Bone metastases are currently incurable. Current treatments are palliative at best, including chemotherapy, radiotherapy, targeted therapies, and anti-resorptive therapies that aim to slow down bone destruction induced by cancer cells [
67]. No real evidence exists that patients with bone metastases would benefit from immune cell-targeted therapies. Some immunotherapies have provided survival benefit for advanced cancer patients, including ipilimumab in metastatic melanoma and sipuleucel-T, an immunotherapy manufactured from a patient’s peripheral blood mononuclear cells in advanced prostate cancer [
68]. New research has provided similar findings, for example for pembrolizumab in bone metastatic prostate cancer [
69]. This highlights that better understanding of immune cells in bone metastasis is needed in order to develop effective therapies.
Currently, immune cells, such as T cells (CD4+, CD8+ and Tregs), MDSCs, macrophages, neutrophils, and NK cells, are targeted for treating bone metastases [
70]. However, most bone metastases have typically low immunogenicity compared to the primary tumor, and therefore, they may respond poorly to immunotherapies. This is currently not well acknowledged, which could be due to a lack of understanding the role of the bone microenvironment and osteo-immuno-oncological interactions in controlling disease growth and resistance to therapies, accompanied with lack of knowledge of using appropriate metastasis models in preclinical research and the low availability of biopsies from bone metastases. It is important to understand how current treatments affect the immune landscape in bone metastatic microenvironment, which is shortly discussed in the last sections of this review.
In the next sections, we will discuss the different immune cell subtypes and their effects on bone metastases, which are also summarized in below.
2.1. T Cells
T cell targeting therapies, including checkpoint inhibitors for PD-1, PD-L1, and CTLA-4, were the first immunotherapies approved for treatment of cancer, and there are currently many other therapies emerging [
118].
T cells in bone metastatic microenvironment comprise CD8+ cytotoxic T cells, CD4+ helper T cells, regulatory T cells (Tregs), and natural killer T (NKT) cells. Interactions between T cells and tumor cells in the bone microenvironment are considered one important reason for metastatic relapse [
119]. Early in the metastatic process, T cells are involved in the formation of pre-metastatic niche in tumor cell arrival in bone. In the pre-metastatic niche, T cells induce osteolysis by increasing expression of pro-osteoclastic cytokines including RANKL [
120]. RANKL seems to be a major regulator in the premetastatic niche, since deletion of RANKL from tumor-specific T cells dismisses the formation of metastases, highlighting the importance of this interaction [
120]. Cancer cells in bone metastases produce factors such as PTHrP, IL-7, and IL-8 that can activate T cells [
71,
121]. In contrast, T cells affect bone cells, resulting in increased cancer-induced osteolysis [
71] and the release of factors such as TGF-β that can suppress proliferation and function of T cells and their anti-tumor effects by increasing conversion of CD4+ T cells to Tregs, creating immunosuppressive microenvironment and increasing the formation of metastases [
71,
72,
73]. Furthermore, the transfer of activated T cells to breast cancer patients with pre-existing tumor-reactive bone marrow T cells causes anti-tumor effects especially in patients with bone metastases [
122]. T cells can also regulate tumor growth in bone independently from interactions with bone cells [
68]. For example, RANK/RANKL interactions with CD4+ T cells and breast cancer cells promotes invasion and dissemination of tumor cells and the formation of bone metastases [
73].
CD4+ and CD8+ T cells have anti-tumor activity in both primary and metastatic tumors [
68]. CD4+ T cells are important in orchestrating immune responses in cancer and in priming and survival of CD8+ T cells. Additionally, CD4+ T cells that encounter mutated genes such as fibronectin are involved in tumor metastasis [
74]. CD8+ T cells are mostly responsible for immune-mediated tumor cell death.
Clinical evidence shows that in ovarian cancer patients, together with increased PD-L1, a high number of intratumoral CD8+ T cells correlates with advanced and metastatic stages. This is further supported by co-culture experiments with ovarian cancer cells and CD8+ T cells, where increased migration, invasion, and expression of metastasis-associated genes such as MMP-9, VEGF, IL-8 and IL-10, and PD-L1 are observed [
74]. In fact, anti-MMP-9 treatment of mice with breast cancer increases immune signature pathways responsible for T cell activation [
123]. Interferon regulatory factor 7, together with NK and CD8+ cells, is associated with longer metastasis-free survival in breast cancer [
75]. A study in a spontaneous melanoma mouse model shows that CD8+ T cells have no effect on primary tumor formation, but they control disease progression and formation of metastases [
76]. In prostate cancer, loss of PTEN in bone metastasis is associated with a low number of CD8+ T cells [
77]. This points towards the initiative to treat patients with early metastatic disease compared to late-stage disease patients. At a late stage, the bone metastatic microenvironment may have a low number of or lacking CD8+ T cells. An alternative option is to develop therapies that increase the number of CD8+ T cells in the bone metastatic microenvironment that could be combined with for example checkpoint inhibitors that affect through CD8+ T cells for better anti-tumor response.
CD4+ T cell subsets, Th17 cells and Tregs increase the growth of bone metastases by increasing RANKL-mediated osteoclastogenesis [
78]. Th17 cells have a role in bone marrow immunity irrespective of tumor presence. The number of Th17 cells is increased via IL-1, IL-6, and TGF-β in multiple myeloma bone disease [
26]. RANKL-expressing Th17 cells can later differentiate into Tregs by exposure to TGF-β and aryl hydrocarbon receptor (AhR) in the bone microenvironment [
78]. A high number of bone marrow Tregs is associated with the development of metastatic disease [
124]. Tregs are immunosuppressive cells whose number is increased in almost all cancer patients, and their presence correlates with poor prognosis [
26,
78]. In ovarian cancer, a high number of Tregs is associated with advanced stage but not survival [
79]. The number of Treg increases in bone metastatic prostate cancer patients and Tregs can also regulate cancer-induced bone resorption [
78]. Treg trafficking to bone marrow is mediated by CXCR4/CXCL12 interactions, and bone marrow is the preferential site for migration, retainment, and function of Tregs. Furthermore, RANKL expressing Tregs can promote tumor cells homing to the bone marrow [
78]. COX2 overexpression leads to increased number of Tregs in primary breast cancer and development of bone metastases [
80].
Bone marrow NKTs can regulate immune responses and reject tumor cells [
26]. In metastatic breast cancer cells, expression of the MHC class I-like molecule CD1b is downregulated, disabling antigen presentation to NKT cells, which leads to decreased anti-tumor responses and the formation of metastases [
81].
2.2. NK Cells
Natural Killer (NK) cells are important mediators in tumor immune surveillance, but their role both in bone physiology and bone metastasis is poorly understood [
125]. NK cells recognize antigen-specific receptors such as NKG2D and DNAM1 expressed on tumor cells [
126] or tumor cells that have downregulated expression of MHC molecules [
78]. The dysfunction of NK cells has been observed in many cancers, including prostate cancer, which may be caused by production of reactive oxygen species (ROS) mainly by tumor cells [
78].
The deletion of NK cells induces tumor growth and the formation of metastases [
78]. In an experimental prostate cancer model, metastasis progression was associated with loss of peripheral NK cells [
82]. IFN-γ signaling seems to be critical for pro-metastatic effects of NK cells. For example, NK cells suppress metastasis via IFN signaling in a preclinical breast cancer model [
75]. Injection of murine IL-12 activates NK cells and decreases metastasis, suggesting that IFN-γ is also needed in NK cell -mediated metastasis via IL-12 [
83]. Anti-metastatic potential of NK cells has also been shown in cancer models, where metastases were diminished by the deletion of NK cells, treatment with IFN-γ neutralizing antibody, and in CD39-deficient models [
127]. In an experimental prostate cancer model, overexpression of IRF7 results in increased bone metastases via IFN-beta and increased activity of NK cells [
128]. NK cells prevent formation of metastasis in a humanized mouse model, but metastases are formed when NK cell-mediated tumor differentiation is blocked with IFN-y or TNF-alpha antibodies [
84]. Furthermore, studies with syngeneic breast cancer models in IFN knockout mice have shown that IFN signaling to hematopoietic system determines metastasis-free survival and responsiveness to circulating NK cells [
85].
There are also other factors that affect NK cell-mediated immune responses. Core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) expressed on tumor cells disturbs NK-mediated immune responses and cancer cell apoptosis in bone metastasis [
78]. The activity of TAM tyrosine kinase receptors regulates NK cells and the formation of metastases in a breast cancer model [
78]. IL-17A is a major regulator of NK cells, and studies in IL-17A knockout mice shows suppression of metastases caused by increased maturation and activity of NK cells [
86]. IL-28R is important for NK cell function and IL-28R knockout mice have an elevated number of metastases [
87]. Studies in NK cell-depleted mice show that treatment with PTHrP neutralizing antibody [
129], the bisphosphonate minodronate [
88], the follistatin inhibitor activin [
130], the VEGF antibody bevacizumab, and the bisphosphonate zoledronic acid [
131] inhibit bone metastasis from lung cancer cells. JAK/STAT pathway is active in breast cancer patients and in preclinical models, but inhibition of JAK signaling increases metastatic tumor burden due to the impairment of NK-mediated anti-tumor activity [
89].
As discussed previously, TGF-β is important both for bone and immune cells. In patients, high TGF-β serum levels correlate with NK cell -mediated immunosuppression and poor clinical outcome [
68]. Inhibition of TGF-β signaling increases the anti-tumor activity of NK cells and prevents the formation of bone metastases in an experimental breast cancer model [
90], but the direct effects between TGF-β and NK cells remain to be studied.
2.3. MDSCs
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells generated in the bone marrow from immature myeloid cells. Normally, immature myeloid cells differentiate into mature myeloid cells including macrophages and dendritic cells. With cancer, this normal differentiation is disturbed by cancer cell-produced immunosuppressive factors, which causes immature myeloid cells to proliferate, differentiate, and become active MDSCs [
78,
86]. Immunosuppressive factors responsible for this include, for example, arginase I, inducible nitric oxidase synthase (iNOS), TNF-α, and TGF-β [
68,
78]. MDSCs accumulate to almost all tumor types and create an immunosuppressive tumor microenvironment by secreting factors such as arginase I [
132], inducible nitric oxidate synthase (iNOS) [
133], TGF-β and IL-10 [
134], as well as other immunosuppressive factors [
135]. MDSCs suppress tumor-directed immune responses by suppressing T cell proliferation, inducing T cell apoptosis, activating Tregs for immunosuppression, and decreasing the activity of NK cells. Cytokines activating metastasis that are linked to MDCS include CCL5, CCL15 [
136], and pro-metastasis proteins such as MMPs and chemoattractants [
91]. Furthermore, monocytic MDSCs are associated with a trend towards decreased survival in patients with liver and bone metastases [
92].
MDSCs can further direct metastasis to the bone. Increased number of MDSCs is associated with increased bone metastasis in a breast cancer animal model [
78]. Interestingly, the number of MDSCs is increased in the metastatic location in a breast cancer model, but after the removal of the tumor, the number of other than granulocytic MDCSs decreases in the metastatic location [
93]. Furthermore, the prevention of tumor accumulation of granulocytic MDSCs leads to anti-metastatic effects in a breast cancer model [
94]. High expression of the TGFβ growth factor family member BMP4 results in inhibition of metastasis [
95]. Inhibition of CXCR4 decreases the number of MDSCs and Tregs, resulting in the progression of bone metastases [
127]. The treatment of mice with a conjugate of paclitaxel and a muramyl dipeptide analogue decreases tumor growth and metastasis in breast and lung cancer models by suppression of MDSCs [
137]. Furthermore, dexamethasone treatment decreases myeloid MDSCs and results in decreased tumor growth [
97].
MDSCs have also an important function in bone, and the number of MDSCs in bone metastasis in higher compared to primary tumors or other metastatic locations [
73]. In a breast cancer mouse model, injection of MDSCs induces reportion in mice with bone metastases but not in healthy mice [
138]. Bone marrow MDSCs can differentiate into osteoclasts [
98] and contribute to cancer-induced bone destruction [
138], therefore contributing to the vicious cycle of bone metastasis. MDSCs also contribute to the production of IL-17 in bone, which can increase osteoclastogenesis via RANKL [
68]. MDSCs express CCR2 and CCL2, which are important for osteoclastogenesis, and cancer cells secrete CCL2, CCL5, and osteopontin, which enhance osteoclast function [
73]. Additionally, MDSCs enhance the growth of bone metastases independently of their ability to differentiate into functional osteoclasts [
73].
MDSCs provide an interesting target for anti-cancer therapy, and currently, there are many therapeutics that affect MDSCs [
139]. From a bone metastasis perspective, these include, for example, zoledronic acid, which decreases the number of circulating MDSCs by inducing their apoptosis, chemokine receptor antagonists of CCR2, CXCR2, and CXCR4, and chemokine inhibitors of CCL2, CXCL5, and CXCL12, which inhibit MDSC accumulation into tumor [
78].
2.4. Macrophages
In tumor microenvironments, macrophages can become tumor-associated macrophages (TAMs) [
140]. There are two different subsets of macrophages that polarize when stimulated by different cytokines in their tissue microenvironment, pro-inflammatory M1/M1-like and anti-inflammatory M2/M2-like macrophages [
78,
141]. M1 macrophages and secrete proinflammatory cytokines such as IL-1, IL-6, IL-12, IL-23, and IFN-γ that can activate T and NK cells [
78]. M2/M2-like macrophage differentiation is influenced by cytokines such as TGF-β and IL-10 [
141], and they secrete anti-inflammatory cytokines such as IL-10, TGF-β, CCL17, CCL18, CCL22, and CCL24, and promote tumorigenesis and later development of metastases [
141].
TAMs are activated by IL-10 and TGF-β, secrete high levels of cytokines that decrease the activation of T cells, and participate in tumor progression and the formation of metastases. For example, BMP4-containing conditioned medium collected from bladder cancer cells increases M2 macrophage differentiation [
99], IL-6 and GM-CSF produced by cancer-associated fibroblasts induce M2 macrophage differentiation [
142], and caspase-1 promotes TAM differentiation by cleaving PPARy, which then interacts with medium-chain acyl-CoA dehydrogenase (MCAD) [
100]. Furthermore, inhibition of caspase-1 or deletion of MCAD decreases tumor growth.
A high number of TAMs correlates with poor clinical outcome in patients in many cancers [
78]. However, a study in osteosarcoma patients without metastases shows increased infiltration of both M1 and M2 macrophages with overall improved survival and reduced metastasis [
101], which might indicate that the balance between M1 and M2 macrophages is important in this aspect. TAMs can be targeted in cancer by depletion, reprogramming, or molecular targeting [
78].
TAMs potentiate bone metastasis [
143], which has been shown for example in lung cancer, where macrophages migrate towards CXCL14 expressing bone-seeking cells more efficiently than towards parental lung cancer cells [
102], and deletion of macrophages suppresses bone metastasis of lung cancer [
103]. In a prostate cancer bone metastasis model, M2 macrophages are observed and treatment with trabectedin reduces M2 macrophages and growth of bone metastases [
104]. A high expression of WNT5A is associated with high levels of CCL2, and BMP6 is commonly observed in prostate cancer cells growing in a bone microenvironment [
144]. Furthermore, deletion of macrophages decreases the castration resistance of prostate cancer cells [
144]. CD169+ macrophages support tumor growth and metastasis in breast cancer [
105], and when they are deleted, the number of CD8+ T cells increases but bone loss remains the same [
105]. Deletion of mTOR signaling complex 2 (mTORC2) from monocytes and their injection to mice in a breast cancer model results in prevention of the monocytes to differentiate to M2 macrophages and formation of distant, including bone, metastases [
106]. Bone marrow-derived macrophages are recruited by tumor chemokines such as CCL2 and CSF-1 or their receptors and promote metastasis to bones. CSF-1/CSF-R1 targeted therapy provides anti-metastatic effects [
78], and CCL2 and CCLR2 signaling causes accumulation of TAMs into tumors, which reduces metastasis in animal models [
78]. However, CCL2-targeted therapy increases metastasis via increased angiogenesis in a breast cancer model [
78].
Macrophages also have effects on cancer-induced formation of new bone. In prostate cancer bone metastasis specimens, CD68+ macrophages are consistently located in the tumor, compared to variable distribution of lymphocytes, and osteal macrophages are located near pathologic new bone [
107]. Deletion of CD169+ macrophages inhibits the formation of pathologic new bone in a prostate cancer model, and deletion of other macrophages than CD169+ increases tumor growth [
107]. Furthermore, CD137+ cells promote the migration of macrophages and monocytes to tumor and further promote their differentiation into osteoclasts, increasing cancer-induced bone destruction in a spontaneous breast cancer model [
145].
2.5. Dendritic Cells
Dendritic cells (DCs) are antigen-presenting cells that regulate T cell responses by increasing their proliferation and activation [
78]. DCs are divided into monocytic DCs (mDCs) and plasmacytoid DCs (pDCs), and pDCs are mainly studied in immuno-oncology [
68].
Circulating DCs migrate to bone marrow and engage with VCAM-2 and selectin-expressing cells. DCs that are infiltrated to bone metastases derived from low-immunogenic tumors are typically immature and lack antigen-presenting capacity [
68]. Furthermore, prostate cancer bone metastatic microenvironment inhibits DC maturation [
68]. RM-1 prostate cancer cells growing in bone marrow decrease DC generation and their capability to increase T cell proliferation [
108]. This might be partially due to the expression of PD-L1 in bone marrow DCs and PD-L1-mediated deactivation of CD8+ T cells [
68]. In breast cancer, DCs purified from cancer patients have a decreased ability to stimulate T cells [
78], and tumor-infiltrating DCs suppress the function of CD8+ T cells via TGF-β, NO, IL-10, VEGF, and arginase I [
78]. The number of pDCs is increased in a breast cancer bone metastasis model [
78]. DCs increase metastasis and decrease survival in Batf3-deficient mice, but they have no effect on primary tumors [
83]. The increase in metastasis via Batf3 requires NK cells and IFN-γ, but not CD4+ or CD8+ T cells. DCs can also recruit immunosuppressive Treg and MDSCs into tumor [
78].
Tumors with high TGF-β expression have less DCs [
109]. TGF-β inhibitor induces maturation of DCs with increased production of IL-12, and can potentially activate DCs into NK cells [
110]. Inhibition of CD115/CSF-1 decreases osteoclast differentiation and prevents monocyte differentiation to M2 macrophages and instead promotes their differentiation into DCs [
146].
Sipuleucel-T is a DC-targeted therapy for castration-resistant prostate cancer (CRPC) patients that provides survival benefit [
68]. DC-based therapeutic approaches are related to vaccines, for example in breast, prostate, and lung cancer, and these vaccines can also prevent the formation of metastases [
78]. DC vaccines also require NK cells and CD8+ cells to function properly [
147]. The prevention of metastases is seen with mature DCs and 5-FU treatment [
78]. Combination of stontium-89 and DC vaccination therapy provides a good response in patients with bone metastases [
111]. Activation of DC with tumor-lysates and combination with anti-CTLA-4 treatment increases the number of CD8+ T cells, decreases the number of Tregs, and inhibits metastatic growth [
112]. However, infiltration of DCs into tumors after injections may be problematic [
148].
2.6. Neutrophils
Mature neutrophils reside in bone marrow and bone marrow regulates their homeostasis via the CXCR4/CXCL12 signaling pathway [
78]. Different chemokines regulate the anti- or pro-tumoral effects of neutrophils [
113], and, for example, the inactivation of atypical chemokine receptors (ACKRs) results in the release of neutrophils from bone marrow that have anti-metastatic activity [
114]. Neutrophils regulate formation of pre-metastatic niche [
115]. Interestingly, lung carcinoma increases bone stromal activity in the pre-metastatic niche by affecting neutrophils that express high levels of SiglecF through osteocalcin-expressing osteoblastic cells [
149]. Neutrophils also have a role in early metastasis development, as a high number of migrating neutrophils with immunosuppressive properties are observed in tumor-bearing mice [
116]. In tumor microenvironments, neutrophils can become tumor-associated neutrophils (TANs) that can be either tumor-inhibiting (N1) or tumor-promoting (N2). TGF-β is an important mediator in differentiating between N1 and N2 neutrophils [
78]. N2 neutrophils promote angiogenesis, dissemination of tumor cells, and metastasis formation, including metastasis to bones [
78]. N2 neutrophils can also release CXCR4, VEGF, and MMP9, which all have well-defined roles in metastasis.
The neutrophil-to-lymphocyte ratio is an independent prognostic marker in patients with bone metastases [
150,
151]. The neutrophil-to-lymphocyte ratio correlates with PSA, being high in patients with bone metastatic prostate cancer [
127]. However, the neutrophil-to-lymphocyte ratio seems to be typically higher in patients with soft tissue metastases [
152]. Furthermore, prostate cancer stimulates recruitment of neutrophils that infiltrate heavily to bone metastatic lesions, and at early stages, neutrophils induce prostate cancer cell apoptosis, but at later stages, they fail to execute cytotoxic responses [
117].