Cancer-Associated Fibroblast-Like Tumor Cells Remodel the Ewing Sarcoma Tumor Microenvironment.

PURPOSE: Despite limited genetic and histologic heterogeneity, Ewing sarcoma (EwS) tumor cells are transcriptionally heterogeneous and display varying degrees of mesenchymal lineage specification in vitro. In this study, we investigated if and how transcriptional heterogeneity of EwS cells contributes to heterogeneity of tumor phenotypes in vivo. EXPERIMENTAL DESIGN: Single-cell proteogenomic-sequencing of EwS cell lines was performed and integrated with patient tumor transcriptomic data. Cell subpopulations were isolated by FACS for assessment of gene expression and phenotype. Digital spatial profiling and human whole transcriptome analysis interrogated transcriptomic heterogeneity in EwS xenografts. Tumor cell subpopulations and matrix protein deposition were evaluated in xenografts and patient tumors using multiplex immunofluorescence staining. RESULTS: We identified CD73 as a biomarker of highly mesenchymal EwS cell subpopulations in tumor models and patient biopsies. CD73+ tumor cells displayed distinct transcriptional and phenotypic properties, including selective upregulation of genes that are repressed by EWS::FLI1, and increased migratory potential. CD73+ cells were distinguished in vitro and in vivo by increased expression of matrisomal genes and abundant deposition of extracellular matrix (ECM) proteins. In epithelial-derived malignancies, ECM is largely deposited by cancer-associated fibroblasts (CAF), and we thus labeled CD73+ EwS cells, CAF-like tumor cells. Marked heterogeneity of CD73+ EwS cell frequency and distribution was detected in tumors in situ, and CAF-like tumor cells and associated ECM were observed in peri-necrotic regions and invasive foci. CONCLUSIONS: EwS tumor cells can adopt CAF-like properties, and these distinct cell subpopulations contribute to tumor heterogeneity by remodeling the tumor microenvironment. See related commentary by Kuo and Amatruda, p. 5002.

Carcinoma-associated fibroblast-like tumor cells remodel the Ewing sarcoma tumor microenvironment.

Tumor heterogeneity is a major driver of cancer progression. In epithelial-derived malignancies, carcinoma-associated fibroblasts (CAFs) contribute to tumor heterogeneity by depositing extracellular matrix (ECM) proteins that dynamically remodel the tumor microenvironment (TME). Ewing sarcomas (EwS) are histologically monomorphous, mesenchyme-derived tumors that are devoid of CAFs. Here we identify a previously uncharacterized subpopulation of transcriptionally distinct EwS tumor cells that deposit pro-tumorigenic ECM. Single cell analyses revealed that these CAF-like cells differ from bulk EwS cells by their upregulation of a matrisome-rich gene signature that is normally repressed by EWS::FLI1, the oncogenic fusion transcription factor that underlies EwS pathogenesis. Further, our studies showed that ECM-depositing tumor cells express the cell surface marker CD73, allowing for their isolation ex vivo and detection in situ. Spatial profiling of tumor xenografts and patient biopsies demonstrated that CD73 + EwS cells and tumor cell-derived ECM are prevalent along tumor borders and invasive fronts. Importantly, despite loss of EWS::FLI1-mediated gene repression, CD73 + EwS cells retain expression of EWS::FLI1 and the fusion-activated gene signature, as well as tumorigenic and proliferative capacities. Thus, EwS tumor cells can be reprogrammed to adopt CAF-like properties and these transcriptionally and phenotypically distinct cell subpopulations contribute to tumor heterogeneity by remodeling the TME.

An Expandable Mechanopharmaceutical Device (2): Drug Induced Granulomas Maximize the Cargo Sequestering Capacity of Macrophages in the Liver.

PURPOSE: Drug-induced liver injuries (DILI) comprise a significant proportion of adverse drug reactions leading to hospitalizations and death. One frequent DILI is granulomatous inflammation from exposure to harmful metabolites that activate inflammatory pathways of immune cells of the liver, which may act as a barrier to isolate the irritating stimulus and limit tissue damage. METHODS: Paralleling the accumulation of CFZ precipitates in the liver, granulomatous inflammation was studied to gain insight into its effect on liver structure and function. A structural analog that does not precipitate within macrophages was also studied using micro-analytical approaches. Depleting macrophages was used to inhibit granuloma formation and assess its effect on drug bioaccumulation and toxicity. RESULTS: Granuloma-associated macrophages showed a distinct phenotype, differentiating them from non-granuloma macrophages. Granulomas were induced by insoluble CFZ cargo, but not by the more soluble analog, pointing to precipitation being a factor driving granulomatous inflammation. Granuloma-associated macrophages showed increased activation of lysosomal master-regulator transcription factor EB (TFEB). Inhibiting granuloma formation increased hepatic necrosis and systemic toxicity in CFZ-treated animals. CONCLUSIONS: Granuloma-associated macrophages are a specialized cell population equipped to actively sequester and stabilize cytotoxic chemotherapeutic agents. Thus, drug-induced granulomas may function as drug sequestering "organoids" -an induced, specialized sub-compartment- to limit tissue damage.

Menin regulates the serine biosynthetic pathway in Ewing sarcoma.

Developmental transcription programs are epigenetically regulated by multi-protein complexes, including the menin- and MLL-containing trithorax (TrxG) complexes, which promote gene transcription by depositing the H3K4me3 activating mark at target gene promoters. We recently reported that in Ewing sarcoma, MLL1 (lysine methyltransferase 2A, KMT2A) and menin are overexpressed and function as oncogenes. Small molecule inhibition of the menin-MLL interaction leads to loss of menin and MLL1 protein expression, and to inhibition of growth and tumorigenicity. Here, we have investigated the mechanistic basis of menin-MLL-mediated oncogenic activity in Ewing sarcoma. Bromouridine sequencing (Bru-seq) was performed to identify changes in nascent gene transcription in Ewing sarcoma cells, following exposure to the menin-MLL interaction inhibitor MI-503. Menin-MLL inhibition resulted in early and widespread reprogramming of metabolic processes. In particular, the serine biosynthetic pathway (SSP) was the pathway most significantly affected by MI-503 treatment. Baseline expression of SSP genes and proteins (PHGDH, PSAT1, and PSPH), and metabolic flux through the SSP were confirmed to be high in Ewing sarcoma. In addition, inhibition of PHGDH resulted in reduced cell proliferation, viability, and tumor growth in vivo, revealing a key dependency of Ewing sarcoma on the SSP. Loss of function studies validated a mechanistic link between menin and the SSP. Specifically, inhibition of menin resulted in diminished expression of SSP genes, reduced H3K4me3 enrichment at the PHGDH promoter, and complete abrogation of de novo serine and glycine biosynthesis, as demonstrated by metabolic tracing studies with 13 C-labeled glucose. These data demonstrate that the SSP is highly active in Ewing sarcoma and that its oncogenic activation is maintained, at least in part, by menin-dependent epigenetic mechanisms involving trithorax complexes. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Physicochemical Basis of Clofazimine-Induced Skin Pigmentation.

Clofazimine is a weakly basic, Food and Drug Administration-approved antibiotic recommended by the World Health Organization to treat leprosy and multi-drug-resistant tuberculosis. Upon prolonged treatment, clofazimine extensively bioaccumulates and precipitates throughout the organism, forming crystal-like drug inclusions (CLDIs). Due to the drug's red color, it is widely believed that clofazimine bioaccumulation results in skin pigmentation, its most common side effect. To test whether clofazimine-induced skin pigmentation is due to CLDI formation, we synthesized a closely related clofazimine analog that does not precipitate under physiological pH and chloride conditions that are required for CLDI formation. Despite the absence of detectable CLDIs in mice, administration of this analog still led to significant skin pigmentation. In clofazimine-treated mice, skin cryosections revealed no evidence of CLDIs when analyzed with a microscopic imaging system specifically designed for detecting clofazimine aggregates. Rather, the reflectance spectra of the skin revealed a signal corresponding to the soluble, free base form of the drug. Consistent with the low concentrations of clofazimine in the skin, these results suggest that clofazimine-induced skin pigmentation is not due to clofazimine precipitation and CLDI formation, but rather to the partitioning of the circulating, free base form of the drug into subcutaneous fat.

Detecting ordered small molecule drug aggregates in live macrophages: a multi-parameter microscope image data acquisition and analysis strategy.

Following prolonged administration, certain orally bioavailable but poorly soluble small molecule drugs are prone to precipitate out and form crystal-like drug inclusions (CLDIs) within the cells of living organisms. In this research, we present a quantitative multi-parameter imaging platform for measuring the fluorescence and polarization diattenuation signals of cells harboring intracellular CLDIs. To validate the imaging system, the FDA-approved drug clofazimine (CFZ) was used as a model compound. Our results demonstrated that a quantitative multi-parameter microscopy image analysis platform can be used to study drug sequestering macrophages, and to detect the formation of ordered molecular aggregates formed by poorly soluble small molecule drugs in animals.

Macrophage-Mediated Clofazimine Sequestration Is Accompanied by a Shift in Host Energy Metabolism.

Prolonged (8 weeks) oral administration of clofazimine results in a profound pharmacodynamic response-bioaccumulation in macrophages (including Kupffer cells) as intracellular crystal-like drug inclusions (CLDIs) with an associated increase in interleukin-1 receptor antagonist production. Notably, CLDI formation in Kupffer cells concomitantly occurs with the formation of macrophage-centric granulomas. Accordingly, we sought to understand the impact of these events on host metabolism using 1H-nuclear magnetic resonance metabolomics. Mice received a clofazimine or vehicle-enriched (sham) diet for at least 8 weeks. At 2 weeks, the antimicrobial activity of clofazimine was evident by changes in urine metabolites. From 2 to 8 weeks, there was a striking change in metabolite levels indicative of a reorientation of host energy metabolism paralleling the onset of CLDI and granuloma formation. This was evidenced by a progressive reduction in urine levels of metabolites involved in one-carbon metabolism with corresponding increases in whole blood, and changes in metabolites associated with lipid, nucleotide and amino acid metabolism, and glycolysis. Although clofazimine-fed mice ate more, they gained less weight than control mice. Together, these results indicate that macrophage sequestration of clofazimine as CLDIs and granuloma formation is accompanied by a profound metabolic disruption in energy homeostasis and one-carbon metabolism.

Clofazimine Biocrystal Accumulation in Macrophages Upregulates Interleukin 1 Receptor Antagonist Production To Induce a Systemic Anti-Inflammatory State.

Clofazimine (CFZ) is a poorly soluble antibiotic and anti-inflammatory drug indicated for the treatment of leprosy. In spite of its therapeutic value, CFZ therapy is accompanied by the formation of drug biocrystals that accumulate within resident tissue macrophages, without obvious toxicological manifestations. Therefore, to specifically elucidate the off-target consequences of drug bioaccumulation in macrophages, we compared the level of inflammasome activation in CFZ-accumulating organs (spleen, liver and lung) in mice after 2 and 8 weeks of CFZ treatment when the drug exists in soluble and insoluble (biocrystalline) forms, respectively. Surprisingly, the results showed a drastic reduction in caspase 1 and interleukin-1ß (IL-1ß) cleavage in the livers of mice treated with CFZ for 8 weeks (8-week-CFZ-treated mice) compared to 2-week-CFZ-treated and control mice, which was accompanied by a 3-fold increase in hepatic IL-1 receptor antagonist (IL-1RA) production and a 21-fold increase in serum IL-1RA levels. In the lung and spleen, IL-1ß cleavage and tumor necrosis factor alpha expression were unaffected by soluble or biocrystal CFZ forms. Functionally, there was a drastic reduction of carrageenan- and lipopolysaccharide-induced inflammation in the footpads and lungs, respectively, of 8-week-CFZ-treated mice. This immunomodulatory activity of CFZ biocrystal accumulation was attributable to the upregulation of IL-1RA, since CFZ accumulation had minimal effect in IL-1RA knockout mice or 2-week-CFZ-treated mice. In conclusion, CFZ accumulation and biocrystal formation in resident tissue macrophages profoundly altered the host's immune system and prompted an IL-1RA-dependent, systemic anti-inflammatory response.

Tumor Repression of VCaP Xenografts by a Pyrrole-Imidazole Polyamide.

Pyrrole-imidazole (Py-Im) polyamides are high affinity DNA-binding small molecules that can inhibit protein-DNA interactions. In VCaP cells, a human prostate cancer cell line overexpressing both AR and the TMPRSS2-ERG gene fusion, an androgen response element (ARE)-targeted Py-Im polyamide significantly downregulates AR driven gene expression. Polyamide exposure to VCaP cells reduced proliferation without causing DNA damage. Py-Im polyamide treatment also reduced tumor growth in a VCaP mouse xenograft model. In addition to the effects on AR regulated transcription, RNA-seq analysis revealed inhibition of topoisomerase-DNA binding as a potential mechanism that contributes to the antitumor effects of polyamides in cell culture and in xenografts. These studies support the therapeutic potential of Py-Im polyamides to target multiple aspects of transcriptional regulation in prostate cancers without genotoxic stress.

A far-red fluorescent probe for flow cytometry and image-based functional studies of xenobiotic sequestering macrophages.

Clofazimine (CFZ) is an optically active, red-colored chemotherapeutic agent that is FDA approved for the treatment of leprosy and is on the World Health Organization's list of essential medications. Interestingly, CFZ massively accumulates in macrophages where it forms crystal-like drug inclusions (CLDIs) after oral administration of the drug in animals and humans. The analysis of the fluorescence spectra of CLDIs formed by resident tissue macrophages revealed that CFZ, when accumulated as CLDIs, undergoes a red shift in fluorescence excitation (from Ex: 540-570 to 560-600 nm) and emission (Em: 560-580 to 640-700 nm) signal relative to the soluble and free-base crystal forms of CFZ. Using epifluorescence microscopy, CLDI(+) cells could be identified, relative to CLDI(-) cells, based on a >3-fold increment in mean fluorescence signal at excitation 640 nm and emission at 670 nm. Similarly, CLDI(+) cells could be identified by flow cytometry, based on a >100-fold increment in mean fluorescence signal using excitation lasers at 640 nm and emission detectors >600 nm. CLDI's fluorescence excitation and emission was orthogonal to that of cell viability dyes such as propidium iodide and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), cellular staining dyes such as Hoechst 33342 (nucleus) and FM 1-43 (plasma membrane), as well as many other fluorescently tagged antibodies used for immunophenotyping analyses. In vivo, >85% of CLDI(+) cells in the peritoneal exudate were F4/80(+) macrophages and >97% of CLDI(+) cells in the alveolar exudate were CD11c(+). Most importantly, the viability of cells was minimally affected by the presence of CLDIs. Accordingly, these results establish that CFZ fluorescence in CLDIs is suitable for quantitative flow cytometric phenotyping analysis and functional studies of xenobiotic sequestering macrophages.

Phagocytosed Clofazimine Biocrystals Can Modulate Innate Immune Signaling by Inhibiting TNFα and Boosting IL-1RA Secretion.

Clofazimine (CFZ) is an FDA-approved leprostatic and anti-inflammatory drug that massively accumulates in macrophages, forming insoluble, intracellular crystal-like drug inclusions (CLDIs) during long-term oral dosing. Interestingly, when added to cells in vitro, soluble CFZ is cytotoxic because it depolarizes mitochondria and induces apoptosis. Accordingly, we hypothesized that, in vivo, macrophages detoxify CFZ by sequestering it in CLDIs. To test this hypothesis, CLDIs of CFZ-treated mice were biochemically isolated and then incubated with macrophages in vitro. The cell biological effects of phagocytosed CLDIs were compared to those of soluble CFZ. Unlike soluble CFZ, phagocytosis of CLDIs did not lead to mitochondrial destabilization or apoptosis. Rather, CLDIs altered immune signaling response pathways downstream of Toll-like receptor (TLR) ligation, leading to enhanced interleukin-1 receptor antagonist (IL-1RA) production, dampened NF-κB activation and tissue necrosis factor alpha (TNFα) production, and ultimately decreased TLR expression levels. In aggregate, our results constitute evidence that macrophages detoxify soluble CFZ by sequestering it in a biocompatible, insoluble form. The altered cellular response to TLR ligation suggests that CLDI formation may also underlie CFZ's anti-inflammatory activity.

Metastatic castration-resistant prostate cancer reveals intrapatient similarity and interpatient heterogeneity of therapeutic kinase targets.

In prostate cancer, multiple metastases from the same patient share similar copy number, mutational status, erythroblast transformation specific (ETS) rearrangements, and methylation patterns supporting their clonal origins. Whether actionable targets such as tyrosine kinases are also similarly expressed and activated in anatomically distinct metastatic lesions of the same patient is not known. We evaluated active kinases using phosphotyrosine peptide enrichment and quantitative mass spectrometry to identify druggable targets in metastatic castration-resistant prostate cancer obtained at rapid autopsy. We identified distinct phosphopeptide patterns in metastatic tissues compared with treatment-naive primary prostate tissue and prostate cancer cell line-derived xenografts. Evaluation of metastatic castration-resistant prostate cancer samples for tyrosine phosphorylation and upstream kinase targets revealed SRC, epidermal growth factor receptor (EGFR), rearranged during transfection (RET), anaplastic lymphoma kinase (ALK), and MAPK1/3 and other activities while exhibiting intrapatient similarity and interpatient heterogeneity. Phosphoproteomic analyses and identification of kinase activation states in metastatic castration-resistant prostate cancer patients have allowed for the prioritization of kinases for further clinical evaluation.

Transcription factors OVOL1 and OVOL2 induce the mesenchymal to epithelial transition in human cancer.

Cell plasticity regulated by the balance between the mesenchymal to epithelial transition (MET) and the opposite program, EMT, is critical in the metastatic cascade. Several transcription factors (TFs) are known to regulate EMT, though the mechanisms of MET remain unclear. We demonstrate a novel function of two TFs, OVOL1 and OVOL2, as critical inducers of MET in human cancers. Our findings indicate that the OVOL-TFs control MET through a regulatory feedback loop with EMT-inducing TF ZEB1, and the regulation of mRNA splicing by inducing Epithelial Splicing Regulatory Protein 1 (ESRP1). Using mouse prostate tumor models we show that expression of OVOL-TFs in mesenchymal prostate cancer cells attenuates their metastatic potential. The role of OVOL-TFs as inducers of MET is further supported by expression analyses in 917 cancer cell lines, suggesting their role as crucial regulators of epithelial-mesenchymal cell plasticity in cancer.

Cyclophosphamide creates a receptive microenvironment for prostate cancer skeletal metastasis.

A number of cancers predominantly metastasize to bone, due to its complex microenvironment and multiple types of constitutive cells. Prostate cancer especially has been shown to localize preferentially to bones with higher marrow cellularity. Using an experimental prostate cancer metastasis model, we investigated the effects of cyclophosphamide, a bone marrow-suppressive chemotherapeutic drug, on the development and growth of metastatic tumors in bone. Priming the murine host with cyclophosphamide before intracardiac tumor cell inoculation was found to significantly promote tumor localization and subsequent growth in bone. Shortly after cyclophosphamide treatment, there was an abrupt expansion of myeloid lineage cells in the bone marrow and the peripheral blood, associated with increases in cytokines with myelogenic potential such as C-C chemokine ligand (CCL)2, interleukin (IL)-6, and VEGF-A. More importantly, neutralizing host-derived murine CCL2, but not IL-6, in the premetastatic murine host significantly reduced the prometastatic effects of cyclophosphamide. Together, our findings suggest that bone marrow perturbation by cytotoxic chemotherapy can contribute to bone metastasis via a transient increase in bone marrow myeloid cells and myelogenic cytokines. These changes can be reversed by inhibition of CCL2.

Inhibition of prostate cancer bone metastasis by synthetic TF antigen mimic/galectin-3 inhibitor lactulose-L-leucine.

Currently incurable, prostate cancer metastasis has a remarkable ability to spread to the skeleton. Previous studies demonstrated that interactions mediated by the cancer-associated Thomsen-Friedenreich glycoantigen (TF-Ag) and the carbohydrate-binding protein galectin-3 play an important role in several rate-limiting steps of cancer metastasis such as metastatic cell adhesion to bone marrow endothelium, homotypic tumor cell aggregation, and clonogenic survival and growth. This study investigated the ability of a synthetic small-molecular-weight nontoxic carbohydrate-based TF-Ag mimic lactulose-L-leucine (Lac-L-Leu) to inhibit these processes in vitro and, ultimately, prostate cancer bone metastasis in vivo. Using an in vivo mouse model, based on intracardiac injection of human PC-3 prostate carcinoma cells stably expressing luciferase, we investigated the ability of Lac-L-Leu to impede the establishment and growth of bone metastasis. Parallel-flow chamber assay, homotypic aggregation assay, modified Boyden chamber assay, and clonogenic growth assay were used to assess the effects of Lac-L-Leu on tumor cell adhesion to the endothelium, homotypic tumor cell aggregation, transendothelial migration, and clonogenic survival and growth, respectively. We report that daily intraperitoneal administration of Lac-L-Leu resulted in a three-fold (P < .05) decrease in metastatic tumor burden compared with the untreated control. Mechanistically, the effect of Lac-L-Leu, which binds and inhibits galectins by mimicking essential structural features of the TF-Ag, was associated with a dose-dependent inhibition of prostate cancer cell adhesion to bone marrow endothelium, homotypic aggregation, transendothelial migration, and clonogenic growth. We conclude that small-molecular-weight carbohydrate-based compounds targeting ß-galactoside-mediated interactions could provide valuable means for controlling and preventing metastatic prostate cancer spread to the skeleton.

Chemical transfection of dye-conjugated microRNA precursors for microRNA functional analysis of M2 macrophages.

MicroRNAs (miRNAs) are short noncoding ribonucleic acids known to affect gene expression at the translational level and there is mounting evidence that miRNAs play a role in the function of tumor-associated macrophages (TAMs). To aid the functional analyses of miRNAs in an in-vitro model of TAMs known as M2 macrophages, a transfection method to introduce artificial miRNA constructs or miRNA molecules into primary human monocytes is needed. Unlike differentiated macrophages or dendritic cells, undifferentiated primary human monocytes have been known to show resistance to lentiviral transduction. To circumvent this challenge, other techniques such as electroporation and chemical transfection have been used in other applications to deliver small gene constructs into human monocytes. To date, no studies have compared these two methods objectively to evaluate their suitability in the miRNA functional analysis of M2 macrophages. Of the methods tested, the electroporation of miRNA-construct containing plasmids and the chemical transfection of miRNA precursor molecules are the most efficient approaches. The use of a silencer siRNA labeling kit (Ambion) to conjugate Cy 3 fluorescence dyes to the precursor molecules allowed the isolation of successfully transfected cells with fluorescence-activated cell sorting. The chemical transfection of these dye-conjugated miRNA precursors yield an efficiency of 37.5 ± 0.6% and a cell viability of 74 ± 1%. RNA purified from the isolated cells demonstrated good quality, and was fit for subsequent mRNA expression qPCR analysis. While electroporation of plasmids containing miRNA constructs yield transfection efficiencies comparable to chemical transfection of miRNA precursors, these electroporated primary monocytes seemed to have lost their potential for differentiation. Among the most common methods of transfection, the chemical transfection of dye-conjugated miRNA precursors was determined to be the best-suited approach for the functional analysis of M2 macrophages.

Activation of urokinase plasminogen activator and its receptor axis is essential for macrophage infiltration in a prostate cancer mouse model.

Macrophages within the tumor microenvironment promote angiogenesis, extracellular matrix breakdown, and tumor cell migration, invasion, and metastasis. Activation of the urokinase plasminogen activator (uPA) and its receptor (uPAR) axis promotes prostate cancer tumorigenicity, invasion, metastasis, and survival within the tumor microenvironment. The link between macrophage infiltration and the uPA/uPAR axis in prostate cancer development has not been established, although it has been reported that uPA plays a critical role inmonocyte and macrophage chemotaxis. In this study, murine prostate cancer RM-1 cells were subcutaneously inoculated into wild-type (WT), uPA(-/-), and uPAR(-/-) mice. Tumor volume was significantly diminished in both uPA(-/-) and uPAR(-/-) mice compared with WT controls. Greater inhibition of tumor volume was also observed in uPA(-/-) mice compared with uPAR(-/-) mice, suggesting the important contribution of stromal-derived uPA to sustain the tumor growth. Immunohistochemical staining revealed that tumors in uPA(-/-) and uPAR(-/-) mice displayed significantly lower proliferative indices, higher apoptotic indices, and less neovascularity compared with the tumors in WT mice. Tumors in uPA(-/-) and uPAR(-/-) mice displayed significantly less macrophage infiltration as demonstrated by F4/80 staining and MAC3(+) cell numbers by flow cytometry compared with the tumors from WT mice. These findings suggest that the uPA/uPAR axis acts in both autocrine and paracrine manners in the tumor microenvironment, and activation of uPA/uPAR axis is essential for macrophage infiltration into prostate tumors.

Erythropoietin couples hematopoiesis with bone formation.

BACKGROUND: It is well established that bleeding activates the hematopoietic system to regenerate the loss of mature blood elements. We have shown that hematopoietic stem cells (HSCs) isolated from animals challenged with an acute bleed regulate osteoblast differentiation from marrow stromal cells. This suggests that HSCs participate in bone formation where the molecular basis for this activity is the production of BMP2 and BMP6 by HSCs. Yet, what stimulates HSCs to produce BMPs is unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we demonstrate that erythropoietin (Epo) activates Jak-Stat signaling pathways in HSCs which leads to the production of BMPs. Critically, Epo also directly activates mesenchymal cells to form osteoblasts in vitro, which in vivo leads to bone formation. Importantly, Epo first activates osteoclastogenesis which is later followed by osteoblastogenesis that is induced by either Epo directly or the expression of BMPs by HSCs to form bone. CONCLUSIONS/SIGNIFICANCE: These data for the first time demonstrate that Epo regulates the formation of bone by both direct and indirect pathways, and further demonstrates the exquisite coupling between hematopoiesis and osteopoiesis in the marrow.

The chemokine CCL2 increases prostate tumor growth and bone metastasis through macrophage and osteoclast recruitment.

CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein-1) has been demonstrated to recruit monocytes to tumor sites. Monocytes are capable of being differentiated into tumor-associated macrophages (TAMs) and osteoclasts (OCs). TAMs have been shown to promote tumor growth in several cancer types. Osteoclasts have also been known to play an important role in cancer bone metastasis. To investigate the effects of CCL2 on tumorigenesis and its potential effects on bone metastasis of human prostate cancer, CCL2 was overexpressed into a luciferase-tagged human prostate cancer cell line PC-3. In vitro, the conditioned medium of CCL2 overexpressing PC-3luc cells (PC-3(lucCCL2)) was a potent chemoattractant for mouse monocytes in comparison to a conditioned medium from PC-3(lucMock). In addition, CCL2 overexpression increased the growth of transplanted xenografts and increased the accumulation of macrophages in vivo. In a tumor dissemination model, PC-3(lucCCL2) enhanced the growth of bone metastasis, which was associated with more functional OCs. Neutralizing antibodies targeting both human and mouse CCL2 inhibited the growth of PC-3(luc), which was accompanied by a decrease in macrophage recruitment to the tumor. These findings suggest that CCL2 increases tumor growth and bone metastasis through recruitment of macrophages and OCs to the tumor site.

CCL2 and interleukin-6 promote survival of human CD11b+ peripheral blood mononuclear cells and induce M2-type macrophage polarization.

CCL2 and interleukin (IL)-6 are among the most prevalent cytokines in the tumor microenvironment, with expression generally correlating with tumor progression and metastasis. CCL2 and IL-6 induced expression of each other in CD11b(+) cells isolated from human peripheral blood. It was demonstrated that both cytokines induce up-regulation of the antiapoptotic proteins cFLIP(L) (cellular caspase-8 (FLICE)-like inhibitory protein), Bcl-2, and Bcl-X(L) and inhibit the cleavage of caspase-8 and subsequent activation of the caspase-cascade, thus protecting cells from apoptosis under serum deprivation stress. Furthermore, both cytokines induced hyperactivation of autophagy in these cells. Upon CCL2 or IL-6 stimulation, CD11b(+) cells demonstrated a significant increase in the mannose receptor (CD206) and the CD14(+)/CD206(+) double-positive cells, suggesting a polarization of macrophages toward the CD206(+) M2-type phenotype. Caspase-8 inhibitors mimicked the cytokine-induced up-regulation of autophagy and M2 polarization. Furthermore, E64D and leupeptin, which are able to function as inhibitors of autophagic degradation, reversed the effect of caspase-8 inhibitors in the M2-macrophage polarization, indicating a role of autophagy in this mechanism. Additionally, in patients with advanced castrate-resistant prostate cancer, metastatic lesions exhibited an increased CD14(+)/CD206(+) double-positive cell population compared with normal tissues. Altogether, these findings suggest a role for CCL2 and IL-6 in the survival of myeloid monocytes recruited to the tumor microenvironment and their differentiation toward tumor-promoting M2-type macrophages via inhibition of caspase-8 cleavage and enhanced autophagy.