) from (C). (H) Western blotting of the indicated mice tumors with antibodies against RNase1. The experiment was repeated a second time with similar results. (I) Measurement of body weight before and after treatment as indicated. The gray box indicates the body weight at the end point (i.e., day 19). Data represent mean ± SD (E, F, and I) or mean ± SEM (B). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, ANOVA test. PD, progressive disease; SD, stable disease; PR, partial response.
Fig 2: RNase1 boosts CD4+ T cell activation. (A) Schematic illustration of expression construct encoding anti-CD3 antibody single-chain fragment variable (aCD3-scFv) fused to the transmembrane domain of CD14. The numbers represent amino-acid residues. CD5L, CD5 leader; VH, variable domain of the heavy chain; VL, variable domain of the light chain. (B) Western blotting of the indicated stable cells with antibodies against CD14 and tubulin as loading control. The experiment was repeated a second time with similar results. (C) Flow cytometric analysis of CD69 and CD3 expression in the indicated BT-549 stable clones cocultured with Jurkat cells for 2 days. (D) Flow cytometric analysis of CD69 expression in the indicated BT-549 stable clones cocultured with Jurkat cells for 2 days. IgG isotype control was performed in the BT-549-Vn cocultured with Jurkat cells. (E and F) Representative flow cytometric images (E) and quantitative analysis (F) of CD69 expression in the indicated BT-549 stable clones cocultured with Jurkat cells combined with or without RNase1 treatment (1 µg/ml) for 24 h. (G-J) Representative flow cytometric images and quantitative analysis of the expression of CD69 and CD4 (G and H) or CD69 and CD8 (I and J) in the indicated BT-549 stable clones cocultured with human PBMCs combined with or without RNase1 treatment (1 µg/ml) for 24 h. IgG isotype control, left panel. Data represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ns, not significant, two-tailed unpaired t test.
Fig 3: Mice expressing RNase1 favor an antitumor TME. (A) A heatmap of different marker intensities in CD45+ tumor-infiltrating immune cell clusters identified by CyTOF data analysis using t-SNE and FlowSOM. (B and C) Annotation of t-SNE plots of immune cells overlaid with color-coded clusters based on marker expression as shown in (A). Mf, macrophage; MDSC, myeloid-derived suppressor cell; NK, natural killer cell; DC, dendritic cell; CD8 T, CD8+ T cell; Mono, monocytes; CD4 T, CD4+ T cell. (D) Box plots of frequency of CD45+ tumor-infiltrating immune cell clusters as indicated. n = 6 mice per group. Box plots indicate minima (lower end of whisker), maxima (upper end of whisker), median (center), 25th percentile (bottom of box), and 75th percentile (top of box). *p < 0.05, **p < 0.01, ***p < 0.001, individually compared with the percentage at the first bar of each cluster (4T1-vector), one-tailed unpaired t test.
Fig 4: RNase1 targets specific subsets of tumor-infiltrating immune cells using a conventional hand-gated strategy. Frequency of the indicated subsets in total CD45+ tumor-infiltrating immune cells, including CD4+ T cells (A), CD8+ T cells (B), T helper cells (C), Treg cells (D), immune checkpoint receptors (E), granulocytic and monocytic MDSCs (F), NK cells (G), M1- and M2-like macrophages (H), and DCs (I). Data represent the mean ± SD. n = 6 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, one-tailed unpaired t test.
Fig 5: RNase1 enhances T cell-mediated cell death in breast cancer cells. (A) Time-course quantitation of T cell-meditated tumor cell-killing assay of dead cells, normalized to that at the zero-time point, in the indicated BT-549 stable cells expressing nuclear-restricted red fluorescent protein cocultured with or without human PBMCs combined with or without RNase1 treatment (1 µg/ml) as indicated. (B) Representative merged images of nuclear-restricted red fluorescent protein (Red nuclei) and green fluorescent caspase 3/7 substrate (Dead cells) from (A) observed at 24 h. Scale bar, 100 µm. Images were captured using the IncuCyte Zoom microscope. (C and D) Time-course quantitation of phase object confluence (C) and dead cells (D), normalized to that at the zero-time point, in the indicated BT-549 stable cells. (E and F) Time-course quantitation of T cell-meditated tumor cell-killing assay of phase object confluence (E) and dead cells (F), normalized to that at the zero-time point, in the indicated BT-549 stable cells cocultured with primary human T cells. (G) Quantitative ratio of T cell-meditated tumor cell-killing assay of dead cells at 70 h in breast cancer cell lines as indicated cocultured with primary human T cells in the presence of EGFR-CD3 bsAb (1 µg/ml) combined with or without RNase1 treatment (1 µg/ml) as indicated. The number of green fluorescent objects were counted and normalized to that at the zero-time point. Data represent mean ± SD (A, C, E, and G) or mean ± SEM (D and F) of two (G) or three (A, C-F) independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001, ns, not significant, ANOVA test (A, C-F), one-tailed unpaired t test (G).
Supplier Page from Sino Biological, Inc. for Human RNase A / Ribonuclease A / RNASE1 Protein (His Tag)