P1-01-02: T Cell Is a Key Player in the Establishment of Cancer Associated Pre-Metastatic Bone Disease.
Authors: A Bonomo, AC Monteiro, AC Leal, AP Alves, T Frusciante, S Braun, RB Azevedo
Journal: Cancer Research (2011)

Abstract Syngeneic mouse models of malignant diseases have the important advantage of allowing studies to be carried out in immunocompetent animals. In this work, the 4T1(metastatic) and 67NR (non-metastatic) sibling cell lines of mammary mouse carcinomas, syngeneic to BALB/c mouse were used to study tumor specific immune response and its impact on bone disease. By day 11 after tumor injection, in the absence of metastasis, pro-osteoclastogenic cytokines IL17F, TNFα, IL-1β and RANKL were present in the BM of 4T1+ mice in contrast to the virtual absence of these in the BM of 67NR+ animals. More important, imaging studies (histomorphometry and microCT) showed that trabecular bone mass is close to maximum loss (around 50%) already by day 11, in the absence of metastasis. These results support the possibility of T cell involvement in pre-metastatic lesion. Indeed, in vitro, BM T cells from 4T1+ mice, but not from 67NR+, could induce OC differentiation in response to tumor antigens ascertained by TRAP enzymatic activity, morphology and osteolytic disk assay. In vivo transfer experiments showed that T cells from 4T1+ mice (Day 11), when transferred into nude mice produce 3 times more IL17F and 4 times more RANKL than donor 67NR+ T cells. Moreover, trabecular bone at day 14 after transfer of 4T1+T cells was already 30% that of normal nude mice in contrast to 100% from 67NR+ T cells. Of note is the fact that these activities are achieved in the absence of the metastatic 4T1 tumor in the nude host, but challenged with either 67NR or protein antigen extract. To address the role of T cell derived IL17F and RANKL, T cells from 4T1 bearing mice (day 11) were silenced with shRNA specific for each of these cytokines and transferred into nude mice. Animals were challenged with soluble tumor antigen. Silencing IL17F had no effect over bone loss. However, RANKL silencing completely inhibited osteolytic lesions. Together, these results indicate that RANKL in T cells mediate the osteolytic lesions and that cancer induced bone disease can be metastasis independent. Since osteolytic lesions are believed to be important to feed the tumor with growth factors in a vicious cycle established in the metastatic niche, we asked if T cells could be preparing the pre-metastatic niche. When 4T1 cells are injected into athymic nude mice, bone metastasis is not observed up to day 24, while by day 20 it is already present in euthymic BALB/c animals, indicating that T cells are critical to prepare the seeding soil for tumor cells within the bone. We are currently investigating the role of T cells in the establishment of metastasis at later time points as well as in the primary tumor growth. Our results strongly suggest that tumor cells, according to its metastatic activity, can modulate T cell activity systemically. Moreover, and not less important, we show that cancer induced bone disease starts before metastatic colonization and is mediated by RANKL expressed in tumor specific T cells. We propose that this is the very first step on the establishment of the “vicious cycle” which will allow tumor growth. We believe this work can open new avenues on the prognostic evaluation and treatment of women with breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-02.