Pten null prostate epithelium promotes localized myeloid-derived suppressor cell expansion and immune suppression during tumor initiation and progression

Abstract

Chronic inflammation is known to be associated with prostate cancer development, but how epithelium-associated cancer-initiating events cross talk to inflammatory cells during prostate cancer initiation and progression is largely unknown. Using the Pten null murine prostate cancer model, we show an expansion of Gr-1(+) CD11b(+) myeloid-derived suppressor cells (MDSCs) occurring intraprostatically immediately following epithelium-specific Pten deletion without expansion in hematopoietic tissues. This MDSC expansion is accompanied by sustained immune suppression. Prostatic Gr-1(+) CD11b(+) cells, but not those isolated from the spleen of the same tumor-bearing mice, suppress T cell proliferation and express high levels of Arginase 1 and iNOS. Mechanistically, the loss of PTEN in the epithelium leads to a significant upregulation of genes within the inflammatory response and cytokine-cytokine receptor interaction pathways, including Csf1 and Il1b, two genes known to induce MDSC expansion and immunosuppressive activities. Treatment of Pten null mice with the selective CSF-1 receptor inhibitor GW2580 decreases MDSC infiltration and relieves the associated immunosuppressive phenotype. Our study indicates that epithelium-associated tumor-initiating events trigger the secretion of inflammatory cytokines and promote localized MDSC expansion and immune suppression, thereby promoting tumor progression.

FIG 1

Pten null murine prostate cancer initiation and progression are marked by persistent immune cell infiltration. (A) Kinetics of prostate cancer initiation and progression in the Pten null murine prostate cancer model. (B, top) Representative immunofluorescence images showing significant CD45⁺ (red) immune cell infiltration surrounding E-cadherin (E-Cad) (green)-positive luminal epithelial cells at the early mPIN (6 weeks) and late adenocarcinoma (16 weeks) stages in mutant (MT) mice compared to wild-type (WT) littermates. (Bottom) FACS analysis demonstrating that levels of CD45⁺ immune cell infiltrates remain elevated throughout all stages of disease progression. (C) Immune cell infiltrates are made up of predominantly Gr-1⁺ CD11b⁺ cells and reach nearly 50% of all CD45⁺ cells by the adenocarcinoma stage. (D, left) The majority of prostate-infiltrating Gr-1⁺ CD11b⁺ cells display a Gr-1^Hi Ly6C^Lo polymorphonuclear (PMN) cell phenotype. (Right) Representative images of cytospun and Giemsa-stained PMN and mononuclear (MO) cell populations. (E) Percentages of various immune cell populations within the CD45⁺ infiltrate in WT and MT mice. Panels B to D show means ± standard errors of the means from a minimum of 5 animals per age/genotype. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

Gr-1⁺ CD11b⁺ cell expansion does not occur in lymph nodes, bone marrow, spleen, or liver of tumor-bearing animals. (A to D) Absolute numbers of Gr-1⁺ CD11b⁺ cells in the draining lymph nodes (A), spleen (B), bone marrow (C), and liver (D) of WT and MT mice throughout disease progression (means ± standard errors of the means from a minimum of 3 animals per age/genotype). (E) The percentage of prostatic BrdU⁺ Gr-1⁺ CD11b⁺ cells is significantly higher in MT animals than in WT littermates. The percentage of spleen and bone marrow BrdU⁺ Gr-1⁺ CD11b⁺ cells remained unchanged between MT and WT littermate tissues (***, P < 0.001). Ten high-power microscopic fields were counted for each organ and genotype (minimum of 3 animals per genotype). n.s., not significant.

FIG 3

PTEN loss in epithelial cells leads to upregulated inflammatory and cytokine-cytokine receptor signaling pathways. (A) GSEA shows enhanced activation of inflammatory response and cytokine-cytokine receptor interaction pathways in vivo in Pten null (MT) prostate epithelial cells. (B) Inflammatory genes upregulated in PTEN null murine prostate epithelial cells significantly overlap those in human prostate cancer samples (P < 0.05). (C, left) Epcam⁺ epithelial cells sorted from MT mice at the mPIN stage express higher levels of Csf1 and Il1b mRNA. Relative gene expression was normalized to levels in sorted WT prostate epithelial cells. (Middle) Csf1 expression is upregulated in two independent PTEN null prostate epithelial cells lines. (Right) IL1b expression is significantly upregulated in human prostate cancer specimens compared to normal prostate tissue (P < 0.05 by two-tailed test). (D) Il1ra (left) and Csf1r (right) gene expression levels are increased in sorted Gr-1⁺ CD11b⁺ cells from prostates of 6- to 8-week-old MT mice. The relative gene expression level was normalized to levels in WT BM (minimum 5 animals per age/genotype for panels C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

Gr-1⁺ CD11b⁺ cells from the prostate but not spleen of tumor-bearing mice can suppress T cell function. (A, left) Gr-1⁺ CD11b⁺ cells sorted from MT prostates have higher expression levels of Arg1 and iNOS than do Gr-1⁺ CD11b⁺ cells from WT prostates. (Right) Gr-1⁺ CD11b⁺ cells sorted from spleens of MT and WT littermate animals revealed no difference in and low levels of Arg1 and iNOS expression. The relative gene expression level for each sample was normalized to levels in WT BM (minimum of 5 animals per genotype). (B) Sorted Gr-1⁺ CD11b⁺ cells from mutant prostates were cocultured with naive CFSE-loaded CD4⁺ or CD8⁺ cells from spleens of wild-type animals at the indicated ratios. CD4⁺ (left) and CD8⁺ (right) T cell proliferations were suppressed by infiltrating Gr-1⁺ CD11b⁺ cells. (C) Identical assays using Gr-1⁺ CD11b⁺ cells from spleens of tumor-bearing mice show an inability to suppress T cell proliferation (means of 3 independent experiments for panels B and C). (D, left) FACS analysis demonstrates increased levels of CD8⁺ immune cell infiltrates as the disease initiates, which then decrease as the disease progresses. (Right) Levels of activated CD8⁺ CD69⁺ T cells decrease during disease progression. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

Prostate-specific Gr-1⁺ CD11b⁺ cell expansion is associated with suppression of dendritic cell and macrophage maturation. (A, left) Mature CD11c⁺ MHCII⁺ dendritic cells are present in both wild-type and mutant prostates, but their levels decrease precipitously in mutant animals as the disease progresses. (Right) Levels of immature CD11c⁺ MHCII⁻ DCs increase in mutant prostates as the disease progresses. (B) Macrophage F4/80 expression shifts away from a mature F4/80^int/high phenotype to an immature F4/80^low phenotype as the disease progresses. (For panels A and B, data are means ± standard errors of the means from a minimum of 4 animals per age/genotype.) (C) The IL10 gene expression level increased in sorted Gr-1⁺ CD11b⁺ cells from the prostate, compared to levels in cells from the spleen and BM of MT mice. The relative gene expression level for each sample was normalized to the values for WT BM (n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

The selective CSF-1 receptor inhibitor GW2580 reduces Gr-1⁺ CD11b⁺ cell infiltration and reverses the immune-suppressive phenotype. (A, left) Stromal reactivity and the desmoplastic response are reduced in the prostates of GW2580-treated animals. (Middle) Invasive adenocarcinoma, as measured by SMA breakdown, is diminished in the prostates of GW2580-treated animals. (Right) Numbers of p-CSFR-1⁺ cells are greatly reduced in the stroma of GW2580-treated prostates. (B) Epithelial and stromal cell Ki67 proliferation indices are reduced following GW2580 treatment. (C) Quantification of stromal p-CSF-1R⁺ cells demonstrates a significant decrease in the percentage of p-CSF-1R⁺ cells following GW2580 treatment. (D) GW2580 treatment alleviates immune-suppressive phenotypes associated with MDSC expansion. Values are represented as fold changes between GW2580- and vehicle-treated MT mice. For panels B to D, there were 6 animals per treatment group; for panels B and C, there were a minimum of 10 high-power microscopic fields/animal. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Macs, macrophages.

FIG 7

Cross talk between epithelial cell-associated tumor-initiating events and inflammatory cells facilitates prostate cancer progression. Epithelial cell Pten loss leads to the upregulation of genes associated with inflammatory response and cytokine-cytokine receptor interaction pathways. Increased cytokine production by Pten null epithelial cells, including CSF-1 and IL-1β production, leads to the recruitment and expansion of MDSCs, which in turn facilitate an immune-suppressive environment and promote tumor progression through the secretion of Arg1, iNOS, and IL-10. Inhibiting immune-responsive pathways, through the use of compounds like GW2580, can decrease immune cell recruitment, alleviate the immune-suppressive environment, delay tumor progression, and potentiate immunotherapeutic modalities. Mac, macrophage.