Myeloid-specific expression of Ron receptor kinase promotes prostate tumor growth

Abstract

Ron receptor kinase (MST1R) is important in promoting epithelial tumorigenesis, but the potential contributions of its specific expression in stromal cells have not been examined. Herein, we show that the Ron receptor is expressed in mouse and human stromal cells of the prostate tumor microenvironment. To test the significance of stromal Ron expression, prostate cancer cells were orthotopically implanted into the prostates of either wild-type or Ron tyrosine kinase deficient (TK(-/-); Mst1r(-/-)) hosts. In TK(-/-) hosts, prostate cancer cell growth was significantly reduced as compared with tumor growth in TK(+/+) hosts. Prostate tumors in TK(-/-) hosts exhibited an increase in tumor cell apoptosis, macrophage infiltration and altered cytokine expression. Reciprocal bone marrow transplantation studies and myeloid cell-specific ablation of Ron showed that loss of Ron in myeloid cells is sufficient to inhibit prostate cancer cell growth. Interestingly, depletion of CD8(+) T cells, but not CD4(+) T cells, was able to restore prostate tumor growth in hosts devoid of myeloid-specific Ron expression. These studies show a critical role for the Ron receptor in the tumor microenvironment, whereby Ron loss in tumor-associated macrophages inhibits prostate cancer cell growth, at least in part, by derepressing the activity of CD8(+) T cells.

Figure 1. Ron is expressed in stromal cells of normal and tumor-bearing prostates and is important for promoting prostate tumor growth

(A) qRT-PCR analysis of Ron mRNA expression in cells isolated from TK+/+ mice. The expression of Ron in whole TK+/+ and TK−/− prostates is shown for comparison. Expression levels are representative of two independent isolations. (B) Ron expression in human prostate tissue specimens. A representative human prostate adenocarcinoma section depicting positive Ron staining in both epithelial (E) and stromal (S) compartments. The horizontal line in the scatter plot represents the mean ± SE. (C) TRAMP-C2Re3 cells were injected into the prostates of TK+/+ and TK−/− mice; tumor weights were determined 30 days post-implantation. (TK+/+, n=36; TK−/−, n=33, *P<0.0001). Red line = average weight (0.18±0.012g) of normal prostates from TK+/+ mice. (D) Hematoxylin and eosin-stained TK+/+ and TK−/− prostate tumor sections showing similar tissue architecture. Scale bar = 100μm.

Figure 2. Characterization of stromal cells in TRAMP-C2Re3 tumors from TK+/+ and TK−/− prostates

(A) Representative F4/80 staining of TRAMP-C2Re3 tumors from TK+/+ and TK−/− mice. Differences in the spatial distribution of macrophages are noted with red arrows depicting tumor peripheral infiltration while green arrows depict intratumoral infiltration. Macrophages were quantitated and the data represents the mean number of F4/80⁺ macrophages per 40X field (five fields per tumor, TK+/+, n=5; TK−/−, n=5, *P<0.05). Scale bar = 50μm. (B) Representative images showing CD31 staining in microvessels and quantification of the microvessel density per tumor area (*P<0.01). Scale bar = 100μm. (C) α-SMA–positive fibroblasts in the tumors and the average intensity of α-SMA expression per 40X field (*P<0.01). Scale bar = 50μm.

Figure 3. Lack of Ron signaling in the host leads to increases in tumor necrosis and apoptosis

(A) Quantification of necrotic areas in TRAMP-C2Re3 tumor sections from TK+/+ and TK−/− hosts. Representative tissue sections are shown. Scale bar = 1cm. (B) Flow analysis for AnnexinV/PI on TRAMP-C2Re3 tumor cells isolated from TK+/+ and TK−/− hosts. A representative flow plot is shown with a histogram depicting data from all experiments (TK+/+, n=4; TK−/−, n=3). Data are the mean percentage of cells in early apoptosis (AnnexinV⁺PI⁻), late apoptosis (AnnexinV⁺PI⁺), dead cells (AnnexinV⁻PI⁺), and live/viable cells (AnnexinV⁻PI⁻) ± SE.

Figure 4. Loss of Ron leads to changes in the inflammatory tumor microenvironment and loss of STAT3 phosphorylation in TAMs

(A) qRT-PCR analyses for iNOS mRNA in TRAMP-C2Re3 tumors from TK+/+ and TK−/− hosts. Data represented as relative change compared to β-glucuronidase (TK+/+, n=5; TK−/−, n=5, *P<0.01). (B) Concentration of serum nitrite from TRAMP-C2Re3 tumor-bearing TK+/+ and TK−/− mice. (TK+/+, n=5; TK−/−, n=5, *P<0.05). (C) Levels of inflammatory cytokines in the serum of tumor-bearing mice. (TK+/+, n=5; TK−/−, n=5, *P<0.05) (D) Gene expression for T-cell regulatory genes depicted as fold change in TK−/− tumors over controls. (E) Western analysis for pSTAT3-Y705 and Total STAT3 in TAMs isolated from orthotopic TRAMP-C2Re3 tumors from TK+/+ and TK−/− mice and is representative of four independent isolations.

Figure 5. TK−/− bone marrow-derived cells decrease the growth of TRAMP-C2Re3 tumors in TK+/+ chimeras

(A) Ron mRNA in bone marrow cells isolated from tumor-bearing TK+/+, TK−/− or chimeric mice. (B) Ron mRNA expression in TAMs isolated from TRAMP-C2Re3 tumors from TK+/+→TK−/− and TK−/−→TK+/+ chimeric mice. (C) Tumor weights determined at day 30 after intraprostatic injection of TRAMP-C2Re3 cells into TK+/+, TK−/− and bone marrow transplantation mice. Tumor progression in both chimeras was delayed compared to TK+/+→TK+/+ mice (*P<0.05). Data are pooled from two independent experiments. (TK+/+, n=7; TK−/−, n=6; TK+/+→TK+/+, n=5; TK−/−→TK−/−, n=5; TK+/+→TK−/−, n=24; TK−/−→TK+/+, n=24; *P<0.05). (D) FACS analysis for tumor immune infiltrates in TRAMP-C2Re3 tumors from TK+/+→TK−/− and TK−/−→TK+/+ chimeric mice. Tumors from TK+/+→TK−/− chimeric mice had significantly more macrophages (CD11b⁺ Gr1⁻ cells) compared to tumors from TK−/−→TK+/+ chimeric mice. Data are mean percentage of cells ± SE (TK+/+→TK−/−, n=2; TK−/−→TK+/+, n=2; *P<0.01).

Figure 6. Loss of Ron signaling in myeloid cells is sufficient to abrogate TRAMP-C2Re3 tumor growth

(A) Ron mRNA in TAMs isolated from TRAMP-C2Re3 tumors from TK^f/f and TK^f/fLysMcre+ mice. Data represented as relative change from two independent TAM isolations. (B) TRAMP-C2Re3 cells were injected into the prostates of TK^f/f and TK^f/fLysMcre+ mice and tumor weight determined after 30 days (TK^f/f, n=15; TK^f/fLysMcre+, n=13; *P<0.001). Data represent two independent experiments. (C) FACS analysis for AnnexinV/PI on epithelial cells isolated from TK^f/f and TK^f/fLysMcre+ mice 23 days post-tumor cell implantation (TK^f/f, n=4; TK^f/fLysMcre+, n=4; *P<0.01). (D) Quantification for F4/80 on TRAMP-C2Re3 tumor sections from TK^f/f and TK^f/fLysMcre+. Data represented as mean F4/80⁺ cells per 40X field. (TK^f/f, n=5; TK^f/fLysMcre+, n=5; *P<0.05). (E) qRT-PCR for iNOS expression in TRAMP-C2Re3 tumors from TK^f/f and TK^f/fLysMcre+ mice. (F) Western analysis of pSTAT3-Y705 and Total STAT3 in TAMs isolated from TRAMP-C2Re3 tumors from TK^f/f and TK^f/fLysMcre+ mice. (G) Western analysis of arginase-1 expression in TAMs isolated from TRAMP-C2Re3 tumors from TK^f/f and TK^f/fLysMcre+ mice. F and G are representative of four independent TAM isolations.

Figure 7. Depletion of CD8+ T-cells restores TRAMP-C2Re3 tumor growth in Ron-deficient mice

(A) Growth curve of subcutaneously injected TRAMP-C2Re3 cells in TK+/+ and TK−/− mice administered with cytotoxic monoclonal antibodies against CD4+ T-cells, CD8+ T-cells, both T-cell subsets, or an isotype control (n=3 per genotype/group). (B) Final tumor volume of TRAMP-C2Re3 cells from TK+/+ and TK−/− mice at 28 days post injection. (C) Growth of TRAMP-C2Re3 cells in control and TK^f/fLysMcre+ mice treated with cytotoxic monoclonal antibodies against CD8+ T-cells or an isotype control (n=3 per genotype/group). (D) Final tumor volumes from control and TK^f/fLysMcre+ mice at 28 days.