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. 2020 Mar 18;12(3):717.
doi: 10.3390/cancers12030717.

Overexpression of Murine Rnaset2 in a Colon Syngeneic Mouse Carcinoma Model Leads to Rebalance of Intra-Tumor M1/M2 Macrophage Ratio, Activation of T Cells, Delayed Tumor Growth, and Rejection

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Overexpression of Murine Rnaset2 in a Colon Syngeneic Mouse Carcinoma Model Leads to Rebalance of Intra-Tumor M1/M2 Macrophage Ratio, Activation of T Cells, Delayed Tumor Growth, and Rejection

Annarosaria De Vito et al. Cancers (Basel). .

Abstract

Human RNASET2 acts as a powerful oncosuppressor protein in in vivo xenograft-based murine models of human cancer. Secretion of RNASET2 in the tumor microenvironment seems involved in tumor suppression, following recruitment of M1-polarized macrophages. Here, we report a murine Rnaset2-based syngeneic in vivo assay. BALB/c mice were injected with parental, empty vector-transfected or murine Rnaset2-overexpressing mouse C51 or TS/A syngeneic cells and tumor growth pattern and immune cells distribution in tumor mass were investigated. Compared to control cells, mouse Rnaset2-expressing C51 cells showed strong delayed tumor growth. CD86+ M1 macrophages were massively recruited in Rnaset2-expressing C51-derived tumors, with concomitant inhibition of MDSCs and CD206+ M2 macrophages recruitment. At later times, a relevant expansion of intra-tumor CD8+ T cells was also observed. After re-challenge with C51 parental cells, most mice previously injected with Rnaset2-expressing C51 cells still rejected C51 tumor cells, suggesting a Rnaset2-mediated T cell adaptive immune memory response. These results point at T2 RNases as evolutionary conserved oncosuppressors endowed with the ability to inhibit cancer growth in vivo through rebalance of intra-tumor M1/M2 macrophage ratio and concomitant recruitment of adaptive anti-tumor CD8+ T cells.

Keywords: C51 colon carcinoma; M1/M2 macrophage ratio; RNASET2; TNFα-producing M1 macrophages; immune T cell responses.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Immunoblot analysis of murine Rnaset2-overexpressing clones in TS/A (panel A) and C51 (panel B) tumor cell lines. The images showing the whole-filter picture from the western blots shown above are provided in Figure S2.
Figure 2
Figure 2
Assessment of in vitro cell proliferation rate (MTT assay) in full length Rnaset2-overexpressing TS/A clones (FL Rnaset2) in comparison to empty vector-transfected TS/A clones (TS/A E) (panel A) and in vivo analysis of tumor growth in BALB/c mice groups (N = six animals in group) injected with TS/A P, TS/A E, and TS/A FL Rnaset2 cell lines (panel B). Survival curves versus time (days) of BALB/c mice groups injected with TS/A P, TS/A E, and TS/A FL Rnaset2 cell lines (panel C).
Figure 3
Figure 3
Assessment of in vitro cell proliferation rate (MTT assay) in full length Rnaset2-overexpressing C51 clones (FL Rnaset2) in comparison to empty vector-transfected C51 clones (C51 E) (panel A); in vivo analysis of tumor growth in BALB/c mice groups (N = six animals in group) injected with C51 P, C51 E, and C51 FL Rnaset2 cell lines (panel B); survival curves versus time (days) of BALB/c mice groups injected with C51 P, C51 E and C51 FL Rnaset2 cell lines (panel C). For MTT assay and tumor growth, an ANOVA statistical analysis was performed assuming p < 0.05 as a threshold value. * p < 0.05; ** p < 0.01.
Figure 4
Figure 4
Tumor growth follow up of ten mice injected with C51 FL Rnaset2 up to about two months and subsequent follow up of tumor growth in four tumor-free of them receiving a C51 P tumor challenge until day 80 from the first C51 FL Rnaset2 injection (panel A); survival curves versus time (days) of BALB/c mice groups injected with C51 P, C51 E, and C51 FL Rnaset2 cell lines (panel B); in vivo analysis of tumor growth with statistical difference at three time points (day 14, 17, and 20) of other groups (N = ten animals in group) of BALB/c mice injected with C51 P, C51 E, and C51 FL Rnaset2 cell lines (panel C). Horizontal bars indicate mean ± SE (Standard error) values for each group. p values of statistically significant differences between the groups connected by lines are also reported. An ANOVA statistical analysis was performed assuming p < 0.05.
Figure 5
Figure 5
Quantification of different key markers of tumor infiltrating immune cells, as follows: CD86+ M1 and CD206+ M2 (panel A), Gr-1 (GR) for granulocytes and CD11b+Gr-1+ for MDSCs (panel B), CD31 for vessels (panel C), and CD4 and CD8 for T cells (panel D) per HMMF in BALB/c mice injected with C51 P, C51 E, and C51 FL Rnaset2 cell lines (N = six animals in group). The mean ± SE are indicated. Immunohistochemistry and immunofluorescence analyses of tumor sections from BALB/c mice injected with C51 P, C51 E, and C51 FL Rnaset2 cell lines at indicated time points are shown. An ANOVA statistical analysis was performed assuming p < 0.05 as a threshold value. * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 6
Figure 6
Cytometric flow assessment after in vitro tumor digestion has been performed to study percentage of intra-tumor CD45+ CD86+ M1 and CD206+ M2 macrophage ratio in BALB/c mice (N = three animals in group) injected with C51 P, C51 E and C51 FL Rnaset2 cell lines (panel A); gating strategy for flow cytometry (FACS) analysis of surface markers in M1 (F4/80+ CD86+) and M2 (F4/80+ CD206+) macrophages (panel B). An ANOVA statistical analysis was performed assuming p < 0.05 as a threshold value. * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 7
Figure 7
Cell cytometric flow assessment in spleens from groups of BALB/c mice (N = three animals in group) at one month after tumor injection, as follows, i.e., receiving C51 P, C51 E tumor cells lines and C51 challenge in rejecting and tumor-free C51 FL Rnaset2 mice. The cytometric flow analysis has been conducted after in vitro tumor digestion to quantify: Percentage of tumor cell infiltration of F4/80+ mature macrophages (panel A); percentage of TNFα-secreting CD11b+ macrophages by intracellular staining, following in vitro 4 h LPS stimulation (panel B); percentage of CD4+ and CD8+ T cells (panel C); and percentage of IFNγ-producing CD4+ and CD8+ T cells by intracellular staining, following in vitro 4 h PMA plus ionomycin stimulation (panel D). An ANOVA statistical analysis was performed assuming p < 0.05 as a threshold value. * p < 0.05; ** p < 0.01; *** p < 0.001.

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