title: S100A4-neutralizing antibody suppresses spontaneous tumor progression, pre-metastatic niche formation and alters T-cell polarization balance.
abstract: BACKGROUND
  The tumor microenvironment plays a determinative role in stimulating tumor progression and metastasis. Notably, tumor-stroma signals affect the pattern of infiltrated immune cells and the profile of tumor-released cytokines. Among the known molecules that are engaged in stimulating the metastatic spread of tumor cells is the S100A4 protein. S100A4 is known as an inducer of inflammatory processes and has been shown to attract T-cells to the primary tumor and to the pre-metastatic niche. The present study aims to examine the immunomodulatory role of S100A4 in vivo and in vitro and assess the mode of action of 6B12, a S100A4 neutralizing antibody.
  
  
  METHODS
  The therapeutic effect of the 6B12 antibody was evaluated in two different mouse models. First, in a model of spontaneous breast cancer we assessed the dynamics of tumor growth and metastasis. Second, in a model of metastatic niche formation we determined the expression of metastatic niche markers. The levels of cytokine expression were assessed using antibody as well as PCR arrays and the results confirmed by qRT-PCR and ELISA. T-cell phenotyping and in vitro differentiation analyses were performed by flow cytometry.
  
  
  RESULTS
  We show that the S100A4 protein alters the expression of transcription factor and signal transduction pathway genes involved in the T-cell lineage differentiation. T-cells challenged with S100A4 demonstrated reduced proportion of Th1-polarized cells shifting the Th1/Th2 balance towards the Th2 pro-tumorigenic phenotype. The 6B12 antibody restored the Th1/Th2 balance. Furthermore, we provide evidence that the 6B12 antibody deploys its anti-metastatic effect, by suppressing the attraction of T-cells to the site of primary tumor and pre-metastatic niche. This was associated with delayed primary tumor growth, decreased vessel density and inhibition of metastases.
  
  
  CONCLUSIONS
  The S100A4 blocking antibody (6B12) reduces tumor growth and metastasis in a model of spontaneous breast cancer. The 6B12 antibody treatment inhibits T cell accumulation at the primary and pre-metastatic tumor sites. The 6B12 antibody acts as an immunomodulatory agent and thus supports the view that the 6B12 antibody is a promising therapeutic candidate to fight cancer.
authors.affiliation: Danish Cancer Society Research Center, 2100, Copenhagen, Denmark. bigru@regionsjaelland.dk.
authors.forename: Birgitte
authors.initials: B
authors.lastname: Grum-Schwensen
authors.affiliation: Institute of Neuroscience and Pharmacology, Faculty of Health Sciences, Copenhagen University, 2200, Copenhagen, Denmark. klingelhofer@sund.ku.dk.
authors.forename: Jörg
authors.initials: J
authors.lastname: Klingelhöfer
authors.affiliation: Danish Cancer Society Research Center, 2100, Copenhagen, Denmark. mkbeck@cbs.dtu.dk.
authors.forename: Mette
authors.initials: M
authors.lastname: Beck
authors.affiliation: Institute of International Health, Immunology and Microbiology, Faculty of Health Sciences, Copenhagen University, 2200, Copenhagen, Denmark. cmenne@sund.ku.dk.
authors.forename: Charlotte Menné
authors.initials: CM
authors.lastname: Bonefeld
authors.affiliation: Danish Cancer Society Research Center, 2100, Copenhagen, Denmark. pkn@cancer.dk.
authors.forename: Petra
authors.initials: P
authors.lastname: Hamerlik
authors.affiliation: Danish Cancer Society Research Center, 2100, Copenhagen, Denmark. perg@cancer.dk.
authors.forename: Per
authors.initials: P
authors.lastname: Guldberg
authors.affiliation: Institute of Neuroscience and Pharmacology, Faculty of Health Sciences, Copenhagen University, 2200, Copenhagen, Denmark. grigorian@sund.ku.dk.
authors.forename: Mariam
authors.initials: M
authors.lastname: Grigorian
authors.affiliation: Danish Cancer Society Research Center, 2100, Copenhagen, Denmark. el@cancer.dk.
authors.forename: Eugene
authors.initials: E
authors.lastname: Lukanidin
authors.affiliation: Institute of Neuroscience and Pharmacology, Faculty of Health Sciences, Copenhagen University, 2200, Copenhagen, Denmark. ambartsumian@sund.ku.dk.
authors.forename: Noona
authors.initials: N
authors.lastname: Ambartsumian
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D057134:Antibodies, Neutralizing / Q000494:pharmacology*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D016207:Cytokines / Q000378:metabolism; D004195:Disease Models, Animal; D018450:Disease Progression; D005260:Female; D008213:Lymphocyte Activation / Q000235:genetics / Q000276:immunology; D018655:Lymphocyte Count; D051379:Mice; D018345:Mice, Knockout; D009362:Neoplasm Metastasis; D009369:Neoplasms / Q000235:genetics / Q000276:immunology* / Q000378:metabolism* / Q000473:pathology; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013154:Spleen / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D059016:Tumor Microenvironment / Q000187:drug effects* / Q000235:genetics / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D016207:Cytokines; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C077591:S100A4 protein, human