In the tumor microenvironment, CD11b+Gr1+ bone marrow–derived cells are a predominant source of protumorigenic factors such as matrix metalloproteinases (MMP), but how distal tumors regulate these cells in the bone marrow is unclear. Here we addressed the hypothesis that the parathyroid hormone–related protein (PTHrP) potentiates CD11b+Gr1+ cells in the bone marrow of prostate tumor hosts. In two xenograft models of prostate cancer, levels of tumor-derived PTHrP correlated with CD11b+Gr1+ cell recruitment and microvessel density in the tumor tissue, with evidence for mediation of CD11b+Gr1+ cell–derived MMP-9 but not tumor-derived VEGF-A. CD11b+Gr1+ cells isolated from mice with PTHrP-overexpressing tumors exhibited relatively increased proangiogenic potential, suggesting that prostate tumor–derived PTHrP potentiates this activity of CD11b+Gr1+ cells. Administration of neutralizing PTHrP monoclonal antibody reduced CD11b+Gr1+ cells and MMP-9 in the tumors. Mechanistic investigations in vivo revealed that PTHrP elevated Y418 phosphorylation levels in Src family kinases in CD11b+Gr1+ cells via osteoblast-derived interleukin-6 and VEGF-A, thereby upregulating MMP-9. Taken together, our results showed that prostate cancer–derived PTHrP acts in the bone marrow to potentiate CD11b+Gr1+ cells, which are recruited to tumor tissue where they contribute to tumor angiogenesis and growth. Cancer Res; 73(22); 6574–83. ©2013 AACR.

The tumor microenvironment provides primary tumor cells to mix with multiple types of stroma such as endothelium, fibroblasts, and immune cells (1). Such heterogeneity of cell populations presents a major impediment for developing a cure for cancer. Increasing evidence supports that stromal cells in the tumor microenvironment not only occupy a significant fraction of the tumor bulk, but also play critical roles in proliferation, invasion, and/or metastasis of tumor cells (2). In this regard, bone is an essential partner for tumor progression, because bone marrow serves as the supplying organ for numerous critical cells in the tumor microenvironment (3, 4). However, it is unclear how tumor cells co-opt the bone and/or bone marrow to facilitate a favorable tumor microenvironment.

Among the bone marrow–derived cells, CD11b+Gr1+ cells [commonly referred to as myeloid-derived suppressor cells (MDSC)] correlate with tumor progression (5). MDSCs were originally investigated for their roles in evasion of host immune surveillance, especially via suppression of T-cell–dependent antitumoral immunity by production of arginase, reactive oxygen species, and inducible nitric oxide synthase (6). Subsequent studies demonstrated that MDSCs are increased in tumor-bearing mice and cancer patients, and infiltrate primary tumor tissue to promote angiogenesis by secreting matrix metalloproteinases (MMP), and also by direct incorporation into tumor endothelium (7, 8). More recently, MDSCs have been shown to play key roles in recovery after radiation therapy (9, 10) and antiangiogenic therapy (11).

In parallel, multiple mechanisms have been proposed to explain the increased recruitment of MDSCs in tumor tissue. Yang and colleagues demonstrated that CXC chemokine ligand (CXCL)-5/CXC receptor (CXCR)-2 and stromal-derived factor-1/CXCR-4 axes recruit circulating MDSCs to tumor tissue (12). More recently, expression of a single integrin (α4β1) promotes MDSC invasion into tumors via activation of phosphatidylinositol 3-kinase (PI3K; ref. 13). However, despite such clear evidence supporting the tumorigenic functions of MDSCs and also the potential mechanisms of recruitment to the tumor tissue, MDSCs are poorly understood about their regulation in the supplying organ (i.e., bone marrow) of the tumor host, and also their potential crosstalk with distant primary tumor cells.

This study was designed to elucidate how CD11b+Gr1+ cells are regulated in the bone marrow of prostate tumor hosts, contributing to tumor growth and angiogenesis. Prostate cancer provides a unique perspective on this process because of its devastating mortality and morbidity associated with its preferential metastasis to the skeleton (14). Accordingly, prostate cancer cells secrete numerous important bone-modulating cytokines, leading to osteoblastic/osteolytic reactions that facilitate growth factor and cytokine release from bone cells and matrix (15). In particular, parathyroid hormone–related protein (PTHrP) is expressed by prostate cancer cells, and stimulates osteoblasts in an endocrine manner to secrete factors such as receptor activator of NF-κB ligand (RANKL), interleukin (IL)-6, C-C chemokine ligand (CCL)-2, and VEGF-A within the bone microenvironment (16–18). Subsequently, PTHrP-induced cytokines have the ability to trigger cascades of unfavorable events (e.g., signaling pathways leading to potentiation of CD11b+Gr1+ bone marrow cells) within the bone marrow, contributing to tumor progression. Overall, the central hypothesis of this study was prostate cancer–derived PTHrP potentiates CD11b+Gr1+ cells within the bone marrow, contributing to angiogenesis and tumor growth.

Cells

Two luciferase-labeled PC-3 clones expressing high and low levels of PTHrP were selected from previously established stable-shRNA clones targeting PTHLH (19), designating PTHrPHi and PTHrPLo, respectively. Ace-1 canine prostate carcinoma cells, expressing undetectable basal levels of PTHrP, were stably transfected with a pcDNA3.1 vector expressing full-length mouse/rat PTHrP (17). An empty-vector transfectant was used as a control. Expression of PTHrP was confirmed from the culture supernatant using an immunoradiometric assay kit (Diagnostic Laboratories). PC-3 clones were regularly authenticated and matched short tandem repeat DNA profiles of the original PC-3 cell line (last tested on August 28, 2012).

Mice and in vivo tumors

All mouse experiments were approved by the Institutional Animal Care and Use Committees of the University of Michigan and Vanderbilt University. For in vivo tumors, 1 × 106 prostate tumor cells were suspended in 100 μL Hank's balanced salt solution and 1:1 mixed with growth factor–reduced Matrigel (BD Biosciences), followed by subcutaneous injection into male athymic mice (Harlan Laboratories) as previously described (20, 21). Mice were regularly monitored for morbidity or tumor growth, and tumor size was calculated using an equation: Volume = ½ × a × b2, where a is the long diameter and b is the short diameter measured with a caliper (22). Anti-human PTHrP 1 to 33 monoclonal antibodies (hybridoma 158) were produced and generously gifted by Dr. R. Kremer (McGill University, Montreal, Quebec, Canada; ref. 18). Mice were treated with anti-PTHrP monoclonal antibody (200 μL) or mouse immunoglobulin G (IgG; Sigma-Aldrich) by every other day intraperitoneal injection for the first 3 weeks, followed by daily injection for 1 week before euthanasia.

Flow cytometry

For analyses of CD11b+Gr1+ cells in the tumor tissue, tumors were mechanically dissociated, followed by digestion in complete RPMI-1640 media supplemented with type I collagenase (5 mg/mL; Sigma-Aldrich). Viable cells were counted and resuspended in fluorescence-activated cell sorting buffer containing combinations of antibodies including FITC-conjugated anti-mouse CD11b, PE-conjugated anti-mouse Gr1, or isotype controls. For analyses or sorting CD11b+Gr1+ cells from the bone marrow, the femoral bone marrow was flushed and dissociated, followed by antibody staining and flow cytometry (23). For analyses of phospho-Y418 Src family kinase (SFK), the bone marrow cells were fixed, permeabilized, stained, and analyzed according to the BD PhosFlow Cell Signaling protocols. All materials were from BD Biosciences.

Immunohistochemistry

Tumors were surgically removed and bisected, a portion fixed in formalin and a portion snap-frozen. Murine endothelial cell–specific CD31/PECAM immunostaining (clone MEC13.3; BD Biosciences) was performed according to a previously described method (24). Rat anti-mouse CD11b (clone M1/70; BD Biosciences) and anti-mouse Ly-6G (clone RB6-8C5; eBioscience) were fluorescently labeled and used to detect CD11b+Gr1+ cells in the tumor tissue. Three to five randomly selected microscopic images per sample were obtained, and positively stained cells were counted using ImageJ software.

Quantitative PCR

mRNA samples were prepared from the bone marrow or tumor tissues using TRIzol reagent (Invitrogen), followed by reverse transcription-quantitative PCR (25). All quantitative PCR probes and reagents were from Applied Biosystems.

Statistical analyses

All in vivo data sets were tested for normality by Shapiro–Wilk test. Statistical analyses were performed by GraphPad Prism software. Student t test or Mann–Whitney U test were used to compare 2 groups and all statistical tests were 2-sided.

Reduction of PTHrP in PC-3 prostate tumors decreased CD11b+Gr1+ bone marrow cell recruitment and angiogenesis

As a first approach to investigate the role of PTHrP in the potential crosstalk between tumor and the bone marrow, the PTHLH gene (encoding PTHrP) was targeted via lentiviral shRNA vectors in PC-3, human prostate cancer cells (19). Two clones expressing high and low levels of PTHrP (961.8 ± 12.8 vs. 457.8 ± 4.1 pg mlL−1 1 × 106 cells−1 48 h−1; measured in the culture supernatant by immunoradiometric assays) were selected and designated PTHrPHi and PTHrPLo, respectively. PTHrP is well known to regulate tumor growth via autocrine, intracrine, and paracrine manners (17–19, 26, 27), hence alterations in the host response (e.g., recruitment of host-derived cells) could simply be secondary to the differences in the tumor size, not in PTHrP expression levels. Therefore, PTHrPLo tumors were grown for a longer period until they reached a similar mean tumor volume as PTHrPHi tumors to circumvent the direct tumor-size effects in the subsequent analyses (Fig. 1A and B). Flow cytometric analyses of the tumor tissues revealed that PTHrPLo tumors had significantly reduced percentages of CD11b+Gr1+ cells in the tumor tissue compared with PTHrPHi tumors (Fig. 1C). Immunohistological analyses showed PTHrP levels correlated with mean vessel density and vessel area of PC-3 tumors (Fig. 1D–F). A well-characterized mechanism of MDSC-dependent tumor angiogenesis is through the expression of MMP-9 (7, 28). Accordingly, tumor tissues were analyzed for expression of host-derived MMP-9 expression as well as tumor-derived VEGF-A (Fig. 1G and H) using species-specific PCR probes. PTHrPLo tumors had significantly reduced host-derived (i.e., murine) Mmp9 expression, whereas no significant reduction in the tumor-derived (i.e., human) VEGFA was observed. Collectively, reduction of PTHrP in PC-3 prostate tumors decreased CD11b+Gr1+ cell recruitment and tumor angiogenesis, in association with reduced expression of host MMP-9 but not of tumor VEGF-A.

Figure 1.

Reduction of PTHrP in PC-3 prostate tumors decreased CD11b+Gr1+ MDSC recruitment and angiogenesis. A, tumor growth curve of PC-3 clones expressing high or low levels of PTHrP (n = 9 for PTHrPHi and n = 10 for PTHrPLo). PTHrPLo tumors were grown for a longer period (57 days) to reach a similar mean tumor volume as PTHrPHi tumors (38 days). B, PTHrPHi and PTHrPLo tumors were surgically dissected on the same day and photographed. Mean tumor volume between the 2 groups was not significantly different (P = 0.68, Student t test). Scale bar, 1 cm. C, percentages of CD11b+Gr1+ double-positive cells in the tumor tissues were analyzed by flow cytometry. D, tumor tissues were sectioned for H&E and murine CD31/PECAM immunohistochemical staining. Original magnification, ×20. Scale bars, 50 μm. E and F, microscopic images were analyzed for tumor mean vessel density (MVD) or CD31+ vascular area with normalization to total nuclear area. G and H, host-derived (i.e., murine) Mmp9 and tumor-derived (i.e., human) VEGFA mRNA levels were measured by quantitative RT-PCR using species-specific probes (n = 9–10 per group). All P values are from Student t test. NS, not significant. Data in all graphs are mean ± SEM.

Figure 1.

Reduction of PTHrP in PC-3 prostate tumors decreased CD11b+Gr1+ MDSC recruitment and angiogenesis. A, tumor growth curve of PC-3 clones expressing high or low levels of PTHrP (n = 9 for PTHrPHi and n = 10 for PTHrPLo). PTHrPLo tumors were grown for a longer period (57 days) to reach a similar mean tumor volume as PTHrPHi tumors (38 days). B, PTHrPHi and PTHrPLo tumors were surgically dissected on the same day and photographed. Mean tumor volume between the 2 groups was not significantly different (P = 0.68, Student t test). Scale bar, 1 cm. C, percentages of CD11b+Gr1+ double-positive cells in the tumor tissues were analyzed by flow cytometry. D, tumor tissues were sectioned for H&E and murine CD31/PECAM immunohistochemical staining. Original magnification, ×20. Scale bars, 50 μm. E and F, microscopic images were analyzed for tumor mean vessel density (MVD) or CD31+ vascular area with normalization to total nuclear area. G and H, host-derived (i.e., murine) Mmp9 and tumor-derived (i.e., human) VEGFA mRNA levels were measured by quantitative RT-PCR using species-specific probes (n = 9–10 per group). All P values are from Student t test. NS, not significant. Data in all graphs are mean ± SEM.

Close modal

Ectopic PTHrP increased the recruitment of CD11b+Gr1+ cells in prostate tumor tissue

An additional prostate tumor model was utilized to establish the causal relationship between PTHrP and CD11b+Gr1+ cells. Ace-1 prostate cancer cells produce predominantly osteoblastic lesions in vivo, a phenotype that recapitulates human prostate cancer more realistically than the majority of currently available prostate cancer cell lines (17, 29). Ace-1 cells, expressing undetectable basal levels of PTHrP, were stably transfected with PTHrP overexpression (designated PTHrP OE) or empty control (designated pcDNA) vectors. In the same approach as the PC-3 tumor model (i.e., growth in differential periods), 2 groups of similarly sized tumors, PTHrP OE and pcDNA control, were produced. To directly examine the effects of systemic PTHrP on CD11b+Gr1+ cell recruitment, one group of mice carrying pcDNA control tumors was treated with recombinant PTHrP (amino acids 1–34, a ligand-binding fragment) for 7 days before harvest (Fig. 2A and Supplementary Fig. S1). Both PTHrP OE and recombinant PTHrP-treated groups had significantly increased CD11b+Gr1+ cells in the tumor tissue compared with pcDNA control tumors (Fig. 2B). Although mice burdened with PTHrP OE tumors had significantly increased percentages of CD11b+Gr1+ cells in the bone marrow (Fig. 2C), recombinant PTHrP treatment failed to show such an increase in the bone marrow. This may be explained by either the different modes of PTHrP administration (i.e., intermittent injection vs. continuous expression) or the reduced duration (7 days) of PTHrP treatment compared with tumor burden (21 days). Immunohistochemical analyses of tumor tissue showed that both PTHrP OE and recombinant PTHrP tumors had significantly increased evidence of angiogenesis (Fig. 2D and Supplementary Fig. S1B). In addition, host-derived Mmp9 expression was significantly increased in PTHrP OE tumor tissue (Fig. 2E), suggesting contribution of the CD11b+Gr1+ cell recruitment, at least in part, to angiogenesis. Collectively, data in Figs. 1 and 2 suggest that prostate cancer-derived PTHrP is a crucial regulator of CD11b+Gr1+ cells.

Figure 2.

Ectopic PTHrP increased CD11b+Gr1+ cells in prostate tumors and in the bone marrow. Ace-1 prostate cancer cells, expressing undetectable basal levels of PTHrP, were engineered to overexpress PTHrP (PTHrP OE) with a vector-alone transfectant control (pcDNA). Cells were implanted subcutaneously in male athymic mice. A, PTHrP OE (n = 10; right) were grown for a shorter period (17 days) to produce similarly sized tumors as pcDNA control tumors (n = 7; grown for 32 days; left). In addition, one group of mice carrying pcDNA tumors (n = 10; middle) was administered recombinant PTHrP (1–34) once daily for 7 days before euthanasia (designated rPTHrP). Mean tumor volume ± SEM (mm3) for each group is indicated. No statistical significance in tumor volume (P > 0.05 by Student t test) in any pair of groups. Scale bar, 1 cm. B and C, percentages of CD11b+Gr1+ cells in the tumor tissues or in the bone marrow were analyzed by flow cytometry. D, tumor tissues were analyzed for quantification of CD31+ vessel area with normalization to total nuclear area (n = 7–10 per group). E, tumor-derived (i.e., canine) MMP9 mRNA levels were measured (n = 7–10 per group). All P values are from Student t test. NS, not significant. Data in all graphs are mean ± SEM.

Figure 2.

Ectopic PTHrP increased CD11b+Gr1+ cells in prostate tumors and in the bone marrow. Ace-1 prostate cancer cells, expressing undetectable basal levels of PTHrP, were engineered to overexpress PTHrP (PTHrP OE) with a vector-alone transfectant control (pcDNA). Cells were implanted subcutaneously in male athymic mice. A, PTHrP OE (n = 10; right) were grown for a shorter period (17 days) to produce similarly sized tumors as pcDNA control tumors (n = 7; grown for 32 days; left). In addition, one group of mice carrying pcDNA tumors (n = 10; middle) was administered recombinant PTHrP (1–34) once daily for 7 days before euthanasia (designated rPTHrP). Mean tumor volume ± SEM (mm3) for each group is indicated. No statistical significance in tumor volume (P > 0.05 by Student t test) in any pair of groups. Scale bar, 1 cm. B and C, percentages of CD11b+Gr1+ cells in the tumor tissues or in the bone marrow were analyzed by flow cytometry. D, tumor tissues were analyzed for quantification of CD31+ vessel area with normalization to total nuclear area (n = 7–10 per group). E, tumor-derived (i.e., canine) MMP9 mRNA levels were measured (n = 7–10 per group). All P values are from Student t test. NS, not significant. Data in all graphs are mean ± SEM.

Close modal

CD11b+Gr1+ cells promoted tumor growth in vivo

The protumorigenic functions of CD11b+Gr1+ cells are relatively well characterized using multiple tumor models (5, 7, 30, 31). To more rigorously examine the effects of CD11b+Gr1+ cells on tumor growth in the prostate tumor model, 2 fractions of bone marrow cells, that is CD11b/Gr1-double positive or negative cells, were isolated and coimplanted with parental Ace-1 tumor cells in vivo (Fig. 3A). Increasing numbers of CD11b+Gr1+ cells mixed with tumor cells correspondingly increased the tumor size within 15 days (Fig. 3B and C). More importantly, Ace-1 tumor coimplanted with 0.5 × 106 CD11b+Gr1+ cells grew significantly larger than tumors coimplanted with the same number of CD11bGr1 cells, suggesting that altered tumor size in Figs. 1 and 2 were secondary to the altered recruitment of CD11b+Gr1+ cells in the tumor tissue.

Figure 3.

CD11b+Gr1+ cells promoted tumor growth in vivo. A, schematic representation of the experiment. Male athymic mice were implanted with PTHrP-overexpressing Ace-1 tumors for 21 days before harvesting femoral bone marrow cells. Flow cytometric sorting resulted in 2 fractions of CD11b/Gr1 double positive or double negative cells, followed by their coinjection with parental Ace-1 cells subcutaneously in male athymic mice (n = 8 per group). B, individual tumor size was measured 15 days after xenograft and plotted on a graph. Dots, individual tumor volumes (mm3). Horizontal lines, medians. P values are from Student t test. C, tumor growth curve was plotted to compare growth kinetics. Data are mean ± SEM. *, P < 0.05 and **, P < 0.01, Student t test.

Figure 3.

CD11b+Gr1+ cells promoted tumor growth in vivo. A, schematic representation of the experiment. Male athymic mice were implanted with PTHrP-overexpressing Ace-1 tumors for 21 days before harvesting femoral bone marrow cells. Flow cytometric sorting resulted in 2 fractions of CD11b/Gr1 double positive or double negative cells, followed by their coinjection with parental Ace-1 cells subcutaneously in male athymic mice (n = 8 per group). B, individual tumor size was measured 15 days after xenograft and plotted on a graph. Dots, individual tumor volumes (mm3). Horizontal lines, medians. P values are from Student t test. C, tumor growth curve was plotted to compare growth kinetics. Data are mean ± SEM. *, P < 0.05 and **, P < 0.01, Student t test.

Close modal

Tumor-derived PTHrP confers increased angiogenic potential to CD11b+Gr1+ cells

To examine whether tumor-derived PTHrP regulates CD11b+Gr1+ cells within the bone marrow of tumor hosts, CD11b+Gr1+ bone marrow cells were isolated from 2 groups of mice bearing either PTHrP-overexpressing or pcDNA control tumors for 3 weeks, resulting in 2 fractions of CD11b+Gr1+ cells (i.e., PTHrP-activated vs. control). Parental Ace-1 tumor cells were mixed with the isolated CD11b+Gr1+ cells and xenografted into male athymic mice (Fig. 4A). Tumors coimplanted with PTHrP-activated CD11b+Gr1+ cells were significantly larger than the tumors with control CD11b+Gr1+ cells (Fig. 4B), potentially because increased MMP-9 and angiogenesis as determined by immunohistochemistry (Fig. 4C and D and Supplementary Fig. S2).

Figure 4.

PTHrP promoted tumorigenic function of CD11b+Gr1+ bone marrow cells. A, schematic representation of the experiment. Two groups of male athymic mice (n = 3 per group) were implanted with PTHrP-overexpressing or control tumors for 21 days before flow cytometric sorting of CD11b+Gr1+ bone marrow cells. Subsequently, parental Ace-1 prostate tumor cells were coinjected with the primed CD11b+Gr1+ cells into male athymic mice (n = 8 per group). B, individual tumor weight was measured and plotted, and photographs of 3 representative tumors are shown. Dots, individual tumor weight (mg). Horizontal lines, median (n = 8 per group). C and D, tumor tissues were analyzed for quantification of CD31+ mean vessel density or vessel area with normalization to total nuclear area. Data are mean ± SEM. All P values are from Student t test.

Figure 4.

PTHrP promoted tumorigenic function of CD11b+Gr1+ bone marrow cells. A, schematic representation of the experiment. Two groups of male athymic mice (n = 3 per group) were implanted with PTHrP-overexpressing or control tumors for 21 days before flow cytometric sorting of CD11b+Gr1+ bone marrow cells. Subsequently, parental Ace-1 prostate tumor cells were coinjected with the primed CD11b+Gr1+ cells into male athymic mice (n = 8 per group). B, individual tumor weight was measured and plotted, and photographs of 3 representative tumors are shown. Dots, individual tumor weight (mg). Horizontal lines, median (n = 8 per group). C and D, tumor tissues were analyzed for quantification of CD31+ mean vessel density or vessel area with normalization to total nuclear area. Data are mean ± SEM. All P values are from Student t test.

Close modal

PTHrP increased expression of phospho-[Y418] Src family kinases in CD11b+Gr1+ cells

The molecular mechanism for the observed PTHrP-dependent CD11b+Gr1+ cell potentiation was subsequently investigated. Recently, Liang and colleagues demonstrated that dasatinib, an SFK inhibitor, suppressed prostate tumor growth as well as the numbers of CD11b+ myeloid cells in tumor tissues (32). Accordingly, the effects of PTHrP administration on SFK in CD11b+Gr1+ cells were investigated. A single administration of PTHrP (1–34) to male athymic mice significantly increased the activating phosphorylation of Tyr-418 residue of SFK in CD11b+Gr1+ cells (Fig. 5A). As SFK activation requires intramolecular conformational changes and interaction with activated receptor kinases via the SH-2 domain, phosphorylation of [Y418] in the SH-2 domain indicates the status of full activation. However, because CD11b+Gr1+ cells do not express receptors for PTHrP (as determined by quantitative reverse transcription (RT)-PCR for Pthr1; Supplementary Fig. S3), phosphorylation of [Y418] SFK was reasoned to be indirect through cytokines from osteoblasts, the predominant cells expressing the PTH/PTHrP receptor (PTH1R) in the bone marrow. Potential candidate cytokines from PTHrP-stimulated osteoblasts included IL-6, VEGF-A, C-C chemokine ligand (CCL)-2, and RANKL (16, 33–35). Therefore, CD11b+Gr1+ cells were isolated from femoral bone marrow and treated with these osteoblastic cytokines. Although all 4 cytokines (IL-6, VEGF-A, CCL-2, and RANKL) have been shown to upregulate SFKs (36–38), only IL-6 and VEGF-A increased the expression of phospho-[Y418] SFK in MDSCs (Fig. 5B).

Figure 5.

PTHrP phosphorylated [Y418] Src family kinases in CD11b+Gr1+ cells. A, male athymic mice (n = 3 per group) were stimulated with a single administration of PTHrP (1–34) or saline control, 8 hours before sacrifice and flow cytometric analyses of phospho-[Y418] Src family kinase expression levels in CD11b+Gr1+ bone marrow cells. Representative histograms from the control group (shaded) and the PTHrP-stimulated group (open) were overlapped to show the intensity of phospho-[Y418] Src expression. CD11b+Gr1+ cells expressing high levels of phospho-[Y418] Src family kinases [indicated by a bracket (M)] were quantified and plotted. Data are mean ± SEM. P < 0.01, Student t test. B, CD11b/Gr1 double positive cells were sorted from the femoral bone marrow of male athymic mice, followed by treatment with saline, IL-6, VEGF-A, CCL-2, RANKL, or PTHrP (all 100 ng/mL for 0.5 × 106 cells) for 1 hour at 37°C (n = 3 per group). Representative histograms (open) were overlapped onto unstimulated controls (shaded) to show the intensity of staining.

Figure 5.

PTHrP phosphorylated [Y418] Src family kinases in CD11b+Gr1+ cells. A, male athymic mice (n = 3 per group) were stimulated with a single administration of PTHrP (1–34) or saline control, 8 hours before sacrifice and flow cytometric analyses of phospho-[Y418] Src family kinase expression levels in CD11b+Gr1+ bone marrow cells. Representative histograms from the control group (shaded) and the PTHrP-stimulated group (open) were overlapped to show the intensity of phospho-[Y418] Src expression. CD11b+Gr1+ cells expressing high levels of phospho-[Y418] Src family kinases [indicated by a bracket (M)] were quantified and plotted. Data are mean ± SEM. P < 0.01, Student t test. B, CD11b/Gr1 double positive cells were sorted from the femoral bone marrow of male athymic mice, followed by treatment with saline, IL-6, VEGF-A, CCL-2, RANKL, or PTHrP (all 100 ng/mL for 0.5 × 106 cells) for 1 hour at 37°C (n = 3 per group). Representative histograms (open) were overlapped onto unstimulated controls (shaded) to show the intensity of staining.

Close modal

Phospho-[Y418] SFK by osteoblastic VEGF-A and IL-6 increased MMP-9 expression in CD11b+Gr1+ cells

To further investigate the functional significance of phospho-[Y418] SFKs in MDSCs, several published markers of CD11b+Gr1+ cell activation were examined in combination with PTHrP-dependent osteoblastic cytokines and an SFK selective inhibitor, PP2 (5, 13, 28). Only VEGF-A and IL-6 increased Mmp9 gene expression, whereas Cxcr2, Cxcr4, or Itgb1 expression remained unaffected in CD11b+Gr1+ cells, and this increase was reversed by PP2 treatment (Fig. 6A–D). Furthermore, to confirm the requirement of osteoblasts in PTHrP-dependent potentiation of CD11b+Gr1+ cells, primary osteoblasts were established from murine calvaria and treated with PTHrP (1–34) or saline for 24 hours and conditioned media harvested (39). CD11b+Gr1+ cells were isolated from femoral bone marrow and stimulated with osteoblast-derived control- or PTHrP-conditioned media in combination with neutralizing antibodies against VEGF-A and/or IL-6. Consistent with the previous data, PTHrP-conditioned media from osteoblast cultures increased Mmp9 gene expression (Fig. 6E) and functional MMP-9 (Fig. 6F) in the MDSCs, and these effects were blocked by anti-VEGF-A and/or anti-IL-6 neutralizing antibodies. Furthermore, the effect of PTHrP-conditioned media on MMP-9 expression was suppressed by PP2 (Fig. 6G).

Figure 6.

Phosphorylation of Src family kinases by osteoblastic VEGF-A and IL-6 increased MMP-9 expression in CD11b+Gr1+ cells. A–D, CD11b+Gr1+ cells were sorted from the femoral bone marrow of male athymic mice via flow cytometry, followed by treatment with saline (control), VEGF-A, IL-6, PTHrP, or CCL-2 (100 ng/mL for 0.5 × 106 cells) in combination with PP2 (an SFK inhibitor; 100 nmol/L) or dimethyl sulfoxide (DMSO) control for 1 hour at 37°C (n = 3 per group). mRNA levels of Mmp9, Cxcr4, Cxcr2, and Itgb1 were determined via quantitative RT-PCR. Data are mean ± SEM. *, P < 0.01 compared with the DMSO control group. No other combination had statistical significance (P > 0.05, Student t test). E–G, conditioned media were collected from primary calvarial osteoblasts stimulated with saline (control) or PTHrP (1–34). Femoral bone marrow CD11b+Gr1+ cells were treated with the control or PTHrP-conditioned media in combination with neutralizing antibodies against VEGF-A and/or IL-6, followed by quantitative PCR (E) for Mmp9 gene expression (n = 3 per group) or zymography (F). Data are mean ± SEM. *, P < 0.05; NS, not significant by Student t test. G, CD11b+Gr1+ cells were treated with control or PTHrP-conditioned media in combination with PP2 or DMSO control, followed by zymography.

Figure 6.

Phosphorylation of Src family kinases by osteoblastic VEGF-A and IL-6 increased MMP-9 expression in CD11b+Gr1+ cells. A–D, CD11b+Gr1+ cells were sorted from the femoral bone marrow of male athymic mice via flow cytometry, followed by treatment with saline (control), VEGF-A, IL-6, PTHrP, or CCL-2 (100 ng/mL for 0.5 × 106 cells) in combination with PP2 (an SFK inhibitor; 100 nmol/L) or dimethyl sulfoxide (DMSO) control for 1 hour at 37°C (n = 3 per group). mRNA levels of Mmp9, Cxcr4, Cxcr2, and Itgb1 were determined via quantitative RT-PCR. Data are mean ± SEM. *, P < 0.01 compared with the DMSO control group. No other combination had statistical significance (P > 0.05, Student t test). E–G, conditioned media were collected from primary calvarial osteoblasts stimulated with saline (control) or PTHrP (1–34). Femoral bone marrow CD11b+Gr1+ cells were treated with the control or PTHrP-conditioned media in combination with neutralizing antibodies against VEGF-A and/or IL-6, followed by quantitative PCR (E) for Mmp9 gene expression (n = 3 per group) or zymography (F). Data are mean ± SEM. *, P < 0.05; NS, not significant by Student t test. G, CD11b+Gr1+ cells were treated with control or PTHrP-conditioned media in combination with PP2 or DMSO control, followed by zymography.

Close modal

Anti-PTHrP monoclonal antibody treatment decreased MDSC recruitment in PC-3 tumors

Finally, to more rigorously determine the causal relationship between PTHrP and MDSC recruitment, mice bearing PTHrPHi PC-3 tumors were treated with nonspecific control IgG or anti-human PTHrP monoclonal antibodies. Anti-PTHrP antibodies significantly suppressed tumor growth, but not to the level of PTHrPLo tumors (Fig. 7A and B). As anti-PTHrP monoclonal antibodies potentially suppress tumor growth via inhibition of autocrine PTHrP effects on tumor cells (Supplementary Fig. S4), tumor tissues were analyzed for MDSC recruitment by immunofluorescence colocalization of CD11b+Gr1+ cells (Fig. 7C and D). Numbers of CD11b+Gr1+ cells were decreased in anti-PTHrP antibody-treated or PTHrPLo tumor tissues, suggesting that reduced PTHrP is causal to decreased MDSCs found in tumor tissues. Serum calcium levels were correlated with PTHrP levels, indicating the functional activity of PTHrP (Fig. 7E). Quantitative RT-PCR analysis in tumor tissues revealed that shRNA-mediated PTHrP knockdown was stable in PC-3 tumor cells during in vivo tumor growth, and the correlation between PTHrP and Mmp9 gene expression (Fig. 7F and G).

Figure 7.

Anti-human PTHrP monoclonal antibody decreased MDSC recruitment in PC-3 tumors. A, tumor growth curve of PC-3 PTHrPHi tumors treated with control IgG or anti-PTHrP monoclonal antibodies (mAb) and PC-3 PTHrPLo tumors (n = 10 per group). Both P values are from linear regression comparison with PC-3 PTHrPHi IgG tumor group. Data are mean ± SEM. B, individual tumor weight was measured upon necropsy and plotted. Dots, individual measurements (mg). Horizontal lines, mean ± SEM (n = 10 per group). C, tumor tissues were sectioned and stained for CD11b (Alexa-Fluor 488), Gr1 (Alexa-Fluor 546), and DAPI. Original magnification, ×40. Scale bars, 50 μm. D, immunofluorescent images were merged and analyzed for CD11b+Gr1+ cell per microscopic field. Three positively stained nonnecrotic tumor areas were randomly selected for quantification (5 tumors/group). E, sera were collected upon necropsy, followed by calcium assay. F and G, PTHLH or Mmp9 mRNA levels in the pulverized tumor tissue were measured by quantitative RT-PCR (n = 10 per group). Data in all bar graphs are mean ± SEM. All P values, unless indicated otherwise, are from Student t test.

Figure 7.

Anti-human PTHrP monoclonal antibody decreased MDSC recruitment in PC-3 tumors. A, tumor growth curve of PC-3 PTHrPHi tumors treated with control IgG or anti-PTHrP monoclonal antibodies (mAb) and PC-3 PTHrPLo tumors (n = 10 per group). Both P values are from linear regression comparison with PC-3 PTHrPHi IgG tumor group. Data are mean ± SEM. B, individual tumor weight was measured upon necropsy and plotted. Dots, individual measurements (mg). Horizontal lines, mean ± SEM (n = 10 per group). C, tumor tissues were sectioned and stained for CD11b (Alexa-Fluor 488), Gr1 (Alexa-Fluor 546), and DAPI. Original magnification, ×40. Scale bars, 50 μm. D, immunofluorescent images were merged and analyzed for CD11b+Gr1+ cell per microscopic field. Three positively stained nonnecrotic tumor areas were randomly selected for quantification (5 tumors/group). E, sera were collected upon necropsy, followed by calcium assay. F and G, PTHLH or Mmp9 mRNA levels in the pulverized tumor tissue were measured by quantitative RT-PCR (n = 10 per group). Data in all bar graphs are mean ± SEM. All P values, unless indicated otherwise, are from Student t test.

Close modal

This study provides new evidence that distant tumors stimulate the bone marrow to increase critical component cells in the tumor microenvironment. In brief, prostate cancer–derived PTHrP circulates to potentiate CD11b+Gr1+ cells within the bone marrow via upregulation of IL-6 and VEGF-A in osteoblasts, contributing to tumor growth and angiogenesis (Fig. 8). As a proposed mechanism of CD11b+Gr1+ cell potentiation, these data demonstrate that PTHrP increased activating phosphorylation of SFKs that subsequently increased Mmp9 gene expression in CD11b+Gr1+ cells, supporting that CD11b+Gr1+ cell-dependent tumor growth is, at least in part, mediated by MMP-9 expression and angiogenesis.

Figure 8.

Proposed model of CD11b+Gr1+ cell activation within the bone marrow of prostate tumor hosts via PTHrP. Prostate tumor–derived PTHrP circulates to stimulate VEGF-A and IL-6 expression by osteoblasts, leading to SFK phosphorylation of CD11b+Gr1+ MDSCs. Activation of SFK confers angiogenic potential of MDSCs via increased MMP-9 expression, contributing to prostate cancer growth and angiogenesis.

Figure 8.

Proposed model of CD11b+Gr1+ cell activation within the bone marrow of prostate tumor hosts via PTHrP. Prostate tumor–derived PTHrP circulates to stimulate VEGF-A and IL-6 expression by osteoblasts, leading to SFK phosphorylation of CD11b+Gr1+ MDSCs. Activation of SFK confers angiogenic potential of MDSCs via increased MMP-9 expression, contributing to prostate cancer growth and angiogenesis.

Close modal

Increasing evidence now clearly supports the critical functions of CD11b+Gr1+ MDSCs in the tumor microenvironment (40). However, the majority of previous studies have been focused either on the roles of MDSCs within the tumor microenvironment (i.e., immune suppression and angiogenesis) or the mechanism of MDSC recruitment to the tumor (41, 42). Given that bone is an essential partner for tumor progression by supplying numerous bone marrow–derived stromal cells, primary tumor cells are speculated to have active mechanisms to interact with the bone/bone marrow (43, 44). The data in this study demonstrate that PTHrP serves as a messenger between the primary tumor and the bone marrow, conferring MDSCs with increased angiogenic potential. PTHrP is a potent bone-modulating cytokine expressed by multiple types of tumor cells such as prostate, breast, lung, and colorectal cancers (45–48). In addition, PTHrP is a key regulator of the “vicious cycle” hypothesis of metastatic tumor–bone interactions (49). However, given that MDSCs are currently considered universally essential components of the tumor microenvironment, not all tumor types express PTHrP, suggesting that PTHrP is not the only factor mediating the interactions between tumor and bone.

The molecular mechanisms of MDSC activation, expansion, and/or mobilization, and ultimately therapeutic approaches targeting the key signaling mechanisms, warrant extensive further investigation. Interestingly, the preliminary studies shown in Supplementary Fig. S5 suggest that PTHrP induces a series of alterations in the bone marrow to mobilize and/or expand MDSCs. Still, questions remain about whether and how PTHrP stimulates differentiation of MDSCs from bone marrow precursors. Nevertheless, this work provides a biological rationale for the clinical application of SFK inhibitors in targeting 2 compartments (i.e., tumor and the microenvironment) simultaneously, of which the mechanism requires further studies. The data in this study demonstrate that activation of SFKs is one of the key signal transduction mechanisms of MDSCs' angiogenic potential, in addition to two other factors, STAT3 and PI3K, that have previously been shown to be implicated in MDSC functions (13, 31). Indeed, SFKs mediate crucial regulatory functions in both tumor cells and stromal cells (e.g., endothelial cells and osteoclasts), suggesting that SFKs are promising therapeutic targets for the suppression of tumor as well as stromal compartments (24, 50).

In conclusion, this study provides evidence that prostate cancers positively regulate the bone marrow microenvironment via PTHrP, IL-6, VEGF-A, and SFKs, thereby increasing the angiogenic potential of CD11b+Gr1+ MDSCs, leading to increased tumor growth.

No potential conflicts of interest were disclosed.

Conception and design: S.I. Park, L.K. McCauley

Development of methodology: S.I. Park, J. Jones, L.K. McCauley

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S.I. Park, C. Lee, A.J. Koh, J.W. Seo, F.N. Soki, S.W. Cho

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.I. Park, A.J. Koh, S.W. Cho, S. Daignault-Newton, L.K. McCauley

Writing, review, and/or revision of the manuscript: S.I. Park, J. Jones, J.W. Seo, F.N. Soki, S. Daignault-Newton, L.K. McCauley

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.I. Park, D. Sadler, L.K. McCauley

Study supervision: L.K. McCauley

The authors thank Drs. E. Keller, R. Taichman, and K. Pienta for valuable discussion; and Dr. R. Kremer for providing anti-PTHrP monoclonal antibody.

This work was financially supported by the Department of Defense Prostate Cancer Research Program (W81XWH-10-1-0546 and WX81XWH-12-1-0348 to S.I. Park) and the National Cancer Institute Program Project (P01CA093900 to L.K. McCauley). Flow cytometric analyses were supported in part by the National Cancer Institute Cancer Center Support (P30CA068485) to Vanderbilt-Ingram Cancer Center.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Hanahan
D
,
Weinberg
RA
. 
Hallmarks of cancer: the next generation
.
Cell
2011
;
144
:
646
74
.
2.
Shiao
SL
,
Ganesan
AP
,
Rugo
HS
,
Coussens
LM
. 
Immune microenvironments in solid tumors: new targets for therapy
.
Gene Dev
2011
;
25
:
2559
72
.
3.
Park
SI
,
Soki
FN
,
McCauley
LK
. 
Roles of bone marrow cells in skeletal metastases: no longer bystanders
.
Cancer Microenviron
2011
;
4
:
237
46
.
4.
Park
SI
,
Liao
J
,
Berry
JE
,
Li
X
,
Koh
AJ
,
Michalski
ME
, et al
Cyclophosphamide creates a receptive microenvironment for prostate cancer skeletal metastasis
.
Cancer Res
2012
;
72
:
2522
32
.
5.
Yang
L
,
Edwards
CM
,
Mundy
GR
. 
Gr-1+CD11b+ myeloid-derived suppressor cells: formidable partners in tumor metastasis
.
J Bone Miner Res
2010
;
25
:
1701
6
.
6.
Youn
J-I
,
Gabrilovich
DI
. 
The biology of myeloid-derived suppressor cells: the blessing and the curse of morphological and functional heterogeneity
.
Eur J Immunol
2010
;
40
:
2969
75
.
7.
Yang
L
,
DeBusk
LM
,
Fukuda
K
,
Fingleton
B
,
Green-Jarvis
B
,
Shyr
Y
, et al
Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis
.
Cancer Cell
2004
;
6
:
409
21
.
8.
Zhao
F
,
Obermann
S
,
Wasielewski von
R
,
Haile
L
,
Manns
MP
,
Korangy
F
, et al
Increase in frequency of myeloid-derived suppressor cells in mice with spontaneous pancreatic carcinoma
.
Immunology
2009
;
128
:
141
9
.
9.
Ahn
G-O
,
Brown
JM
. 
Influence of bone marrow-derived hematopoietic cells on the tumor response to radiotherapy: experimental models and clinical perspectives
.
Cell Cycle
2009
;
8
:
970
6
.
10.
Ahn
G-O
,
Tseng
D
,
Liao
C-H
,
Dorie
MJ
,
Czechowicz
A
,
Brown
JM
. 
Inhibition of Mac-1 (CD11b/CD18) enhances tumor response to radiation by reducing myeloid cell recruitment
.
Proc Natl Acad Sci U S A
2010
;
107
:
8363
8
.
11.
Shojaei
F
,
Wu
X
,
Malik
AK
,
Zhong
C
,
Baldwin
ME
,
Schanz
S
, et al
Tumor refractoriness to anti-VEGF treatment is mediated by CD11b+Gr1+ myeloid cells
.
Nat Biotechnol
2007
;
25
:
911
20
.
12.
Yang
L
,
Huang
J
,
Ren
X
,
Gorska
AE
,
Chytil
A
,
Aakre
M
, et al
Abrogation of TGF beta signaling in mammary carcinomas recruits Gr-1+CD11b+ myeloid cells that promote metastasis
.
Cancer Cell
2008
;
13
:
23
35
.
13.
Schmid
MC
,
Avraamides
CJ
,
Dippold
HC
,
Franco
I
,
Foubert
P
,
Ellies
LG
, et al
Receptor tyrosine kinases and TLR/IL1Rs unexpectedly activate myeloid cell PI3kγ, a single convergent point promoting tumor inflammation and progression
.
Cancer Cell
2011
;
19
:
715
27
.
14.
Dayyani
F
,
Gallick
GE
,
Logothetis
CJ
,
Corn
PG
. 
Novel therapies for metastatic castrate-resistant prostate cancer
.
J Natl Cancer Inst
2011
;
103
:
1665
75
.
15.
Weilbaecher
KN
,
Guise
TA
,
McCauley
LK
. 
Cancer to bone: a fatal attraction
.
Nat Rev Cancer
2011
;
11
:
411
25
.
16.
Li
X
,
Loberg
R
,
Liao
J
,
Ying
C
,
Snyder
LA
,
Pienta
KJ
, et al
A destructive cascade mediated by CCL2 facilitates prostate cancer growth in bone
.
Cancer Res
2009
;
69
:
1685
92
.
17.
Liao
J
,
Li
X
,
Koh
AJ
,
Berry
JE
,
Thudi
N
,
Rosol
TJ
, et al
Tumor expressed PTHrP facilitates prostate cancer-induced osteoblastic lesions
.
Int J Cancer
2008
;
123
:
2267
78
.
18.
Li
J
,
Karaplis
AC
,
Huang
DC
,
Siegel
PM
,
Camirand
A
,
Yang
XF
, et al
PTHrP drives breast tumor initiation, progression, and metastasis in mice and is a potential therapy target
.
J Clin Invest
2011
;
121
:
4655
69
.
19.
Park
SI
,
McCauley
LK
. 
Nuclear localization of parathyroid hormone-related peptide confers resistance to anoikis in prostate cancer cells
.
Endocr Relat Cancer
2012
;
19
:
243
54
.
20.
Park
SI
,
Kim
SJ
,
McCauley
LK
,
Gallick
GE
. 
Pre-clinical mouse models of human prostate cancer and their utility in drug discovery
.
Curr Protoc Pharmacol
2010
;
Chapter 14:Unit14.15
.
21.
Jung
Y
,
Shiozawa
Y
,
Wang
J
,
McGregor
N
,
Dai
J
,
Park
SI
, et al
Prevalence of prostate cancer metastases after intravenous inoculation provides clues into the molecular basis of dormancy in the bone marrow microenvironment
.
Neoplasia
2012
;
14
:
429
39
.
22.
Tomayko
MMM
,
Reynolds
CPC
. 
Determination of subcutaneous tumor size in athymic (nude) mice
.
Cancer Chemother Pharmacol
1989
;
24
:
148
54
.
23.
Pirih
FQ
,
Michalski
MN
,
Cho
SW
,
Koh
AJ
,
Berry
JE
,
Ghaname
E
, et al
Parathyroid hormone mediates hematopoietic cell expansion through interleukin-6
.
PLoS One
2010
;
5
:
e13657
.
24.
Park
SI
,
Zhang
J
,
Phillips
KA
,
Araujo
JC
,
Najjar
AM
,
Volgin
AY
, et al
Targeting SRC family kinases inhibits growth and lymph node metastases of prostate cancer in an orthotopic nude mouse model
.
Cancer Res
2008
;
68
:
3323
33
.
25.
Novince
CM
,
Koh
AJ
,
Michalski
MN
,
Marchesan
JT
,
Wang
J
,
Jung
Y
, et al
Proteoglycan 4, a novel immunomodulatory factor, regulates parathyroid hormone actions on hematopoietic cells
.
Am J Pathol
2011
;
179
:
2431
42
.
26.
Dougherty
KM
,
Blomme
EA
,
Koh
AJ
,
Henderson
JE
,
Pienta
KJ
,
Rosol
TJ
, et al
Parathyroid hormone-related protein as a growth regulator of prostate carcinoma
.
Cancer Res
1999
;
59
:
6015
22
.
27.
Tovar
Sepulveda VA
,
Falzon
M
. 
Parathyroid hormone-related protein enhances PC-3 prostate cancer cell growth via both autocrine/paracrine and intracrine pathways
.
Regul Pept
2002
;
105
:
109
20
.
28.
Ahn
G-O
,
Brown
JM
. 
Matrix metalloproteinase-9 is required for tumor vasculogenesis but not for angiogenesis: role of bone marrow-derived myelomonocytic cells
.
Cancer Cell
2008
;
13
:
193
205
.
29.
LeRoy
BE
,
Thudi
NK
,
Nadella
MVP
,
Toribio
RE
,
Tannehill-Gregg
SH
,
van Bokhoven
A
, et al
New bone formation and osteolysis by a metastatic, highly invasive canine prostate carcinoma xenograft
.
Prostate
2006
;
66
:
1213
22
.
30.
Abe
F
,
Dafferner
AJ
,
Donkor
M
,
Westphal
SN
,
Scholar
EM
,
Solheim
JC
, et al
Myeloid-derived suppressor cells in mammary tumor progression in FVB Neu transgenic mice
.
Cancer Immunol Immunother
2010
;
59
:
47
62
.
31.
Kujawski
M
,
Kortylewski
M
,
Lee
H
,
Herrmann
A
,
Kay
H
,
Yu
H
. 
Stat3 mediates myeloid cell-dependent tumor angiogenesis in mice
.
J Clin Invest
2008
;
118
:
3367
77
.
32.
Liang
W
,
Kujawski
M
,
Wu
J
,
Lu
J
,
Herrmann
A
,
Loera
S
, et al
Antitumor activity of targeting SRC kinases in endothelial and myeloid cell compartments of the tumor microenvironment
.
Clin Cancer Res
2010
;
16
:
924
35
.
33.
Huang
YF
,
Harrison
JR
,
Lorenzo
JA
,
Kream
BE
. 
Parathyroid hormone induces interleukin-6 heterogeneous nuclear and messenger RNA expression in murine calvarial organ cultures
.
Bone
1998
;
23
:
327
32
.
34.
Lowik
CW
,
van der Pluijm
G
,
Bloys
H
,
Hoekman
K
,
Bijvoet
OL
,
Aarden
LA
, et al
Parathyroid hormone (PTH) and PTH-like protein (PLP) stimulate interleukin-6 production by osteogenic cells: a possible role of interleukin-6 in osteoclastogenesis
.
Biochem Biophys Res Commun
1989
;
162
:
1546
52
.
35.
Esbrit
P
,
Alvarez-Arroyo
MV
,
De Miguel
F
,
Martin
O
,
Martinez
ME
,
Caramelo
C
. 
C-terminal parathyroid hormone-related protein increases vascular endothelial growth factor in human osteoblastic cells
.
J Am Soc Nephrol
2000
;
11
:
1085
92
.
36.
Sabbota
AL
,
Kim
H-RC
,
Zhe
X
,
Fridman
R
,
Bonfil
RD
,
Cher
ML
. 
Shedding of RANKL by tumor-associated MT1-MMP activates Src-dependent prostate cancer cell migration
.
Cancer Res
2010
;
70
:
5558
66
.
37.
Hallek
M
,
Neumann
C
,
Schaffer
M
,
Danhauser-Riedl
S
,
Bubnoff von
N
,
de Vos
G
, et al
Signal transduction of interleukin-6 involves tyrosine phosphorylation of multiple cytosolic proteins and activation of Src-family kinases Fyn, Hck, and Lyn in multiple myeloma cell lines
.
Exp Hematol
1997
;
25
:
1367
77
.
38.
Inngjerdingen
M
,
Torgersen
KM
,
Maghazachi
AA
. 
Lck is required for stromal cell-derived factor 1α (CXCL12)-induced lymphoid cell chemotaxis
.
Blood
2002
;
99
:
4318
25
.
39.
Jin
R
,
Sterling
JA
,
Edwards
JR
,
DeGraff
DJ
,
Lee
C
,
Park
SI
, et al
Activation of NF-κB signaling promotes growth of prostate cancer cells in bone
.
Wang
QJ
,
editor
.
PLoS One
2013
;
8
:
e60983
.
40.
Youn
J-I
,
Kumar
V
,
Collazo
M
,
Nefedova
Y
,
Condamine
T
,
Cheng
P
, et al
Epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer
.
Nat Immunol
2013
:
14
:
211
20
.
41.
Coffelt
SB
,
Lewis
CE
,
Naldini
L
,
Brown
JM
,
Ferrara
N
,
De Palma
M
. 
Elusive identities and overlapping phenotypes of proangiogenic myeloid cells in tumors
.
Am J Pathol
2010
;
176
:
1564
76
.
42.
Shojaei
F
,
Zhong
C
,
Wu
X
,
Yu
L
,
Ferrara
N
. 
Role of myeloid cells in tumor angiogenesis and growth
.
Trends Cell Biol
2008
;
18
:
372
8
.
43.
Li
X
,
Koh
AJ
,
Wang
Z
,
Soki
FN
,
Park
SI
,
Pienta
KJ
, et al
Inhibitory effects of megakaryocytic cells in prostate cancer skeletal metastasis
.
J Bone Miner Res
2011
;
26
:
125
34
.
44.
Li
X
,
Liao
J
,
Park
SI
,
Koh
AJ
,
Sadler
WD
,
Pienta
KJ
, et al
Drugs which inhibit osteoclast function suppress tumor growth through calcium reduction in bone
.
Bone
2011
;
48
:
1354
61
.
45.
Kremer
R
,
Li
J
,
Camirand
A
,
Karaplis
AC
. 
Parathyroid hormone related protein (PTHrP) in tumor progression
.
Adv Exp Med Biol
2011
;
720
:
145
60
.
46.
Zhang
H
,
Yu
C
,
Dai
J
,
Keller
JM
,
Hua
A
,
Sottnik
JL
, et al
Parathyroid hormone-related protein inhibits DKK1 expression through c-Jun-mediated inhibition of β-catenin activationof the DKK1 promoter in prostate cancer
.
Oncogene
2013 Jun 10
.
47.
McCauley
LK
,
Martin
TJ
. 
Twenty-five years of PTHrP progress: from cancer hormone to multifunctional cytokine
.
J Bone Miner Res
2012
;
27
:
1231
9
.
48.
Cho
SW
,
Pirih
FQ
,
Koh
AJ
,
Michalski
M
,
Eber
MR
,
Ritchie
K
, et al
The soluble interleukin-6 receptor is a mediator of hematopoietic and skeletal actions of parathyroid hormone
.
J Biol Chem
2013
;
288
:
6814
25
.
49.
Guise
TA
,
Yin
JJ
,
Taylor
SD
,
Kumagai
Y
,
Dallas
M
,
Boyce
BF
, et al
Evidence for a causal role of parathyroid hormone-related protein in the pathogenesis of human breast cancer-mediated osteolysis
.
J Clin Invest
1996
;
98
:
1544
9
.
50.
Kim
MP
,
Park
SI
,
Kopetz
S
,
Gallick
GE
. 
Src family kinases as mediators of endothelial permeability: effects on inflammation and metastasis
.
Cell Tissue Res
2009
;
335
:
249
59
.