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.
Using the 4T1 model of experimental breast cancer we had recently shown that cancer induced bone disease starts before metastatic colonization and is mediated by RANKL expressed by tumor specific T cells. The role of anti-tumor B cell immune response in the context of cancer induced bone disease has never been investigated. There is evidence in the literature that B cells are good prognostic markers for metastatic breast cancer. B cells have an intimate relationship with bone cells as they differentiate from HSC present on endosteal surfaces; cross-talk with skeletal system through the RANK-RANKL-OPG signaling axis; and produce OPG, a decoy receptor of RANKL. Here we used the BALB/c derived 4T1 (metastatic) and 67NR (non-metastatic) sibling cell lines of mammary mouse carcinomas. By day 7, 14 and 21 after tumor injection, B220+ BM B cells from 67NR+ animals produce high amounts of OPG in vitro in contrast to B220+ BM B cells of 4T1+ mice. In vitro, BM B cells from 67NR+ mice, but not from 4T1+, could inhibit the RANKL dependent, anti-4T1 T cell mediated-OC differentiation ascertained by TRAP enzymatic activity, morphology and osteolytic disk assay. Transference of BM B cells from 67NR+ mice together with 4T1 tumor cells to BALB/c mice led to inhibition of osteoclastogenesis, increased numbers of bone lining cells and mesenchyme stem cell. Besides acting directly on the bone remodeling system, these B cells also modulated T cell activity evidenced by diminished RANKL and IL-17F production. All the anti-osteolytic and pro-osteogenic activity of B cells modulate and inhibits the pre-metastatic niche formation. Indeed transference of such cells to 4T1 animals inhibited LN and BM metastatic colonization. We conclude that 67NR induced OPG+ B cells can inhibit pro-osteoclastic / pre-metastatic activity of tumor induced T cells, favoring a bone metastasis free-phenotype. These findings have implications, not only for the understanding of the direct contribution of B cells in the control of bone metastasis but also it might be a promising prognostic tool for predicting cancer-induced bone metastasis. Citation Format: Bonomo A, Monteiro AC, Leal AC, Fontão AP, Spinetti E, Balduino A. OPG+ bone marrow B cells induced by non-metastatic tumors inhibit the pre-metastatic bone niche induced by T cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-24.
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