During tumor progression, immune system phagocytes continually clear apoptotic cancer cells in a process known as efferocytosis. However, the impact of efferocytosis in metastatic tumor growth is ...unknown. In this study, we observed that macrophage-driven efferocytosis of prostate cancer cells in vitro induced the expression of proinflammatory cytokines such as CXCL5 by activating Stat3 and NF-κB(p65) signaling. Administration of a dimerizer ligand (AP20187) triggered apoptosis in 2 in vivo syngeneic models of bone tumor growth in which apoptosis-inducible prostate cancer cells were either coimplanted with vertebral bodies, or inoculated in the tibiae of immunocompetent mice. Induction of 2 pulses of apoptosis correlated with increased infiltration of inflammatory cells and accelerated tumor growth in the bone. Apoptosis-induced tumors displayed elevated expression of the proinflammatory cytokine CXCL5. Likewise, CXCL5-deficient mice had reduced tumor progression. Peripheral blood monocytes isolated from patients with bone metastasis of prostate cancer were more efferocytic compared with normal controls, and CXCL5 serum levels were higher in metastatic prostate cancer patients relative to patients with localized prostate cancer or controls. Altogether, these findings suggest that the myeloid phagocytic clearance of apoptotic cancer cells accelerates CXCL5-mediated inflammation and tumor growth in bone, pointing to CXCL5 as a potential target for cancer therapeutics.
Cellular subpopulations in the bone marrow play distinct and unexplored functions in skeletal homeostasis. This study delineated a unique role of osteal macrophages in bone and parathyroid hormone ...(PTH)-dependent bone anabolism using murine models of targeted myeloid-lineage cell ablation. Depletion of c-fms ⁺ myeloid lineage cells via administration of AP20187 in the macrophage Fas-induced apoptosis (MAFIA) mouse model reduced cortical and trabecular bone mass and attenuated PTH-induced trabecular bone anabolism, supporting the positive function of macrophages in bone homeostasis. Interestingly, using a clodronate liposome model with targeted depletion of mature phagocytic macrophages an opposite effect was found with increased trabecular bone mass and increased PTH-induced anabolism. Apoptotic cells were more numerous in MAFIA versus clodronate-treated mice and flow cytometric analyses of myeloid lineage cells in the bone marrow showed that MAFIA mice had reduced CD68 ⁺ cells, whereas clodronate liposome-treated mice had increased CD68 ⁺ and CD163 ⁺ cells. Clodronate liposomes increased efferocytosis (clearance of apoptotic cells) and gene expression associated with alternatively activated M2 macrophages as well as expression of genes associated with bone formation including Wnt3a , Wnt10b , and Tgfb1 . Taken together, depletion of early lineage macrophages resulted in osteopenia with blunted effects of PTH anabolic actions, whereas depletion of differentiated macrophages promoted apoptotic cell clearance and transformed the bone marrow to an osteogenic environment with enhanced PTH anabolism. These data highlight a unique function for osteal macrophages in skeletal homeostasis.
Tumor cells secrete factors that modulate macrophage activation and polarization into M2 type tumor-associated macrophages, which promote tumor growth, progression, and metastasis. The mechanisms ...that mediate this polarization are not clear. Macrophages are phagocytic cells that participate in the clearance of apoptotic cells, a process known as efferocytosis. Milk fat globule- EGF factor 8 (MFG-E8) is a bridge protein that facilitates efferocytosis and is associated with suppression of proinflammatory responses. This study investigated the hypothesis that MFG-E8-mediated efferocytosis promotes M2 polarization. Tissue and serum exosomes from prostate cancer patients presented higher levels of MFG-E8 compared with controls, a novel finding in human prostate cancer. Coculture of macrophages with apoptotic cancer cells increased efferocytosis, elevated MFG-E8 protein expression levels, and induced macrophage polarization into an alternatively activated M2 phenotype. Administration of antibody against MFG-E8 significantly attenuated the increase in M2 polarization. Inhibition of STAT3 phosphorylation using the inhibitor Stattic decreased efferocytosis and M2 macrophage polarization in vitro, with a correlating increase in SOCS3 protein expression. Moreover, MFG-E8 knockdown tumor cells cultured with wild-type or MFG-E8-deficient macrophages resulted in increased SOCS3 expression with decreased STAT3 activation. This suggests that SOCS3 and phospho-STAT3 act in an inversely dependent manner when stimulated by MFG-E8 and efferocytosis. These results uncover a unique role of efferocytosis via MFG-E8 as a mechanism for macrophage polarization into tumor-promoting M2 cells.
To delineate the role of SDF‐1 and CXCR4 in metastatic prostate cancer (CaP), positive correlations were established between SDF‐1 levels and tumor metastasis. Neutralization of CXCR4 limited the ...number and the growth of intraosseous metastasis in vivo. Together, these in vivo metastasis data provide critical support that SDF‐1/CXCR4 plays a role in skeletal metastasis.
Introduction: Previously we determined that the stromal‐derived factor‐1 (SDF‐1)/CXCR4 chemokine axis is activated in prostate cancer (CaP) metastasis to bone. To delineate the role of SDF‐1/CXCR4 in CaP, we evaluated SDF‐1 levels in a variety of tissues and whether neutralization of SDF‐1 prevented metastasis and/or intraosseous growth of CaPs.
Materials and Methods: SDF‐1 levels were established in various mouse tissues by ELISA, immunohistochemistry, and in situ hybridization. To assess the role of SDF‐1/CXCR4 in metastasis, bone metastases were established by administering CaP cells into the left cardiac ventricle of nude animals in the presence or absence of neutralizing CXCR4 antibody. The effect of SDF‐1 on intraosseous growth of CaP cells was determined using intratibial injections and anti‐CXCR4 antibodies and peptides.
Results: There was a positive correlation between the levels of SDF‐1 and tissues in which metastatic CaP lesions were observed. SDF‐1 levels were highest in the pelvis, tibia, femur, liver, and adrenal/kidneys compared with the lungs, tongue, and eye, suggesting a selective effect. SDF‐1 staining was generally low or undetectable in the center of the marrow and in the diaphysis. SDF‐1 mRNA was localized to the metaphysis of the long bones nearest to the growth plate where intense expression was observed near the endosteal surfaces covered by osteoblastic and lining cells. Antibody to CXCR4 significantly reduced the total metastatic load compared with IgG control‐treated animals. Direct intratibial injection of tumor cells followed by neutralizing CXCR4 antibody or a specific peptide that blocks CXCR4 also decreased the size of the tumors compared with controls.
Conclusions: These data provide critical support for a role of SDF‐1/CXCR4 in skeletal metastasis. Importantly, these data show that SDF‐1/CXCR4 participate in localizing tumors to the bone marrow for prostate cancer.
Parathyroid hormone (PTH) is currently the only FDA-approved anabolic drug to treat osteoporosis, and is systemically administered through daily injections. A new local pulsatile PTH delivery device ...was developed from biodegradable polymers to expand the application of PTH from systemic treatment to spatially controlled local bone defect regeneration in this work. This is the first time that local pulsatile PTH delivery has been demonstrated to promote bone regeneration via enhanced bone remodeling. The biodegradable delivery device was designed to locally deliver PTH in a preprogrammed pulsatile manner. The PTH delivery was utilized to facilitate the regeneration of a bone defect spatially defined with a cell-free biomimetic nanofibrous (NF) scaffold. The local pulsatile PTH delivery (daily pulse for 21 days) not only promoted the regeneration of a critical-sized bone defect with negligible systemic side effects in a mouse model, but also advantageously achieved higher quality regenerated bone than the standard systemic PTH injection. These results demonstrate a promising and novel pulsatile PTH delivery device for spatially defined local bone regeneration.
Apoptosis and efficient efferocytosis are integral to growth, development, and homeostasis. The heterogeneity of these mechanisms in different cells across distinct tissues renders it difficult to ...develop broadly applicable in vivo technologies. Here, we introduced a novel inducible caspase-9 (iCasp9) mouse model which allowed targeted cell apoptosis and further facilitated investigation of concomitant efferocytosis. We generated iCasp9
mice with conditional expression of chemically inducible caspase-9 protein that is triggered in the presence of Cre recombinase. In vitro, bone marrow cells from iCasp9
mice showed expression of the iCasp9 protein when transduced with Cre-expressing adenovirus. Treatment of these cells with the chemical dimerizer (AP20187/AP) resulted in iCasp9 processing and cleaved caspase-3 upregulation, indicating successful apoptosis induction. The in vivo functionality and versatility of this model was demonstrated by crossing iCasp9
mice with CD19-Cre and Osteocalcin (OCN)-Cre mice to target CD19
B cells or OCN
bone-lining osteoblasts. Immunofluorescence and/or immunohistochemical staining in combination with histomorphometric analysis of EGFP, CD19/OCN, and cleaved caspase-3 expression demonstrated that a single dose of AP effectively induced apoptosis in CD19
B cells or OCN
osteoblasts. Examination of the known efferocytes in the target tissues showed that CD19
cell apoptosis was associated with infiltration of dendritic cells into splenic B cell follicles. In the bone, where efferocytosis remains under-explored, the use of iCasp9 provided direct in vivo evidence that macrophages are important mediators of apoptotic osteoblast clearance. Collectively, this study presented the first mouse model of iCasp9 which achieved selective apoptosis, allowing examination of subsequent efferocytosis. Given its unique feature of being controlled by any Cre-expressing mouse lines, the potential applications of this model are extensive and will bring forth more insights into the diversity of mechanisms and cellular effects induced by apoptosis including the physiologically important efferocytic process that follows.
Bone marrow macrophages stimulate skeletal wound repair and osteoblastic bone formation by poorly defined mechanisms. Specialized proresolving mediators of inflammation drive macrophage efferocytosis ...(phagocytosis of apoptotic cells) and resolution, but little is known regarding this process in the bone marrow. In this study, metabololipidomic profiling via liquid chromatography mass spectrometry revealed higher levels of specialized proresolving mediators in the bone marrow relative to the spleen. The endocrine and bone anabolic agent parathyroid hormone increased specialized proresolving mediator levels, including resolvins (Rvs), in bone marrow. Human and murine primary macrophages efferocytosed apoptotic osteoblasts in vitro, and RvD1 and RvD2 (10 pM-10 nM) enhanced this process. These findings support a unique profile of specialized lipid mediators in bone marrow that contribute to a feedback system for resolution of inflammation and maintenance of skeletal homeostasis.
Aberrant DNA methylation of CpG islands has been widely observed in human colorectal tumors and is associated with gene silencing when it occurs in promoter areas. A subset of colorectal tumors has ...an exceptionally high frequency of methylation of some CpG islands, leading to the suggestion of a distinct trait referred to as 'CpG island methylator phenotype', or 'CIMP'. However, the existence of CIMP has been challenged. To resolve this continuing controversy, we conducted a systematic, stepwise screen of 195 CpG island methylation markers using MethyLight technology, involving 295 primary human colorectal tumors and 16,785 separate quantitative analyses. We found that CIMP-positive (CIMP+) tumors convincingly represent a distinct subset, encompassing almost all cases of tumors with BRAF mutation (odds ratio = 203). Sporadic cases of mismatch repair deficiency occur almost exclusively as a consequence of CIMP-associated methylation of MLH1 . We propose a robust new marker panel to classify CIMP+ tumors.
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