Abstract
Myeloid-derived suppressor cells (MDSCs) are bone marrow (BM)-derived immunosuppressive cells in the tumor microenvironment, but the mechanism of MDSC mobilization from the BM remains unclear. We investigated how BM stromal cell activation by PTH1R contributes to MDSC mobilization. PTH1R activation by parathyroid hormone (PTH) or PTH-related peptide (PTHrP), a tumor-derived counterpart, mobilized monocytic (M-) MDSCs from murine BM without increasing immunosuppressive activity. In vitro cell-binding assays demonstrated that α4β1 integrin and vascular cell adhesion molecule (VCAM)-1, expressed on M-MDSCs and osteoblasts, respectively, are key to M-MDSC binding to osteoblasts. Upon PTH1R activation, osteoblasts express VEGF-A and IL6, leading to Src family kinase phosphorylation in M-MDSCs. Src inhibitors suppressed PTHrP-induced MDSC mobilization, and Src activation in M-MDSCs upregulated two proteases, ADAM-17 and MMP7, leading to VCAM1 shedding and subsequent disruption of M-MDSC tethering to osteoblasts. Collectively, our data provide the molecular mechanism of M-MDSC mobilization in the bones of tumor hosts.
Similar content being viewed by others
Introduction
Antitumoral T-cell immunity in the tumor microenvironment is counteracted by several types of immunosuppressive bone marrow-derived cells, such as tumor-associated macrophages, tumor-associated neutrophils and myeloid-derived suppressor cells (MDSCs).1,2,3 Although these cells have important functions in tumor tissue, key questions remain unanswered, such as how bone marrow-derived immune cells are regulated and/or mobilized within the bone marrow of tumor hosts. The aim of this study was to elucidate the molecular mechanism of MDSC mobilization within the bone marrow of breast cancer patients and mouse models.
MDSCs are a subset of immature myeloid-lineage bone marrow cells in tumor tissue and are known for their T-cell suppressive activity by expressing arginase 1 (Arg1), transforming growth factor (TGF) β, inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), etc.4 Numerous lines of evidence clearly support the existence and function of MDSCs in human cancer patients and murine tumor models.5,6,7 Two major subpopulations of MDSCs, polymorphonuclear (PMN-, or granulocytic, G-) and monocytic (M-) types, are currently accepted. G-MDSCs are more prevalent and suppress T-cell responses in an antigen-specific manner, whereas M-MDSCs are more suppressive on a per cell basis and in both antigen-specific and antigen-nonspecific manners. Notwithstanding recent exponentially increasing publications on MDSCs, the generation and development of MDSCs in cancer patients remain a crucial topic of further investigation.8,9 The current hypothesis of MDSC development is a two-stage model based on two distinct yet interconnected groups of signals, e.g., expansion of immature myeloid cells in the bone marrow and subsequent activation/acquisition of suppressive activity.10 Several chemokines and cytokines, particularly those regulating myelopoiesis and differentiation of myeloid cells, have been shown to expand MDSCs. For example, multiple groups independently reported that granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF) and/or interleukin (IL) 6 increase MDSC accumulation in tumor tissue.11,12,13 In addition, tumor-derived factors, including C-C chemokine ligand (CCL) 2, transforming growth factor (TGF)-β, IL-4, etc., have been demonstrated to increase MDSCs.8,14 Stomach-specific IL-1β overexpression increased MDSC accumulation during gastric cancer initiation and progression.15 However, very little is known about how cytokines/chemokines trigger molecular pathways leading to MDSC mobilization within the bone marrow. The majority of previous studies on the mechanism of MDSC mobilization focused on the intrinsic effects of MDSCs, i.e., via receptors expressed on the cell surface of MDSCs. Svoronos et al. demonstrated that estrogen receptor α expressed by human and murine bone marrow myeloid precursors activates the signal transducer and activation of transcription (STAT) 3 pathway via Janus kinase (JAK) 2 and Src nonreceptor tyrosine kinase.16 Activation of the cannabinoid receptors CB1 and CB2 expressed on immune cells mobilized functional MDSCs, contributing to the immunomodulatory effects of cannabinoids.17 Stimulator of interferon genes (STING) and the type I interferon pathway induce M-MDSC accumulation in tumor tissue via C-C chemokine receptor (CCR) 2.18
Given that MDSCs originate in the bone marrow of tumor hosts, stromal cells comprising the bone microenvironment are thought to participate in the initial phase of MDSC development. We have previously demonstrated that prostate tumor-derived parathyroid hormone-related peptide (PTHrP) correlates with MDSC accumulation in prostate tumor tissues, a process that involves activation of the PTHrP receptor, PTH1R, in osteoblasts.19 Briefly, prostate tumor-derived PTHrP activates osteoblasts, the main cell type expressing PTH1R in the bone microenvironment, leading to the expression of vascular endothelial growth factor (VEGF)-A and IL6. Consequently, MDSCs gain increased angiogenic potential to contribute to prostate tumor growth and angiogenesis. In addition to the functional activation of MDSCs, we observed that PTHrP rapidly increases MDSCs in the circulation of tumor hosts, suggesting that osteoblastic PTH1R activation potentially contributes to the expansion and/or mobilization of MDSCs. In this manuscript, we further delved into the molecular mechanism of PTH1R-dependent MDSC mobilization in tumor hosts using in vitro cell binding assays, in vivo models and human breast cancer patient-derived MDSCs.
Results
PTHrP increased the number of M-, but not G-, MDSCs in the circulation
To investigate the specific effects of PTHrP and to rule out many other tumor-derived factors that potentially affect MDSCs, we first infused recombinant PTHrP (amino acids 1-34, a PTH1R receptor-binding fragment) or control PBS diluent continuously for three weeks using in vivo subcutaneous implantable pumps in tumor-naïve mice (Fig. 1a). In parallel, subcutaneous or intratibial 4T1 tumor-bearing mice were used as a positive controls for MDSC induction (Fig. 1b–f). Flow cytometric analyses show that PTHrP administration significantly increased the number of M-MDSCs, but not G-MDSCs, in both blood circulation and the femoral bone marrow. The increase was statistically significant compared with the no-treatment or PBS-treated control groups but was less prominent compared with murine subcutaneous or intratibial 4T1 tumor-bearing mice, suggesting that PTHrP is not the only but one of the M-MDSC mobilizing factors. Because MDSCs are functionally defined as T-cell-suppressive immature myeloid cells, we performed a T-cell suppression assay to confirm that MDSCs isolated from PTHrP-infused mice were functional. Fig. 1g shows that M-MDSCs isolated from the PTHrP-treated mice suppressed in vitro T-cell proliferation compared with MDSCs isolated from the PBS-treated control mice. The T-cell suppressive activity was less prominent than that of MDSCs isolated from the subcutaneous 4T1 tumor-bearing mice. We subsequently tested whether PTH (amino acids 1–34, a receptor-binding fragment), whose receptor, PTH1R, is shared with PTHrP, also increases M- and G-MDSCs in vivo. A single subcutaneous administration of both PTH(1-34) or PTHrP(1-34) significantly increased M-, but not G-, MDSCs in the circulation of mice within 24 h. In contrast, AMD3100 (plerixafor, a CXCR4 inhibitor) and granulocyte-macrophage colony stimulating factor (GM-CSF), two clinically used hematopoietic stem cell mobilizing agents, did not increase M- or G-MDSCs, indicating that M-MDSC mobilization is specific to PTH1R activation (Fig. 1h, i). We also examined whether PTH or PTHrP increases other types of hematopoietic lineage cells. Neither PTH(1-34) nor PTHrP(1-34) affected granulocytes, lymphocytes (B cells and CD3, 4, 8 or NK T cells) or white blood cell (WBC) differential counting (Supplemental Fig. 2) in mice, indicating that PTH1R activation specifically increases M-MDSCs. Figure 1j, k shows that the PTHrP(1-34)-induced M-MDSC increase in the circulation occurred rapidly within 12 h after injection and reached a nadir at the 48-hour time point, suggesting that the rapid increase is potentially attributable to mobilization, not proliferation, of M-MDSCs.
PTHrP did not increase the immunosuppressive activity of M-MDSCs in tumor-bearing mice
MDSCs exist only in pathologic conditions such as inflammation and cancer, but the experiments in Fig. 1a–k were performed in tumor-naïve mice, and M-MDSCs isolated from PTHrP-infused tumor-naïve mice were immunosuppressive. We thus re-examined the effects of PTHrP in tumor-bearing mice. We chose B16F10 murine melanoma cells because of undetectable endogenous PTHrP expression. B16F10 tumor-bearing mice were continuously infused with recombinant PTHrP(1-34) or PBS for two weeks, followed by flow cytometric analysis (Fig. 1l–p). The PTHrP-treated mice had significantly increased numbers of M- but not G-MDSCs in the circulation. However, contrary to the data from tumor-naïve mice shown in Fig. 1d, PTHrP did not increase M-MDSCs in the bone marrow. We reasoned that MDSCs in the bone marrow had already maximally expanded in response to the 2-week subcutaneous tumor burden, and the effects of exogenous PTHrP addition did not generate further noticeable changes (Fig. 1o).
We next tested whether the T-cell-suppressive activity of MDSCs, a hallmark of functional MDSCs, was affected by PTHrP treatment. In Fig. 1q–r, in the in vitro T-cell suppression assay with MDSCs isolated from two-week PTHrP- or PBS-infused mice carrying B16-F10 tumors, MDSCs suppressed T-cell proliferation, while PTHrP treatment did not augment T-cell suppressive function in tumor-bearing mice. These findings may seem to contradict the data in Fig. 1g showing that PTHrP treatment increased the immunosuppressive activity of M-MDSCs in tumor-naïve mice, but we reasoned that the effects of PTHrP on the immunosuppressive activity of MDSCs may not be prominent in tumor-bearing mice because PTHrP may not be the only tumor-derived factor for MDSC function, and cytokines and growth factors released by tumor cells may increase and mask the effects of PTHrP. In contrast, PTHrP-dependent M-MDSC mobilization was evident in both tumor-naïve and tumor-bearing mice, supporting the validity of our work. Collectively, we conclude that PTHrP mobilizes M-MDSCs and increases the immunosuppressive activity of M-MDSCs, while the effect on immunosuppressive activity is not prominent in tumor-bearing mice. We conclude that PTHrP(1-34) induced MDSC mobilization without enhancing the immunosuppressive functions of M-MDSCs.
Activation of osteoblasts via PTH1R ligands released M-MDSCs binding to osteoblasts in vitro
The data in Fig. 1 collectively demonstrate that PTH1R activation by PTHrP or PTH specifically increases M-MDSCs in the circulation. In addition, the kinetics of the PTHrP-induced increase in MDSCs (Fig. 1h–k) suggest that the rapid increase in M-MDSCs (i.e., within 12 h after PTHrP injection) is mediated by mobilization of M-MDSCs from the bone marrow into circulation rather than by proliferation and/or expansion of M-MDSCs in the bone marrow. Given that PTH1R is predominantly expressed by osteoblasts in bone, we subsequently hypothesized that interactions between osteoblasts and M-MDSCs are disrupted by PTH1R activation, leading to disengagement of M-MDSCs from the bone marrow into the circulation. To test this hypothesis, we reconstituted osteoblast-MDSC interactions in vitro and tested the effects of PTH or PTHrP (Fig. 2a and Fig. S3a). Microscopic images in Fig. 2b, c show that human or murine M-MDSCs bound to the cultured osteoblast monolayer. The addition of PTHrP(1-34) or PTH(1-34) reduced the binding between M-MDSCs and osteoblasts in a dose-dependent manner, while PTHrP(7-34, a nonreceptor binding fragment) or AMD-3100 did not reduce the binding. Moreover, the effects of PTHrP(1-34) or PTH(1-34) on M-MDSC and osteoblast binding release were inhibited by SQ22356, an inhibitor of adenylate cyclase downstream of PTH1R. Conversely, forskolin, an adenylate cyclase activator, released M-MDSCs and osteoblast binding, which was blocked by the addition of SQ22356 (Fig. 2d–f, Fig. S3b and c and Movie S1). We further confirmed whether activation of PTH1R, a common receptor for PTH and PTHrP, is essential for the binding release of M-MDSCs from osteoblasts. Indeed, PTH1R knockdown in osteoblasts significantly blunted the effects of PTHrP(1-34) or PTH(1-34) on MDSC-osteoblast binding release (Fig. 2g–j). Overall, the data in Fig. 2 support that PTH1R is critical to the effects of PTH or PHTrP on M-MDSC mobilization.
Electron microscopy demonstrates physical binding between M-MDSCs and osteoblasts via cell adhesion
We further confirmed the specificity of M-MDSC and osteoblast binding by electron microscopy. Correlative light and electron microscopy (CLEM) images in Fig. 3a–d show physical binding between osteoblasts (adherent cells) and M-MDSCs (green). Three M-MDSCs that seemingly appear not to bind directly to osteoblasts under a fluorescence microscope (Fig. 3a) indeed bind to the cell body of osteoblasts under an electron microscope (Fig. 3b–d). Furthermore, serial ultrathin sections of M-MDSCs and osteoblasts confirmed that M-MDSC and osteoblast binding occurs through cellular adhesion, not simple colocalization (Fig. 3f). These data support that M-MDSCs bind to osteoblasts via cell adhesion molecules.
M-MDSCs reside in the endosteal surface of bone marrow via VCAM1 and Integrin β1
Based on the data in Figs. 1–3, we reasoned that M-MDSCs are bound to osteoblasts in the bone marrow of tumor hosts, and the binding becomes disengaged upon PTH1R activation by tumor-derived PTHrP. We subsequently searched for cell-to-cell adhesion molecules associated with M-MDSC and osteoblast binding. Very late antigen (VLA)-4 is a dimer of integrin α4 (CD49D) and β1 (CD29) subunits and is expressed by hematopoietic lineage cells, including hematopoietic stem cells (HSCs) and monocytes. Stromal-derived factor (SDF)1, also known as C-X-C motif chemokine (CXCL)12, activates VLA4, leading to conformational changes and binding to vascular cell adhesion molecule (VCAM)1, the primary ligand of VLA4. The VLA-4 and VCAM1 axis regulates HSC homing, dormancy and mobilization.20,21 Recently, the Varner group showed that VLA4 is expressed by MDSCs, playing key roles in recruitment to tumor tissues, angiogenesis, and immunosuppressive activity.22,23,24,25 We subsequently tested whether the VLA-4 (α4β1 integrin) and VCAM1 axis plays a role in MDSC mobilization from the bone marrow into the circulation.
The Fig. 4a immunofluorescence images of the tibia of Balb/c female mice carrying 4T1 breast cancer revealed that alkaline phosphatase (ALP)-positive osteoblasts on the endosteal surface colocalized with CD11b+Ly6C+ M-MDSCs. In addition, a fraction of CD11b+ cells (including MDSCs) located adjacent to the endosteal lining osteoblasts (Fig. 4b) and endosteal lining osteoblasts expressed VCAM1 (Fig. 4c). In our subsequent in vitro cell binding assay, anti-VCAM1 or anti-integrin β1 neutralizing antibodies blocked M-MDSC and osteoblast binding dose-dependently (Fig. 4e, f), supporting that VCAM1 (expressed on osteoblasts) and VLA-4 (integrin α4β1, expressed on M-MDSCs) mediate the binding between osteoblasts and M-MDSCs.
Binding between M-MDSCs and bone marrow stromal cells is dependent on VCAM1 expression, but PTH1R ligand (PTH or PTHrP)-induced release is dependent on PTH1R expression
Notwithstanding the in vivo histological data in Fig. 4, VCAM-1 expression is not confined to the endosteal lining osteoblasts, and not all M-MDSCs in the bone marrow bind to osteoblasts in vivo or in human patients. We subsequently examined whether M-MDSCs potentially bind to other cell types expressing VCAM1 (e.g., osteoclasts and fibroblasts). Figure 5a, b, d demonstrates that cells in the bone marrow, such as endothelial cells, osteoclasts, and fibroblasts, express VCAM-1, but only osteoblasts express PTH1R. In vitro cell binding assays (Fig. 5c, e) showed that only PTH/PTHrP stimulation released M-MDSC binding from osteoblasts, supporting that MDSC-bone marrow stromal cell binding release is dependent on PTH1R activation. Collectively, these data indicate that a fraction of M-MDSCs bound to the endosteal surface in the bone marrow are mobilized by PTH1R activation in osteoblasts, which potentially is not the only mechanism for M-MDSC mobilization in cancer patients. Thus, the anti- and protumoral immunity balance is likely not regulated by one gold standard mechanism; instead, the balance is regulated by multiple overlapping yet compensatory mechanisms.
Ectopic expression of VCAM1 confers M-MDSC binding capability
We further confirmed the role of VCAM1 and integrin β1 in osteoblast-M-MDSC binding. MCF7 breast cancer cells that do not express VCAM1 were transfected with a VCAM1 overexpression vector, and ectopic expression was confirmed by Western blotting and immunofluorescence imaging (Fig. 6a). Supplemental Fig. 4a, b shows that murine M-MDSCs bind to VCAM1-overexpressing but not to parental MCF7 cells. In addition, an in vitro cell binding assay showed that anti-VCAM1 and/or anti-integrin β1 antibodies blocked M-MDSC binding to VCAM1-overexpressing but not parental MCF7 cells (Fig. 6b, c). In contrast, an in vitro cell binding assay (Fig. 6d) showed that ectopic expression of VCAM1 in MCF-7 cells confers M-MDSC binding capacity, but the binding is not reversed by rhPTH(1-34) or rhPTHrP(1-34), supporting that M-MDSC binding to bone marrow stromal cells is dependent on VCAM1 and integrin β1 expression but that the binding release is dependent on PTH1R expression.
Src family kinase activation in M-MDSCs is required for releasing M-MDSCs from osteoblasts
We have previously demonstrated that prostate cancer-derived PTHrP induces activating phosphorylation of Src family kinase (SFK) at tyrosine residue 419 (Y419) in MDSCs via vascular endothelial growth factor (VEGF)-A and interleukin (IL)-6 expressed by osteoblasts, leading to increased proangiogenic and protumorigenic functions of MDSCs.19 We subsequently tested whether SFK activation also plays a role in M-MDSC mobilization. First, we examined whether PTHrP-activated osteoblasts induce pY419 of SFK in M-MDSCs. Murine splenocytes and femoral bone marrow-derived M- and G-MDSCs were isolated from 4T1 tumor-bearing mice by flow cytometry, followed by treatment with 24-hour PTHrP(1-34)- or PBS control-conditioned media from murine calvarial osteoblast cultures. Fig. 7a shows that SFK specifically in M-MDSCs, but not in G-MDSCs or splenocytes, was activated by PTHrP-conditioned media from osteoblasts. In addition, PTHrP-conditioned media decreased the binding between osteoblasts and M-MDSCs in vitro, which was effectively reversed by PP2, an SFK-specific pharmacologic inhibitor (Fig. 7b). In our subsequent in vivo experiment using dasatinib, a tyrosine kinase inhibitor for SFK in clinical use, suppression of SFK effectively inhibited PTHrP(1-34)-induced M-MDSC mobilization in mice (Fig. 7c), suggesting that activating phosphorylation of SFK in M-MDSCs is essential for disengagement from osteoblasts. Furthermore, the effects of PTHrP-conditioned media on disrupting osteoblast-M-MDSC binding were inhibited by the addition of anti-VEGF-A or anti-IL6 neutralizing antibodies (Fig. 7d, e). Indeed, in our subsequent in vivo experiment (Fig. 7f), anti-VEGF-A and/or anti-IL6 antibodies suppressed PTHrP(1-34)-induced M-MDSC mobilization in vivo, supporting that PTHrP-induced VEGF-A and IL6 contribute to M-MDSC mobilization. However, we did not observe statistical significance in the VEGF-A alone treatment group due to two outliers. These data support that SFK activation in M-MDSCs by PTHrP-induced osteoblastic cytokines such as VEGF-A and IL6 is important in M-MDSC mobilization.
PTHrP-induced M-MDSC mobilization is dependent on a disintegrin and metalloproteinase (ADAM) 17 and matrix metalloprotease (MMP) 7
We subsequently sought to determine the downstream mediators of pY419 SFK in M-MDSCs contributing to the disruption of M-MDSC and osteoblast binding. Human or murine M-MDSCs were isolated and stimulated for two hours with PBS- or PTHrP-conditioned media from human or murine osteoblasts, respectively, followed by reverse transcription-quantitative polymerase chain reaction (RT‒qPCR) for candidate protease genes, including ADAM17, MMP2, 7, 9, ELANE and CTSB (cathepsin B). Interestingly, ADAM17 and MMP7 were significantly increased in both murine and human M-MDSCs by PTHrP-conditioned media compared with no-treatment or PBS-conditioned media controls (Fig. 8a, b). In addition, VEGF-A and IL6 treatment increased ADAM17 and MMP7 protein expression in M-MDSCs (Fig. 8c, d). SFK suppression by PP2 decreased ADAM17 and MMP7 expression (Fig. 8e). These data collectively demonstrate that osteoblastic cytokines such as VEGF-A and IL6 induced by PTH1R activation result in ADAM17 and MMP7 expression in M-MDSCs via pY419 SFK. We subsequently performed a functional assay to test whether ADAM17 and MMP7 inhibition suppresses PTH/PTHrP-induced osteoblast-M-MDSC binding release. Figure 8f–j demonstrates that TAPI-1, an inhibitor of ADAM17 and MMPs, reversed PTHrP(1-34)- or PTH(1-34)-induced MDSC release from osteoblasts in vitro and the PTHrP(1-34)-induced M-MDSCs increase in the circulation of mice.
Discussion
The present study demonstrated the molecular mechanism underlying how tumor cells exploit the skeletal system to augment MDSCs, which are important protumorigenic immune cells in the tumor microenvironment. As illustrated in Fig. 9, tumor-derived PTHrP circulates and triggers PTH1R on osteoblasts, leading to the expression of VEGF-A and IL6. As a result, M-MDSCs become activated via phosphorylation of SFK and express proteases such as MMP7 and ADAM17 to disengage the VLA4-VCAM1 axis-dependent tethering between M-MDSCs and osteoblasts. Mobilized M-MDSCs subsequently become available to the tumor tissue via circulation, contributing to escape from antitumoral immunity and tumor progression. The skeletal system is an essential partner for tumor progression because diverse subsets of stromal cells comprising the tumor microenvironment, such as immune cells, endothelial progenitor cells, tumor-associated macrophages, and tumor-associated fibroblasts, commonly originate in the bone marrow. Thus, stromal cells in the bone marrow, such as osteoblasts, are speculated to play active roles in supplying bone marrow-derived cells to the tumor microenvironment, but the precise mechanism remains to be elucidated. The present study focused on how the monocytic subset of MDSCs is mobilized from the bone marrow by tumor-derived factors. The majority of studies on MDSC development are centered on the recruitment of circulating MDSCs to tumor tissue or on the mechanism of immunosuppressive activities, and only a fraction of studies have investigated MDSC mobilization from the bone marrow into the circulation. As briefly summarized in the above introduction section, many cytokines and tumor-derived factors, mostly involved in HSC mobilization and/or myelopoiesis, potentially contribute to MDSC mobilization. However, the majority of studies are only observational, lack precise molecular mechanisms and are often confused with terms such as mobilization vs. expansion.13,16,26,27 In the present study, we employed in vivo mouse models and in vitro cell-binding assays using human patient MDSCs to show the molecular mechanisms of M-MDSC tethering and release to/from osteoblasts.
The applicability of our findings is multifaceted. Therapeutic strategies targeting MDSCs are currently under extensive investigation, and MDSC inhibition will significantly enhance the efficacy of immune checkpoint inhibitors. Numerous lines of evidence support that the number and function of MDSCs positively correlate with cancer progression as well as the reduced efficacy of immune checkpoint inhibitors.28 Currently, therapeutic targets to suppress MDSCs include pathways involved in MDSC expansion (e.g., STAT3 or prostaglandin E2 inhibitors), activation (e.g., inhibitors of interferon-γ, IL6, IL-1β, TNFα, etc.), metabolic pathways of MDSCs (e.g., hypoxia-inducible factor 1α, lactate accumulation, fatty acid oxidation, etc.), MDSC infiltration (e.g., Toll-like receptor 9, STAT3, VEGF-A, etc.), and MDSC depletion (e.g., cytotoxic chemotherapeutic agents and tyrosine kinase inhibitors such as nilotinib, dasatinib, sorafenib and ibrutinib) (refer to a review article29 written by Dr. Larry Kwak for more details). In contrast, none of the currently targeted pathways aims to shut down the MDSC supply from storage, the most fundamental step of MDSC development. The data in the present study support that multiple approaches using anti-PTHrP antibodies, protease inhibitors specific for ADAM17 or MMP7, and tyrosine kinase inhibitors suppressing SFK and/or VEGFR potentially block M-MDSC production in the bone marrow and consequently enhance the therapeutic efficacy of immune checkpoint inhibitors. PTHrP is a well-known factor for malignancy-induced hypercalcemia and tumor-bone interactions in breast cancer bone metastasis30,31; thus, anti-PTHrP antibodies have long been used to suppress cancer progression and metastasis.32,33,34,35 However, anti-PTHrP antibodies have never been tested in combination with immune checkpoint inhibitors in cancer patients or murine tumor models, which is an important direction for further studies. Likewise, dasatinib, a selective SFK inhibitor, was shown to significantly reduce PTHrP-induced M-MDSC mobilization in vivo (Fig. 7c), supporting that SFK tyrosine kinase inhibitors could be repositioned to suppress MDSCs and to enhance cancer immunotherapy. Previously, dasatinib was shown to reduce tumor-infiltrating myeloid cells.36 In addition, Sun et al. demonstrated that inhibition of SFK by dasatinib reduced MDSCs in a head and neck cancer mouse model and that among nine family members of SFK, Lyn is an important therapeutic target in MDSCs.37 Indeed, the same group subsequently showed that combinatorial treatment with dasatinib and anti-CTLA4 antibody reduced tumor growth and MDSC numbers in a murine tumor model.38 Data in our present study not only logically extended and confirmed the previous findings but also provided the precise molecular mechanism of how SFK inhibitors could synergistically cooperate with immune checkpoint blockade.
Our proposed molecular mechanism of MDSC mobilization, however, raises many more important questions. First, while MDSCs are considered universally important stromal cells in the tumor microenvironment, PTHrP is not expressed by every tumor type. Thus, we reason that additional tumor-derived factors or mechanisms mediating tumor-bone interactions and MDSC mobilization warrant extensive further investigation. Second, given that recombinant human PTH (teriparatide, Forteo ®) is currently used as a bone anabolic agent for osteoporotic patients and that PTH and PTHrP share the common receptor PTH1R, increased PTH levels in the circulation due to rhPTH treatment or hyperparathyroidism will potentially increase MDSCs in the blood and reduce T-cell immunity. Questions such as whether hyperparathyroidism patients or those who receive rhPTH have an increased number and/or activity of MDSCs will be worth addressing in large clinical cohorts. If the increased level of PTH is found to be associated with increased MDSCs, the patients who receive rhPTH should be thoroughly screened for cancer because MDSCs can fuel the growth of occult or microscopic tumor cells. Third, the roles of proteolytic cleavage and shedding of VCAM1 and/or VLA-4 in the mobilization of MDSCs and other types of hematopoietic lineage cells need further investigation. The proposed mechanism in this paper is supported by assays using multiple inhibitors, including TAPI-1, an ADAM17 inhibitor. Garton et al. showed ADAM17-dependent shedding of VCAM1 and measurement of soluble VCAM1 in the culture supernatant.39 Lévesque et al. further demonstrated that proteolytic cleavage of VCAM1 expressed by bone marrow stromal cells is an essential step of hematopoietic progenitor cell mobilization in response to G-CSF40 and that hematopoietic mobilizing agents such as G-CSF and cyclophosphamide increase proteases such as neutrophil elastase and cathepsin G, transforming the bone marrow into a highly proteolytic environment.41 Our study added further details on the VCAM1 proteolytic cleavage-dependent regulation of M-MDSCs in the bone marrow of tumor hosts. In contrast, further evidence, such as detection of cleaved VCAM1 in the bone marrow flush or in the circulation, will strengthen the interpretation. In conclusion, the data in this study explain how M-MDSCs, essential bone marrow-derived cells in the TME, are regulated in the bones of cancer patients and provide a scientific basis for novel therapeutic strategies suppressing M-MDSCs and boosting immune checkpoint inhibitors.
Materials and methods
Flow cytometric analysis and sorting
Murine blood samples were collected from the retro-orbital sinus, and bone marrow cells were collected by flushing hindlimb long bones. Erythrocytes were lysed by ACK lysis buffer, followed by staining with anti-mouse monoclonal antibodies including CD16/CD32 (Mouse Fc Block™), CD45 (30-F11), CD11b (M1/70), Gr-1 (RB6-8C5), Ly-6C (AL-21), Ly-6G (1A8), CCR2 (SA203G11), CD3 (17A2), CD4 (RM4-5), CD8 (53-6.7), CD49b (DX5) and B220 (RA3-6B2). BioGems™ Viability Dye 506 or 780 was used to exclude dead cells. Flow cytometric analysis was performed using BD LSRFortessa™ X-20 or FACSCanto™ II cytometers and FlowJo™ software version 10.6., and flow cytometric sorting was performed using a BD FACSAria™ III sorter and FACSDIVA™ software version 8.0. Gating strategies are presented in Fig. 1b and Fig. S1.
Human blood samples
Peripheral blood mononuclear cells (PBMCs) of female breast cancer patients were obtained from an ongoing clinical study. The study was approved by the institutional review board of the Korea University Medical Center Anam Hospital (No. ED17183). Briefly, whole blood samples were collected from female breast cancer patients who visited the outpatient clinic of the Korea University Anam Hospital, Seoul, Korea, from October 2017 to October 2019. PBMCs were isolated by Ficoll-Paque™ density-gradient centrifugation, followed by washing and resuspension in FACS buffer (phosphate-buffered saline (PBS) with 2% fetal bovine serum (FBS) and 2 mmol·L−1 EDTA). Cells were stained with anti-human monoclonal antibodies, including CD45 (HI30), CD11b (ICFR44), CD14 (MφP9) and CCR2 (K036C2), followed by flow cytometric sorting (BD FACSAria™ II). All patients signed written informed consent before enrollment, and the study was conducted in accordance with the International Conference on Harmonization Good Clinical Practice guidelines and the provisions of the Declaration of Helsinki.
Cells
Murine primary calvarial osteoblasts were isolated as previously described.42,43,44 Briefly, the calvariae of 2~5-day-old C57BL6/J mouse pups were dissected and subjected to serial digestion with alpha modification minimal essential media (αMEM) with type 1 collagenase (125 units per mL) and 1× penicillin/streptomycin antibiotics for 15 min each. After the first and second fractions were discarded, the third and fourth fractions were plated and expanded in complete αMEM, followed by cryopreservation in CELLBANKER® cell freezing media (Amsbio). Cells were thawed and used without passaging more than once. Thermolabile SV40 large T antigen-transformed hFOB 1.19 human fetal osteoblasts cells (ATCC) were cultured in Dulbecco’s modified Eagle’s media (DMEM)/F-12 media at restrictive temperature for experiments and were used without passaging more than three times. HUVECs (ATCC) were cultured and expanded in Endothelial Cell Growth Medium 2 (produced by and purchased from PromoCell). NIH3T3 fibroblasts were cultured and expanded in complete DMEM. For murine primary osteoclasts, monocytes were isolated from the bone marrow flush of tumor-naïve Balb/C mice by gradient centrifugation, followed by treatment with αMEM with M-CSF (30 ng·mL−1) and receptor activator of nuclear factor kappa B ligand (RANKL, 50 ng·mL−1) for four days. PTH1R knockdown osteoblasts were established by transducing primary calvarial osteoblasts or hFOB1.19 cells with PTH1R-targeting shRNA lentiviral particles (Santa Cruz Biotechnology SC-36327-V). Scrambled-sequence shRNA lentiviral particles were used as controls. VCAM1-overexpressing MCF7 cells were generated by transfecting wild-type MCF7 cells with a VCAM1 overexpression vector (Origene RC209761). Cells were regularly confirmed to be free of mycoplasma by PCR tests and authenticated for matching short tandem repeat DNA profiles of the original cell lines.
Mouse models
All animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea University Medical Center. Mouse tumor models were established as previously described.19,44 Briefly, luciferase- and tdTomato-labeled 4T1 murine breast cancer cells (gifted by Dr. Eun Kyoung Choi, Asan Medical Center, Seoul, Korea) were injected intratibially (1 × 104 cells per 20 µL) or subcutaneously (5 × 104 cells per 100 µL) into 7-week-old female Balb/C mice. Osteocalcin (Ocn)-Cre:ROSAmT/mG reporter mice were generated by crossing mT/mG mice (Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo/J, The Jackson Laboratory Strain No. 007576) with Ocn-Cre mice (B6N.FVB-Tg(BGLAP-cre)1Clem/J, The Jackson Laboratory Strain No. 019509). B16F10 murine melanoma cells were injected subcutaneously into 7-week-old C57BL6 mice or Ocn-Cre:ROSAmT/mG mice. For continuous PTHrP stimulation, Alzet® osmotic pumps releasing 80 μg·kg−1 per day recombinant human (rh) PTHrP (amino acids 1–34) were surgically transplanted into the subcutaneous space of 7-week-old male mice.19 For analysis of inhibitors of M-MDSC mobilization, mice were intraperitoneally administered anti-IL6 antibody (10 mg·kg−1), anti-VEGF-A antibody (5 mg·kg−1), TAPI-1 (2 mg·kg−1), and/or dasatinib (15 mg·kg−1, P.O) prior to 30 min of rhPTHrP (1–34. 80 μg·kg−1) subcutaneous administration.
T-cell suppression assay
Murine CD3+ T cells were isolated from the spleens of tumor-naïve C57BL6 or Balb/C mice using T Cell Enrichment Columns (R&D Systems, MTCC-25) and stained with carboxyfluorescein succinimidyl ester (CFSE, 1 μmol·L−1). Murine M-MDSCs were isolated from the bone marrow of B16F10 tumor-bearing C57BL6 and 4T1 tumor-bearing Balb/c mice or Alzet osmotic pump-implanted Balb/c mice using a Mouse MDSC Isolation Kit (Miltenyi Biotec 130-094-538). CD3+ T-cell purity after column enrichment was confirmed to be higher than 75% (Fig. S1c). T cells and MDSCs were cocultured in the presence of IL-2 (10 IU per mL) and Dynabeads® CD3/CD28 T-cell activators (bead:T-cell ratios = 0.5:1 or 0.2:1) for three days, followed by flow cytometric quantification of proliferating T cells.
Isolation of M-MDSCs
Human or murine M-MDSCs were isolated by flow cytometric cell sorting (BD FACSAria II) from the PBMCs of human breast cancer patients or the spleens or tibiae of tumor-bearing mice.
In vitro cell binding assay
An in vitro osteoblast-MDSC binding assay was performed by modifying a previously published hematopoietic stem cell-osteoblast binding assay.45,46 Briefly, human or murine osteoblasts (2.5 × 103 or 1 × 104, respectively) were seeded on 96-well flat-bottom black-wall polystyrene plates and incubated overnight. Human or murine M-MDSCs were sorted by flow cytometry and stained with CFSE. Subsequently, M-MDSCs were added to the osteoblast monolayer culture and incubated for 30 min in a 5% CO2 37 °C incubator with experimental treatments (e.g., PTH or PTHrP). Unbound cells were washed with PBS, and fluorescence intensity (excitation 485 nm, emission 515 nm) was measured with a plate reader. The fluorescence intensity of the PBS-treated control group was considered 100% binding, and a reduction in fluorescence intensity was interpreted as reduced cell binding (%).
Fluorescence and confocal microscopy
For DiD cell membrane staining, adherent cells (osteoblasts and MCF7 cells) were trypsinized and resuspended in serum-free complete media (106 cells in 1 mL of media), followed by the addition of VibrantTM DiD Cell-Labeling Solution (5 μL) and incubation for 20 min at 37 °C. Cells were plated on confocal dishes. The next day, MDSCs were sorted by flow cytometry and stained with CFSE. If needed, nuclear counterstaining was performed using 4’,6-diamidino-2-phenylindole (DAPI) or Hoechst 33342. Fluorescence or confocal microscopic images were captured using an EVOS FL AUTO2 (Thermo Fisher) or confocal microscope (LSM800 or LSM900, Zeiss), respectively. For in vivo histological images, hindlimbs from B16F10 tumor-bearing C57BL6 mice or Ocn-Cre:ROSAmT/mG reporter mice were dissected and fixed with 4% (w/v) paraformaldehyde for 3 days, followed by decalcification in 14% EDTA for 3 days and 30% sucrose in PBS for 1 day. For cryosectioning, tissues were then embedded in Tissue-Tek® OCT compound, and 5 μm thick sections were cut and mounted onto microscopic slides. For immunohistochemistry, sections were stained with anti-CD11b (M1/70), anti-Ly6C (ER-MP20), anti-alkaline phosphatase (ALP, Abcam Catalog No. 224335, polyclonal) and anti-VCAM1 (Genetax Catalog No. GTX12133, polyclonal) antibodies.
Electron microscopy
M-MDSCs isolated from 4T1 tumor-bearing mice were cocultured with adherent murine calvarial osteoblasts on a confocal dish, fixed with 2.5% glutaraldehyde solution overnight at 4 °C and washed in PBS. Fluorescence images were captured by a widefield microscope (EVOS M7000, Thermo Fisher). Subsequently, the specimen was postfixed with 1% osmium tetroxide. After washing with distilled water, the specimen was gradually dehydrated with increasing concentrations of ethanol series (50%, 70%, 90%, and 100%) and hexamethyldisilazane (HMDS) series (3:1, 1:1 and 1:3 ethanol:HMDS, and 100% HMDS). After drying in air, the specimen was mounted on SEM stubs followed by platinum coating at 15 mA for 60 s using a sputter coater (E-1045, Hitachi). Surface images were observed using SEM (TeneoVS, FEI) in SE mode at 10 kV and 0.1 nA with an ETD detector at the same location where fluorescence images were taken.
Statistical analysis
Statistical analysis was performed using GraphPad Prism™ version 8.0. All data were tested for normality by the Shapiro‒Wilk test, and Student’s t test (for normally distributed samples) or the Mann–Whitney U test (for nonparametric analysis) was used to compare groups. One-way ANOVA with multiple group comparisons analysis was performed to compare normally distributed multiple groups. All statistical analyses were two-sided.
References
Jeong, H. M., Cho, S. W. & Park, S. I. Osteoblasts are the centerpiece of the metastatic bone microenvironment. Endocrinol. Metab. (Seoul.) 31, 485–492 (2016).
Buenrostro, D., Park, S. I. & Sterling, J. A. Dissecting the role of bone marrow stromal cells on bone metastases. Biomed. Res. Int. 2014, 875305 (2014).
Park, S. I., Soki, F. N. & McCauley, L. K. Roles of bone marrow cells in skeletal metastases: no longer bystanders. Cancer Microenviron. 4, 237–246 (2011).
Gabrilovich, D. I. Myeloid-derived suppressor cells. Cancer Immunol. Res. 5, 3–8 (2017).
Gros, A. et al. Myeloid cells obtained from the blood but not from the tumor can suppress T-cell proliferation in patients with melanoma. Clin. Cancer Res. 18, 5212–5223 (2012).
Weide, B. et al. Myeloid-derived suppressor cells predict survival of patients with advanced melanoma: comparison with regulatory T cells and NY-ESO-1- or Melan-A-Specific T Cells. Clin. Cancer Res. 20, 1601–1609 (2014).
Lim, J.-Y. et al. Ex vivo generated human cord blood myeloid-derived suppressor cells attenuate murine chronic graft-versus-host diseases. Biol. Blood Marrow Transpl. 24, 2381–2396 (2018).
Marvel, D. & Gabrilovich, D. I. Myeloid-derived suppressor cells in the tumor microenvironment: expect the unexpected. J. Clin. Invest. 125, 3356–3364 (2015).
Bronte, V. et al. Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards. Nat. Commun. 7, 12150 (2016).
Veglia, F., Perego, M. & Gabrilovich, D. Myeloid-derived suppressor cells coming of age. Nat. Immunol. 19, 108–119 (2018).
Dolcetti, L. et al. Hierarchy of immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by GM-CSF. Eur. J. Immunol. 40, 22–35 (2010).
Marigo, I. et al. Tumor-induced tolerance and immune suppression depend on the C/EBPbeta transcription factor. Immunity 32, 790–802 (2010).
Luyckx, A. et al. G-CSF stem cell mobilization in human donors induces polymorphonuclear and mononuclear myeloid-derived suppressor cells. Clin. Immunol. 143, 83–87 (2012).
Lee, C.-R., Lee, W., Cho, S. K. & Park, S.-G. Characterization of multiple cytokine combinations and TGF-β on differentiation and functions of myeloid-derived suppressor cells. Int. J. Mol. Sci. 19, 869 (2018).
Tu, S. et al. Overexpression of interleukin-1beta induces gastric inflammation and cancer and mobilizes myeloid-derived suppressor cells in mice. Cancer Cell 14, 408–419 (2008).
Svoronos, N. et al. Tumor cell-independent estrogen signaling drives disease progression through mobilization of myeloid-derived suppressor cells. Cancer Discov. 7, 72–85 (2017).
Hegde, V. L., Nagarkatti, M. & Nagarkatti, P. S. Cannabinoid receptor activation leads to massive mobilization of myeloid-derived suppressor cells with potent immunosuppressive properties. Eur. J. Immunol. 40, 3358–3371 (2010).
Liang, H. et al. Host STING-dependent MDSC mobilization drives extrinsic radiation resistance. Nat. Commun. 8, 1736–10 (2017).
Park, S. I. et al. Parathyroid hormone-related protein drives a CD11b+Gr1+ cell-mediated positive feedback loop to support prostate cancer growth. Cancer Res. 73, 6574–6583 (2013).
Rettig, M. P., Ansstas, G. & DiPersio, J. F. Mobilization of hematopoietic stem and progenitor cells using inhibitors of CXCR4 and VLA-4. Leukemia 26, 34–53 (2012).
Shiozawa, Y., Pienta, K. J. & Taichman, R. S. Hematopoietic stem cell niche is a potential therapeutic target for bone metastatic tumors. Clin. Cancer Res. 17, 5553–5558 (2011).
Schmid, M. C. et al. PI3-kinase γ promotes Rap1a-mediated activation of myeloid cell integrin α4β1, leading to tumor inflammation and growth. PLoS One 8, e60226 (2013).
Schmid, M. C. et al. Combined blockade of integrin-α4β1 plus cytokines SDF-1α or IL-1β potently inhibits tumor inflammation and growth. Cancer Res. 71, 6965–6975 (2011).
Jin, H., Su, J., Garmy-Susini, B., Kleeman, J. & Varner, J. Integrin alpha4beta1 promotes monocyte trafficking and angiogenesis in tumors. Cancer Res. 66, 2146–2152 (2006).
Foubert, P., Kaneda, M. M. & Varner, J. A. PI3Kγ activates integrin α4 and promotes immune suppressive myeloid cell polarization during tumor progression. Cancer Immunol. Res. 5, 957–968 (2017).
Xu, J. et al. Tpl2 protects against fulminant hepatitis through mobilization of myeloid-derived suppressor cells. Front. Immunol. 10, 1980 (2019).
Hawila, E. et al. CCR5 directs the mobilization of CD11b+Gr1+Ly6Clow polymorphonuclear myeloid cells from the bone marrow to the blood to support tumor development. Cell Rep. 21, 2212–2222 (2017).
Park, S.-M. & Youn, J.-I. Role of myeloid-derived suppressor cells in immune checkpoint inhibitor therapy in cancer. Arch. Pharm. Res. 42, 560–566 (2019).
Liu, Y. et al. Targeting myeloid-derived suppressor cells for cancer immunotherapy. Cancer Immunol. Immunother. 67, 1181–1195 (2018).
McCauley, L. K. & Martin, T. J. Twenty-five years of PTHrP progress: from cancer hormone to multifunctional cytokine. J. Bone Miner. Res. 27, 1231–1239 (2012).
Li, J. et al. PTHrP drives breast tumor initiation, progression, and metastasis in mice and is a potential therapy target. J. Clin. Invest. 121, 4655–4669 (2011).
Sato, K. et al. Passive immunization with anti-parathyroid hormone-related protein monoclonal antibody markedly prolongs survival time of hypercalcemic nude mice bearing transplanted human PTHrP-producing tumors. J. Bone Miner. Res. 8, 849–860 (1993).
Sato, K., Onuma, E., Yocum, R. C. & Ogata, E. Treatment of malignancy-associated hypercalcemia and cachexia with humanized anti-parathyroid hormone-related protein antibody. Semin. Oncol. 30, 167–173 (2003).
Iguchi, H., Aramaki, Y., Maruta, S. & Takiguchi, S. Effects of anti-parathyroid hormone-related protein monoclonal antibody and osteoprotegerin on PTHrP-producing tumor-induced cachexia in nude mice. J. Bone Min. Metab. 24, 16–19 (2006).
Camirand, A., Fadhil, I., Luco, A.-L., Ochietti, B. & Kremer, R. B. Enhancement of taxol, doxorubicin and zoledronate anti-proliferation action on triple-negative breast cancer cells by a PTHrP blocking monoclonal antibody. Am. J. Cancer Res. 3, 500–508 (2013).
Liang, W. et al. Antitumor activity of targeting SRC kinases in endothelial and myeloid cell compartments of the tumor microenvironment. Clin. Cancer Res. 16, 924–935 (2010).
Mao, L. et al. Inhibition of SRC family kinases reduces myeloid-derived suppressor cells in head and neck cancer. Int. J. Cancer 140, 1173–1185 (2017).
Yu, G.-T. et al. Inhibition of SRC family kinases facilitates anti-CTLA4 immunotherapy in head and neck squamous cell carcinoma. Cell. Mol. Life Sci. 75, 4223–4234 (2018).
Garton, K. J. et al. Stimulated shedding of vascular cell adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor-alpha-converting enzyme (ADAM 17). J. Biol. Chem. 278, 37459–37464 (2003).
Lévesque, J. P., Takamatsu, Y., Nilsson, S. K., Haylock, D. N. & Simmons, P. J. Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor. Blood 98, 1289–1297 (2001).
Lévesque, J.-P. et al. Mobilization by either cyclophosphamide or granulocyte colony-stimulating factor transforms the bone marrow into a highly proteolytic environment. Exp. Hematol. 30, 440–449 (2002).
Datta, N. S., Chen, C., Berry, J. E. & McCauley, L. K. PTHrP signaling targets cyclin D1 and induces osteoblastic cell growth arrest. J. Bone Miner. Res. 20, 1051–1064 (2005).
Koh, A. J., Beecher, C. A., Rosol, T. J. & McCauley, L. K. 3‘,5’-Cyclic adenosine monophosphate activation in osteoblastic cells: effects on parathyroid hormone-1 receptors and osteoblastic differentiation in vitro. Endocrinology 140, 3154–3162 (1999).
Lee, C. et al. Dual targeting c-met and VEGFR2 in osteoblasts suppresses growth and osteolysis of prostate cancer bone metastasis. Cancer Lett. 414, 205–213 (2018).
Jung, Y. et al. Annexin II expressed by osteoblasts and endothelial cells regulates stem cell adhesion, homing, and engraftment following transplantation. Blood 110, 82–90 (2007).
Shiozawa, Y. et al. Annexin II/annexin II receptor axis regulates adhesion, migration, homing, and growth of prostate cancer. J. Cell. Biochem. 105, 370–380 (2008).
Acknowledgements
The authors thank the Korea University Medical Center (KUMC) Flow Cytometry Core Service Laboratory and the KUMC Cancer Precision Medicine Diagnosis and Treatment Enterprise (K-MASTER) for assistance with flow cytometry and the Advanced Analysis Center at the Korea Institute of Science and Technology, Seoul (Dr. Kyung Eun Lee) for assistance with electron microscopy.
Funding
This work was in part supported by the National R&D Program for Cancer Control, the Ministry of Health and Welfare, the Republic of Korea (HA17C0040 to SIP); the National Research Foundation of the Republic of Korea (2018R1D1A1B07050329 and 2020R1A2C1012966 to SIP; and 2020R1F1A1076996 to SPJ); and the Korea University Research Grants (SIP).
Author information
Authors and Affiliations
Contributions
S.I.P conceived the original idea, designed the experiments, supervised the project and wrote the manuscript. E.J.L and K.J.L performed the experiments. K.H.P provided clinical samples. K.H.P and S.P.J contributed to the final version of the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Lee, E.J., Lee, K.J., Jung, S. et al. Mobilization of monocytic myeloid-derived suppressor cells is regulated by PTH1R activation in bone marrow stromal cells. Bone Res 11, 22 (2023). https://doi.org/10.1038/s41413-023-00255-y
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41413-023-00255-y