*3.3.2 Role of breast-tumor-primed T and B cells in bone pre-metastatic niche formation*

Primary breast cancer has been shown to "prepare" distant organs for tumor cell colonization even before their arrival [171, 192, 193]. Immune cells such as macrophages [194, 195], DCs [196], neutrophils [197], and T cells [64, 195, 198, 199] are associated with the formation of the pre-metastatic niches, highlighting the importance of basic mechanisms responsible for tumor cells distant establishment [171, 200]. Accordingly, it has been found that cells of the immune system acting as pro-tumor cells are enriched in the pre-metastatic niches and support cancer cell seeding via paracrine signaling and/or by suppressing anti-tumor immune cells [171, 172, 200, 201].

Particularly, our group previously showed that spontaneous bone metastases development, originated from 4 T1 triple negative breast tumor model, depends on RANKL production by tumor primed CD3+ T cells [64]. This conclusion was achieved by adoptive T cell transference to nude mice, which shows that 4 T1 primed T cells, in the total absence of tumor cells, induce a pre-metastatic osteolytic disease [64]. Moreover, inhibition of RANKL production (using shRNA) in fresh tumor-primed T cells does not generate osteolytic disease and the associated bone pre-metastatic niche. Consequently, development of bone metastases is completely absent. Taking together, we proposed an extra step to Mundy's vicious cycle where initial bone consumption, mediated by pre-metastatic CD3<sup>+</sup> T cells, generates a rich microenvironment that license further colonization of the bone cavity by the metastatic clones [64]. Once the initial seeding of the bone tissue is achieved, tumor cells shall continue the osteolytic process on their own, feeding themselves through the vicious cycle established within the bone microenvironment [64].

As pre-metastatic osteolytic disease happens much before metastatic colonization, it is not known how the tumor Ag would get to the BM to be recognized by T cells. This is important because T cells' effector functions depend on peptide recognition complexed to MHC molecule, a function better exerted by DCs. Since DCs can carry Ag from peripheral tissues via lymphatics to LNs, and also travel from the peripheral tissue into the blood and to the BM [202, 203], we envisage at least two nonexclusive possibilities for Ag presentation and recognition: (i) cancer-derived exosomes could travel to the bone cavity and provide tumor Ags to be processed and presented by local resident DCs [204, 205] and/or (ii) DCs loaded with tumor Ags at the primary tumor or at the tumor draining LNs, can migrate to the BM where Ag presentation would take place [203, 206]. Moreover, it is already known that BM can prime naive T cells and recruit effector T cells, also serving as a site for CD4+ and CD8+ T cells proliferation [202].

In addition, DCs display a high developmental and functional plasticity depending on local factors and stimuli encountered during their differentiation and maturation, providing a multitude of necessary signals for shaping immune responses [207–210]. Plasticity can also allow DCs to develop into other cell types, among them OCs (DC-OC), what is not unexpected considering their same origin from common myelopoietic stem cell progenitors [211–213]. Indeed, for the last 15 years, it has been reported that immature DCs can develop into OCs *in vitro* and *in vivo*, when cultured with osteoclastogenic factors, M-CSF and RANKL or RA synovial fluids containing pro-osteoclastogenic cytokines [212, 214, 215]. Independently of the presence of DCs at bone resorptive sites during inflammatory conditions [211, 216–221], their direct contribution to bone resorption, either as APCs, keeping osteoclastogenic Th17 T cells locally activated, or overcoming their own phenotype differentiating into OCs mature functional phenotype, has yet to be solved. Indeed, it has been confirmed that multinucleated giant cells expressing markers of DCs and OCs are located next to the bone in inflammatory bone disease [222].

In fact, we recently addressed the role of DCs in breast-tumor-derived bone metastases context [223, 224]. We showed that DC-OC differentiation is induced by

### *Perspective Chapter: Breast-Tumor-Derived Bone Pre-Metastatic Disease – Interplay... DOI: http://dx.doi.org/10.5772/intechopen.107278*

RANKL, either recombinant or produced by specific-tumor T cells [224], and they can act as both an APC for 4 T1 tumor-specific T cells and as an OC-like cell (DC-OC), amplifying the osteolytic phenomena before bone tumor colonization [224]. Furthermore, it is already known that the pretreatment of DCs with high levels of RANKL leads to enhancement of [76] and augments their ability to stimulate T cell proliferation [225–227]. Therefore, we can suppose that RANKL-enriched environment setup by osteoclastogenic CD3+ T cells located inside the BM probably contributes to a higher DC survival ratio, which in turn would support T cells' activities in promoting the pre-metastatic niche formation [224]. Additionally, DC-OCs, but not BM-OCs, are incredibly good in activating T cell proliferation and cytokine secretion [224], and secrete high amounts of IL-23, which in turn boosts IL-17 and RANKL production by T cells, feeding the positive osteoclastogenic loop of adaptive T cell immunity [224]. This positive loop has IL-23 as one limiting step since blocking IL-23 with monoclonal antibody inhibits T cell IL-17 and RANKL production [224]. Adding more information to our work, recent data published [228] showed that monocytederived macrophages, rather than bone-residing macrophages, are critical for breastcarcinoma-derived bone metastases outgrowth *in vivo*, in IL-4R and CCR2-dependent manners [228].

More recently, we described that 67NR non-metastatic tumor cells—an *in situ* breast carcinoma sibling of 4 T1 tumor cell line, can modify distant sites promoting bone physiological alterations, increasing in trabecular bone mass on day 11 post-tumor implant [229]. This observation was associated with an expansion of the osteoblastic lineage cells accompanied by a reduction of OCs numbers [229]. Moreover, CD8+ T cells express an anti-osteoclastogenic cytokine milieu enriched by IFN-γ, IL-10 and low levels of RANKL, and the frequency of BM-derived CD8+ FoxP3<sup>+</sup> regulatory T cells, as defined as potent suppressors of osteoclastogenesis, was also increased in such animals [229]. This milieu was capable to suppress 4 T1 tumor-specific CD4+ T cells phenotype *in vivo* and *in vitro* and strongly inhibited bone metastases establishment, restoring trabecular bone mass volume [229]. We concluded that the 67NR+ tumor derived CD8<sup>+</sup> T cells phenotypes, either contributing to bone homeostasis and/or control of 4 T1 breast tumor pre-metastatic disease, interfere with OCs and OBs activities inside BM. Our study highlights the opposing roles of subverted tumor CD4+ and CD8+ T cell subtypes in directing breast cancer progression and bone metastases establishment. Furthermore, this likely reflects the fact that modification of the distant bone site by 67NR breast tumor disfavors premetastatic bone niche formation [229].
