Tumour-infiltrating regulatory T cells stimulate mammary cancer metastasis through RANKL-RANK signalling

Abstract

Inflammatory mechanisms influence tumorigenesis and metastatic progression even in cancers whose aetiology does not involve pre-existing inflammation or infection, such as breast and prostate cancers. For instance, prostate cancer metastasis is associated with the infiltration of lymphocytes into advanced tumours and the upregulation of two tumour-necrosis-factor family members: receptor activator of nuclear factor-κB (RANK) ligand (RANKL) and lymphotoxin. But the source of RANKL and its role in metastasis have not been established. RANKL and its receptor RANK control the proliferation of mammary lobuloalveolar cells during pregnancy through inhibitor of nuclear factor-κB (IκB) kinase-α (IKK-α), a protein kinase that is needed for the self-renewal of mammary cancer progenitors and for prostate cancer metastasis. We therefore examined whether RANKL, RANK and IKK-α are also involved in mammary/breast cancer metastasis. Indeed, RANK signalling in mammary carcinoma cells that overexpress the proto-oncogene Erbb2 (also known as Neu), which is frequently amplified in metastatic human breast cancers, was important for pulmonary metastasis. Metastatic spread of Erbb2-transformed carcinoma cells also required CD4(+)CD25(+) T cells, whose major pro-metastatic function was RANKL production. Most RANKL-producing T cells expressed forkhead box P3 (FOXP3), a transcription factor produced by regulatory T cells, and were located next to smooth muscle actin (SMA)(+) stromal cells in mouse and human breast cancers. The dependence of pulmonary metastasis on T cells was replaceable by exogenous RANKL, which also stimulated pulmonary metastasis of RANK(+) human breast cancer cells. These results are consistent with the adverse impact of tumour-infiltrating CD4(+) or FOXP3(+) T cells on human breast cancer prognosis and suggest that the targeting of RANKL-RANK can be used in conjunction with the therapeutic elimination of primary breast tumours to prevent recurrent metastatic disease.

Figures

Figure 1. RANK-signaling in mammary carcinoma cells enhances metastasis

a, MMTV-ErbB2/Rank+/+/Fvb and MMTV-ErbB2/Rank+/−/Fvb mice were sacrificed 8 weeks after tumor onset. RANK expression in primary tumors was analyzed by immunoblotting (each lane a tumor from a different mouse, upper left) and quantitated by densitometry of relative to E-cadherin (bottom left; means +/− s.e.m.; n=6). Lung metastasis multiplicity was analyzed by H&E staining of lungs from MMTV-ErbB2/Rank+/+/Fvb (n=10) and MMTV-ErbB2/Rank+/−/Fvb (n=13) mice. Means +/− s.e.m. and data for individual mice are shown (right panel). b, PCaM cells from MMTV-ErbB2/Fvb mice were isolated and grafted (1×106 cells) into the #2 mammary glands of FVB/N mice subjected to twice weekly intra-tumoral injections of BSA or recombinant RANKL (80 μg/kg), starting one week post-inoculation. At day 35, lungs were isolated, sectioned, H&E stained, and metastatic lesions counted. Shown are means +/− s.e.m. (n=6) as well as values for individual mice. c, MT2 cells (1×106 ) were grafted into #2 mammary glands of FVB/N or MMTV-ErbB2/Fvb mice treated with BSA or RANKL and analyzed for pulmonary metastases as in a. Data are presented as incidence and multiplicity of pulmonary metastases (mean +/− s.e.m.; n=7–8). d, Mice were inoculated with 1×106 shRANK-transduced or control MT2 cells and lung metastases were measured 56 days later. Data are presented as incidence and multiplicity of pulmonary metastases (means +/− s.e.m.; n=6). *: P<0.05; **: P<0.01.

Figure 2. Expression of RANKL in mammary tumors depends on CD4+ T cells

a, Mammary glands of indicated mouse strains were inoculated with 1×106 MMTV-ErbB2 PCaM cells. After 56 days, tumors were isolated, fixed, paraffin embedded and sectioned. Parallel sections were stained with RANKL- and CD5-specific antibodies and counterstained with hematoxylin. Panels show the stroma- and carcinoma-containing regions separated by a line, when relevant. Arrows indicate CD5+ and RANKL+ cells. b–f, Rankl mRNA expression in tumors. b, MT2 cells were transplanted into the #2 mammary glands of indicated mice. After 56 days, tumors were excised and Rankl mRNA was quantitated by q-RT-PCR and normalized to cyclophilin mRNA. Results are means +/− s.e.m. (n=3). c, Freshly-isolated MMTV-ErbB2 PCaM cells were transplanted as above into indicated mice. After 56 days, tumors were isolated and Rankl mRNA was quantified. Results are means +/−s.e.m. (n=3). d, MT2 cells were transplanted into mock-, B cell-, CD4+ T cell- or CD8+ T cell-reconstituted Rag1−/− mice, 3 days after reconstitution. Rankl mRNA in tumors was analyzed as above. Means +/− s.e.m. (n=3). e, Spontaneous MMTV-ErbB2 tumors were dissociated into single cell suspensions. Tumor-infiltrating lymphocytes were enriched by positive selection and Rankl mRNA was quantified as above. Means +/− s.e.m. (n=3). f, Tumor-infiltrating B cells and CD4+ T cells were purified from MT2 formed tumors and analyzed for Rankl mRNA. Means +/− s.e.m. (n=3). *: P<0.05; **: P<0.01. g, Parallel sections of tumors raised in WT mice as in a. were stained with FoxP3 (brown)-, RANKL (brown)- and SMA (blue)-specific antibodies without counterstaining. Arrows: FoxP3+ and RANKL+ cells. The stroma and carcinoma regions are separated by a line. h, Tumor-infiltrating CD4+CD25+ and CD4+CD25− T cells were purified and analyzed for Rankl and FoxP3 mRNA expression as above. Means +/− s.e.m. (n=3). *: P<0.05; **: P<0.01. i, Cancer associated fibroblasts (CAF) and PCaM cells were purified from MMTV-ErbB2 tumors. Chemokine mRNAs were quantified by qRT-PCR as above. Means +/− s.e.m. (n=3). **: P<0.01. j, Sectioned MMTV-ErbB2 tumors were stained with CCL5- (brown) and FoxP3-(blue) specific antibodies. k, The indicated cell types were plated onto multiwell plates at 2X105 cells per well with 3 μg/ml rat IgG or CCL5 antibody. Splenocytes from tumor-bearing MMTV-ErbB2 mice were added to the upper compartment of Boyden chambers. After 24 hrs, T cells in the bottom compartment were quantified by flow cytometry. CAF1 and CAF2, two independent preparations. Means +/− s.e.m. (n=3). *, P<0.05. l, Rat IgG, CCL5, or CCL22 antibodies (2 mg/kg) were i.p. injected into FVB/N females bearing MT2 tumors twice weekly. After one week, tumors were excised and Cd25 mRNA was quantified by q-RT-PCR and normalized to cyclophilin mRNA. Results are means +/− s.e.m. (n=4). *, P<0.05. CA, carcinoma region.

Figure 3. Tumor infiltrating CD4+ T cells stimulate pulmonary metastasis

a–b, Freshly-isolated MMTV-ErbB2 PCaM cells (1×106) were transplanted into the #2 mammary glands of indicated mice. a, Lung metastasis incidence and multiplicity were determined 56 days later. Means +/− s.e.m. (n=4–6). Each point represents a value from a single mouse. b, Pulmonary metastasis in indicated mice inoculated with PCaM cells was quantified by qRT-PCR analyses of lung RNA with MMTV-ErbB2 specific primers. Means +/− s.e.m. (n=4). c, MT2 cells were transplanted as above. After 56 days, pulmonary metastasis incidence and multiplicity were quantified. Means +/− s.e.m. (n=6). d, MT2 cells were transplanted into Rag1−/− mice reconstituted with indicated cell types, 3 days after reconstitution. After 35 days, lung metastasis incidence and multiplicity were quantified. Means +/− s.e.m. (n=3). e, MT2-inoculated Rag1−/− mice were treated with BSA or RANKL (80 μg/kg) twice weekly, starting one week post-inoculation. After 35 days, lung metastasis incidence and multiplicity were determined. Means +/− s.e.m. (n=6–7). f, MT2 cells were transplanted into Rag1−/− mice reconstituted with indicated cell types as in d. Lung metastases were measured by Q-RT-PCR of ErbB2 mRNA on day 56 post-inoculation. Each plot represents ErbB2 mRNA expression from an independent littermate group. Means +/− s.e.m. (n=3). g, MT2 cells were transplanted into Rag1−/− littermate females reconstituted with PBS, CD4+CD25− or CD4+CD25+ cell, as above. After one week, the CD4+CD25+ reconstituted mice were treated with control Fc or RANK-Fc (2.5 mg/kg) twice weekly. Pulmonary metastasis multiplicity was calculated on day 35 post-inoculation. Means +/− s.e.m. (n=4–5). *: P<0.05; **: P<0.01.

Figure 4. RANKL in human breast cancer

a, RANK expression in human breast cancer cell lines was analyzed by immunoblotting. b, ZR-75-1 cells were transplanted into the #2 mammary glands of Nude mice. Mice were treated with BSA or RANKL (80 μg/kg) twice weekly, starting one week post-inoculation. After 84 days, lung metastasis incidence and multiplicity were determined. Means +/− s.e.m. (n=6). c, Maspin mRNA in ZR-75-1-transplanted tumors treated with BSA or RANKL was analyzed by qRT-PCR. Means +/− s.e.m. (n=6). **: P<0.01. d, Human breast cancer sections were stained with FoxP3- and RANKL-specific antibodies and counterstained with hematoxylin. Arrows: FoxP3+ and RANKL+ cells. CA, carcinoma region. e, Human breast cancer sections were stained with FoxP3- (brown) and SMA- (blue) specific antibodies without counterstaining. f, Tissue arrays containing 50 samples each of ductal carcinoma in situ (DCIS), invasive ductal carcinoma without metastasis(IDC), IDC with metastasis (IDC-Met), and lymph node macro-metastasis (LN-Met) were analyzed for RANKL-expressing cells. Samples were considered positive when at least two cells/field (200x) were RANKL+.