PLoS One 2013 Jul 18;8(7):e68171. doi: 10.1371/journal.pone.0068171

T cells induce pre-metastatic osteolytic  disease and help bone metastases establishment in a mouse model of metastatic breast cancer

Ana Carolina Monteiro1,5, Ana Carolina Leal1, Triciana Gonçalves-Silva1,5,6, Ana Carolina T. Mercadante1,5, Fabiola Kestelman2, Sacha Braun Chaves3, Ricardo Bentes Azevedo3, João P. Monteiro4 and Adriana Bonomo 1,5,6

1Experimental Medicine Program, Brazilian National Cancer Institute, Rio de Janeiro, BRAZIL; 2HCIII, Brazilian National Cancer Institute, Rio de Janeiro, Brazil; 3University of Brasilia, Institute of Biology, Brasilia, Brazil; 4Lymphocyte Biology Section, Laboratory of Systems Biology/NIAID/NIH, USA, 5Laboratory on Thymus Research, Oswaldo Cruz Institute, FIOCRUZ, Brazil and 6Immunology Department, Microbiology Institute Prof. Paulo de Góes, Federal University of Rio de Janeiro, Brazil.

Corresponding author:

Adriana Bonomo, Department of Immunology, Paulo de Góes Microbiology Institute, Federal Universisty of Rio de Janeiro, CCS/BLI/Sl. I2 062 – Ilha do Fundão, Rio de Janeiro – RJ – 22.290-140, Phone: +55 21 25626748; Fax: +55 21 25608344. Email : abonomo@micro.ufrj.br

 

ABSTRACT

Bone metastases, present in 70% of patients with metastatic breast cancer, lead to skeletal disease, fractures and intense pain, which are all believed to be mediated by tumor cells (Mundy, 2002 and Roodman, 2004). Engraftment of tumor cells is supposed to be preceded by changes in the target tissue to create a permissive microenvironment, the pre-metastatic niche, for the establishment of the metastatic foci (Fidler, 2003 and Joyce and Pollard, 2009). In bone metastatic niche, metastatic cells stimulate bone consumption resulting in the release of growth factors that feed the tumor, establishing a vicious cycle between the bone remodeling system and the tumor itself (Mundy, 2002 and Roodman, 2004). Yet, how the pre-metastatic niches arise in the bone tissue remains unclear. In this study, we showed that tumor-specific T cells induced osteolytic bone disease before bone colonization. T cells pro-metastatic activity was correlated with a pro-osteoclastogenic cytokine profile, including RANKL, a master regulator of osteoclastogenesis. In vivo inhibition of RANKL from tumor-specific T cells completely blocked bone loss and metastasis. Our results unveiled an unexpected role for RANKL-derived from T cells in setting the pre-metastatic niche and promoting tumor spread. We believe this information can bring new possibilities for the development of prognostic and therapeutic tools based on modulation of T cell activity for prevention and treatment of bone metastasis.

PMID: 23935856

 

SUPPLEMENTARY

Although immune activity is classically linked to anti-tumor activity several reports were published in the past linking immunity to tumor progression (Hirsch and Iversen, 1961; Martinez, 1964 and Yunis et al., 1969). In our work we showed that indeed this can be the case. Using a mouse model of breast cancer (4T1), we demonstrated that RANKL production by tumor-primed CD4+ T cells is required for development of bone metastasis. We reached this conclusion by first showing that the metastatic 4T1 tumor, but not its non-metastatic 67NR sibling, induced production of pro-osteoclastogenic cytokines, including IL-17F and RANKL by CD4+ T cells. Production of such cytokines leading to OC formation and activation, and osteolytic disease, was observed even before tumor cells had colonized the bone cavity, suggesting that CD4+ T cells prepare the metastatic niche for further establishment of tumor cells in the model used. Inhibition of RANKL production by tumor-primed CD4+ T cells protected mice from osteolytic disease and, surprisingly, completely abolished the development of bone metastases (Figure 1).

We believe that the characterization of T cell-induced pre-metastatic osteolytic disease adds an extra step to the vicious cycle hypothesis (Figure 2). Tumor cells are believed to establish themselves in the bone marrow through mechanisms that culminate in the release of growth factors from the bone matrix as a consequence of osteoclast activity.  Our results suggested that in the presence of metastatic tumors, antigen-specific T cells are primed and acquire a pro-osteoclastogenenic phenotype. Following their migratory pattern, tumor-specific primed T cells expressing RANKL migrate to the bone cavity, before tumor cells colonize it, and once there they stimulate the differentiation and activation of OCs. Pre-metastatic T cell mediated bone consumption generates a rich environment that will allow the colonization of the bone cavity by the metastatic clones. Once initial seeding of the bone tissue is achieved, the tumor cells can continue the osteolytic process on their own, feeding themselves through the vicious cycle established with the bone microenvironment.

Altogether, our results unveiled an uncommon perspective of tissue-specific immune activation leading to progression of cancer and identify T cells as a major player in pre-metastatic osteolytic disease and development of bone metastasis.

 

REFERENCES

Fidler IJ. (2003) The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited. Nat Rev Cancer 3:453-458.

Hirsch HM, Iversen I. (1961) Accelerated development of spontaneous mammary tumors in mice pretreated with mammary tumor tissue and adjuvant. Cancer Res 21:752-760.

Joyce JA, Pollard JW. (2009) Microenvironmental regulation of metastasis. Nat Rev Cancer 9:239-52.

Martinez C. (1964) Effect of early thymectomy on development of mammary tumors in mice. Nature 203:1188.

Mundy GR. (2002) Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer 2:584-593.

Roodman GD. (2004) Mechanisms of bone metastasis. N Engl J Med 350:1655–1664.

Yunis EJ, Martinez C, Smith J, Stutman O, Good RA. (1969) Spontaneous mammary adenocarcinoma in mice: influence of thymectomy and reconstitution with thymus grafts or spleen cells. Cancer Res 29:174-178.

 

Adriana Bonomo-Fig1

Figure 1: T cell-induced osteoclastogenesis and bone loss requires expression of RANKL in T cells which “helps” metastatic colonization (A) LN cells obtained from 4T1 tumor-bearing BALB/c mice (T 4T1), 11 days after tumor injection, were transfected with shRNA for IL17F (shIL17-F), RANKL (shRANKL), scramble (scr), or both, RANKL and IL17F (Db sh), transferred i. v. into athymic nude mice and challenged with soluble tumor antigen (sAg). Bone sections from recipient mice were prepared and the number of TRAP+ OCs/mm of bone surface was determined. (B) High resolution µCT of iliac bones from the different groups of nude mice transferred with the indicated T cells. Results shown are representative of two experiments with 5 mice/group). *p≤0.05; **p≤0.001. (C-D) Number of metastatic clones in the LNs and iliac BMs was assessed by clonogenic metastatic assay in the recipient mice on day 12 and 28. Nude, non-reconstituted control; T 4T1; reconstitution with 4T1 T cells; sh scr, sh Scramble T 4T1; sh RANKL, sh RANKLT 4T1. Results shown are representative of two experiments with 6 mice/group). **p≤0.001.

 

Adriana Bonomo-Fig2Figure 2: RANKL+ tumor-specific T cells prepare the bone pre-metastatic niche. After being stimulated (1) and modulated (2) by tumor cells T cells migrate to the bone marrow (3). When inside the bone marrow niche, T cell derived RANKL stimulate osteoclastogenesis (4) with bone consumption before tumor bone colonization (5). This initial bone loss induced by T cells in response to tumor antigen, prepares the bone marrow niche to receive tumor cells (6). Once inside the marrow, tumor cells will be able to establish themselves comfortable (7), at the expense of the pre-metastatic niche already set by T cell derived RANKL in response to tumor stimulation.

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