VEGF-A and Tenascin-C produced by S100A4+ stromal cells are important for metastatic colonization - PubMed (original) (raw)

. 2011 Sep 20;108(38):16002-7.

doi: 10.1073/pnas.1109493108. Epub 2011 Sep 12.

Hikaru Sugimoto, Vesselina G Cooke, Brian A MacDonald, Ankit I Mehta, Valerie S LeBleu, Rajan Dewar, Rafael M Rocha, Ricardo R Brentani, Murray B Resnick, Eric G Neilson, Michael Zeisberg, Raghu Kalluri

Affiliations

VEGF-A and Tenascin-C produced by S100A4+ stromal cells are important for metastatic colonization

Joyce T O'Connell et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Increased numbers of S100A4(+) cells are associated with poor prognosis in patients who have cancer. Although the metastatic capabilities of S100A4(+) cancer cells have been examined, the functional role of S100A4(+) stromal cells in metastasis is largely unknown. To study the contribution of S100A4(+) stromal cells in metastasis, we used transgenic mice that express viral thymidine kinase under control of the S100A4 promoter to specifically ablate S100A4(+) stromal cells. Depletion of S100A4(+) stromal cells significantly reduced metastatic colonization without affecting primary tumor growth. Multiple bone marrow transplantation studies demonstrated that these effects of S100A4(+) stromal cells are attributable to local non-bone marrow-derived S100A4(+) cells, which are likely fibroblasts in this setting. Reduction in metastasis due to the loss of S100A4(+) fibroblasts correlated with a concomitant decrease in the expression of several ECM molecules and growth factors, particularly Tenascin-C and VEGF-A. The functional importance of stromal Tenascin-C and S100A4(+) fibroblast-derived VEGF-A in metastasis was established by examining Tenascin-C null mice and transgenic mice expressing Cre recombinase under control of the S100A4 promoter crossed with mice carrying VEGF-A alleles flanked by loxP sites, which exhibited a significant decrease in metastatic colonization without effects on primary tumor growth. In particular, S100A4(+) fibroblast-derived VEGF-A plays an important role in the establishment of an angiogenic microenvironment at the metastatic site to facilitate colonization, whereas stromal Tenascin-C may provide protection from apoptosis. Our study demonstrates a crucial role for local S100A4(+) fibroblasts in providing the permissive "soil" for metastatic colonization, a challenging step in the metastatic cascade.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Ablation of S100A4+ stromal cells inhibits metastatic colonization of orthotopically implanted 4T1 cancer cells. S100A4+ stromal cells (green) with DAPI nuclear counterstain (blue) in normal mammary tissue vs. 4T1 mammary tumor tissue (A) and normal lung tissue vs. 4T1 metastatic lung tissue (B) from S100A4-GFP transgenic mice. (Scale bars: 50 μm.) Primary tumor volume (C) and percent metastatic area in lung tissue (D) of GCV-treated S100A4-tk mice (n = 10) and control littermates (n = 10) 24 d after orthotopic implantation of 4T1 cancer cells. (E) Representative H&E-stained lung sections with dotted lines encircling metastatic lesions. (Scale bars: 400 μm.) Quantification of BrdU+ (F) and TUNEL+ (G) cancer cells within metastatic nodules. (H) Microvessel density was quantified by CD31 staining (red) with DAPI nuclear counterstain (blue) in primary tumors and within metastatic nodules. (Scale bars: 20 μm.) Mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

Fig. 2.

Ablation of S100A4+ stromal cells inhibits metastatic colonization of i.v. inoculated 4T1 cancer cells. (A) Percent metastatic area in lung tissue of GCV-treated S100A4-tk mice (n = 6) and control littermates (n = 6). Representative H&E-stained lung sections are displayed. Arrows point to metastatic lesions. (Scale bars: 160 μm.) Quantification of BrdU+ (B) and TUNEL+ (C) cancer cells within metastatic nodules. (D) Quantification of microvessel density by CD31 staining (red) with DAPI nuclear counterstain (blue) within metastatic nodules. Dotted lines encircle metastatic lesions. (Scale bars: 20 μm.) Mean ± SEM. **P < 0.01; ***P < 0.001.

Fig. 3.

Fig. 3.

Bone marrow-derived S100A4+ stromal cells do not functionally contribute to metastatic colonization. (A) S100A4+ (green) and CD45+ (red) stromal cells in metastatic lung tissue of S100A4-GFP mice and WT mice bearing an S100A4-GFP bone marrow (BM) transplant. White arrows point to S100A4-GFP+ stromal cells; yellow arrowheads point to S100A4-GFP+/CD45+ stromal cells. (Scale bars: 20 μm.) (B) Quantification of metastatic area in WT mice transplanted with WT bone marrow (WT BM > WT, n = 13), S100A4-tk mice transplanted with S100A4-tk bone marrow (TK BM > TK, n = 13), WT mice transplanted with S100A4-tk bone marrow (TK BM > WT, n = 10), and S100A4-tk mice transplanted with WT bone marrow (WT BM > TK, n = 12). Representative H&E-stained lung sections are displayed with dotted lines encircling metastatic lesions. (Scale bars: 50 μm.) Mean ± SEM. *P < 0.05.

Fig. 4.

Fig. 4.

Loss of stromal Tenascin-C inhibits metastatic colonization. (A) Representative images of the cancer cell marker luciferase (red) and Tenascin-C (green) as well as S100A4-tk (red) and Tenascin-C (green) double staining within metastatic lung tissue of control and GCV-treated S100A4-tk mice. (Scale bars: 20 μm.) The bar graph summarizes the relative Tenascin-C (TN-C) antibody staining between the two groups. (B) Percent metastatic area and microvessel density in lung tissue of TN-C KO (n = 4) and WT (Wildtype) littermates (n = 6) 4 d after i.v. inoculation of 4T1 cancer cells. Representative H&E-stained lung sections. (Scale bars: 50 μm.) CD31 staining (red) with DAPI nuclear counterstain (blue). (Scale bars: 20 μm.) Metastatic lesions are encircled by dotted lines. (C) Primary tumor volume of TN-C KO (n = 6) and WT littermates (n = 4) 24 d after orthotopic implantation of 4T1 cancer cells. Mean ± SEM. *P < 0.05; **P < 0.01.

Fig. 5.

Fig. 5.

S100A4+ stromal cells provide VEGF-A to the metastatic microenvironment to support metastatic colonization. (A) Representative images and quantification of VEGF-A (green) and S100A4-tk (red) immunostaining in metastatic lung tissue of control and GCV-treated S100A4-tk mice. (Scale bars: 20 μm.) (B) Primary tumor volume of S100A4-Cre;VEGF-Aflox/flox mice and control littermates 24 d after 4T1 orthotopic implantation. Representative H&E-stained lung sections and quantification of metastatic area from S100A4-Cre;VEGF-Aflox/flox (S100A4-Cre;VEGF-Af/f) mice and control littermates after 4T1 orthotopic (C) and 4T1 i.v. (D) injections (n = 5–8 in each group). (Scale bars: C, 50 μm; D, 100 μm). (E) Microvessel density was quantified by CD31 staining (red) with DAPI nuclear counterstain (blue) within metastatic lesions of these mice. (Scale bars: 50 μm.) Dotted lines encircle metastatic lesions. Mean ± SEM. **P < 0.01; ***P < 0.001.

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