The HGF/c-MET Pathway Is a Driver and Biomarker of VEGFR-inhibitor Resistance and Vascular Remodeling in Non-Small Cell Lung Cancer.

Purpose: Resistance to VEGFR inhibitors is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). We investigated the cellular mechanisms mediating resistance of NSCLCs to VEGFR tyrosine kinase inhibitors.Experimental Design: We generated murine models of human NSCLC and performed targeted inhibition studies with the VEGFR TKIs cediranib and vandetanib. We used species-specific hybridization of microarrays to compare cancer (human) and stromal (mouse) cell transcriptomes of TKI-sensitive and -resistant tumors. We measured tumor microvascular density and vessel tortuosity to characterize the effects of therapy on the tumor vascular bed. Circulating cytokine and angiogenic factor levels in patients enrolled in VEGFR TKI trials were correlated with clinical outcomes.Results: Murine xenograft models of human lung adenocarcinoma were initially sensitive to VEGFR TKIs, but developed resistance to treatment. Species-specific microarray analysis identified increased expression of stromal-derived hepatocyte growth factor (HGF) as a candidate mediator of TKI resistance and its receptor, c-MET, was activated in cancer cells and tumor-associated stroma. A transient increase in hypoxia-regulated molecules in the initial response phase was followed by adaptive changes resulting in a more tortuous vasculature. Forced HGF expression in cancer cells reduced tumor sensitivity to VEGFR TKIs and produced tumors with tortuous blood vessels. Dual VEGFR/c-MET signaling inhibition delayed the onset of the resistant phenotype and prevented the vascular morphology alterations. In patients with cancer receiving VEGFR TKIs, high pretreatment HGF plasma levels correlated with poorer survival.Conclusions: HGF/c-MET pathway mediates VEGFR inhibitor resistance and vascular remodeling in NSCLC. Clin Cancer Res; 23(18); 5489-501. ©2017 AACR.

Treatment of experimental human breast cancer and lung cancer brain metastases in mice by macitentan, a dual antagonist of endothelin receptors, combined with paclitaxel.

BACKGROUND: We recently demonstrated that brain endothelial cells and astrocytes protect cancer cells from chemotherapy through an endothelin-dependent signaling mechanism. Here, we evaluated the efficacy of macitentan, a dual endothelin receptor (ETAR and ETBR) antagonist, in the treatment of experimental breast and lung cancer brain metastases. METHODS: The effect of macitentan on astrocyte- and brain endothelial cell-mediated chemoprotective properties was measured in cytotoxic assays. We compared survival of mice bearing established MDA-MB-231 breast cancer or PC-14 non-small cell lung cancer (NSCLC) brain metastases that were treated with vehicle, macitentan, paclitaxel, or macitentan plus paclitaxel. Cell division, apoptosis, tumor vasculature, and expression of survival-related proteins were assessed by immunofluorescent microscopy. RESULTS: Cancer cells and tumor-associated endothelial cells expressed activated forms of AKT and MAPK in vehicle- and paclitaxel-treated groups in both metastasis models, but these proteins were downregulated in metastases of mice that received macitentan. The survival-related proteins Bcl2L1, Gsta5, and Twist1 that localized to cancer cells and tumor-associated endothelial cells in vehicle- and paclitaxel-treated tumors were suppressed by macitentan. Macitentan or paclitaxel alone had no effect on survival. However, when macitentan was combined with paclitaxel, we noted a significant reduction in cancer cell division and marked apoptosis of both cancer cells and tumor-associated endothelial cells. Moreover, macitentan plus paclitaxel therapy significantly increased overall survival by producing complete responses in 35 of 35 mice harboring brain metastases. CONCLUSIONS: Dual antagonism of ETAR and ETBR signaling sensitizes experimental brain metastases to paclitaxel and may represent a new therapeutic option for patients with brain metastases.

Dual Metronomic Chemotherapy with Nab-Paclitaxel and Topotecan Has Potent Antiangiogenic Activity in Ovarian Cancer.

There is growing recognition of the important role of metronomic chemotherapy in cancer treatment. On the basis of their unique antiangiogenic effects, we tested the efficacy of nab-paclitaxel, which stimulates thrombospondin-1, and topotecan, which inhibits hypoxia-inducible factor 1-α, at metronomic dosing for the treatment of ovarian carcinoma. In vitro and in vivo SKOV3ip1, HeyA8, and HeyA8-MDR (taxane-resistant) orthotopic models were used to examine the effects of metronomic nab-paclitaxel and metronomic topotecan. We examined cell proliferation (Ki-67), apoptosis (cleaved caspase-3), and angiogenesis (microvessel density, MVD) in tumors obtained at necropsy. In vivo therapy experiments demonstrated treatment with metronomic nab-paclitaxel alone and in combination with metronomic topotecan resulted in significant reductions in tumor weight (62% in the SKOV3ip1 model, P < 0.01 and 96% in the HeyA8 model, P < 0.03) compared with vehicle (P < 0.01). In the HeyA8-MDR model, metronomic monotherapy with either cytotoxic agent had modest effects on tumor growth, but combination therapy decreased tumor burden by 61% compared with vehicle (P < 0.03). The greatest reduction in MVD (P < 0.05) and proliferation was seen in combination metronomic therapy groups. Combination metronomic therapy resulted in prolonged overall survival in vivo compared with other groups (P < 0.001). Tube formation was significantly inhibited in RF-24 endothelial cells exposed to media conditioned with metronomic nab-paclitaxel alone and media conditioned with combination metronomic nab-paclitaxel and metronomic topotecan. The combination of metronomic nab-paclitaxel and metronomic topotecan offers a novel, highly effective therapeutic approach for ovarian carcinoma that merits further clinical development.

Macitentan, a Dual Endothelin Receptor Antagonist, in Combination with Temozolomide Leads to Glioblastoma Regression and Long-term Survival in Mice.

PURPOSE: The objective of the study was to determine whether astrocytes and brain endothelial cells protect glioma cells from temozolomide through an endothelin-dependent signaling mechanism and to examine the therapeutic efficacy of the dual endothelin receptor antagonist, macitentan, in orthotopic models of human glioblastoma. EXPERIMENTAL DESIGN: We evaluated several endothelin receptor antagonists for their ability to inhibit astrocyte- and brain endothelial cell-induced protection of glioma cells from temozolomide in chemoprotection assays. We compared survival in nude mice bearing orthotopically implanted LN-229 glioblastomas or temozolomide-resistant (LN-229(Res) and D54(Res)) glioblastomas that were treated with macitentan, temozolomide, or both. Tumor burden was monitored weekly with bioluminescence imaging. The effect of therapy on cell division, apoptosis, tumor-associated vasculature, and pathways associated with cell survival was assessed by immunofluorescent microscopy. RESULTS: Only dual endothelin receptor antagonism abolished astrocyte- and brain endothelial cell-mediated protection of glioma cells from temozolomide. In five independent survival studies, including temozolomide-resistant glioblastomas, 46 of 48 (96%) mice treated with macitentan plus temozolomide had no evidence of disease (P < 0.0001), whereas all mice in other groups died. In another analysis, macitentan plus temozolomide therapy was stopped in 16 mice after other groups had died. Only 3 of 16 mice eventually developed recurrent disease, 2 of which responded to additional cycles of macitentan plus temozolomide. Macitentan downregulated proteins associated with cell division and survival in glioma cells and associated endothelial cells, which enhanced their sensitivity to temozolomide. CONCLUSIONS: Macitentan plus temozolomide are well tolerated, produce durable responses, and warrant clinical evaluation in glioblastoma patients.

Modulation of the cancer cell transcriptome by culture media formulations and cell density.

We investigated how varying the composition of cell culture formulations and growing cancer cells at different densities might affect tumor cell genotype. Specifically, we compared gene expression profiles generated by human MDA­MB­231 breast cancer cells cultured in different media [minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), or Roswell Park Memorial Institute (RPMI)­1640 medium] containing different concentrations of fetal bovine serum (FBS) or different sera (equine or bovine) that were grown at different cell densities. More than 2,000 genes were differentially modulated by at least a 2­fold difference when MDA­MB­231 cancer cells were 90% confluent and compared with cultures that were 50% confluent. Altering the concentration of serum produced an even more pronounced effect on MDA­MB­231 cancer cell gene expression in that 2,981 genes were differentially expressed in a comparison between cells cultured in 0.1% FBS and same cell density cultures that were maintained in 10% FBS. A comparison between MDA­MB­231 cancer cells that were 90% confluent in MEM, DMEM, or RPMI­1640 media, all containing 10% FBS, resulted in 8,925 differentially expressed genes. Moreover, one­quarter (25.6%) of genes from our genome­wide expression analysis were expressed at significantly different levels by cells grown in MEM, DMEM, or RPMI­1640 media. Genes associated with epithelial­mes-enchymal transition (EMT) were among the genes that were differentially modulated by cells grown in different cell culture formulations and these genes were verified at the protein level. Collectively, these results underscore the importance of accurate reporting and maintenance of uniform culture conditions to ensure reproducible results.

Antiangiogenic Therapy with Human Apolipoprotein(a) Kringle V and Paclitaxel in a Human Ovarian Cancer Mouse Model.

INTRODUCTION: The present study compared the effect of combination therapy using human apolipoprotein(a) kringle V (rhLK8) to conventional chemotherapy with paclitaxel for human ovarian carcinoma producing high or low levels of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: Human ovarian carcinoma cells producing high (SKOV3ip1) or low (HeyA8) levels of VEGF were implanted into the peritoneal cavity of female nude mice. Seven days later, mice were randomized into four groups: control (vehicle), paclitaxel [5 mg/kg, weekly intraperitoneal (i.p.) injection], rhLK8 (50 mg/kg, daily i.p. injection), or the combination of paclitaxel and rhLK8. Mice were treated for 4 weeks and examined by necropsy. RESULTS: In mice implanted with SKOV3ip1 cells, rhLK8 treatment had no significant effect on tumor incidence or the volume of ascites but induced a significant decrease in tumor weight compared with control mice. Paclitaxel significantly reduced tumor weight and ascites volume, and combination treatment with paclitaxel and rhLK8 had an additive therapeutic effect. Similarly, in HeyA8 mice, the effect of combination treatment on tumor weight and tumor incidence was statistically significantly greater than that of paclitaxel or rhLK8 alone. Immunohistochemical analysis showed a significant decrease in microvessel density and a marked increase of apoptosis in tumor and tumor-associated endothelial cells in response to combination treatment with paclitaxel and rhLK8. CONCLUSION: Collectively, these results suggest that antiangiogenic therapy with rhLK8 in combination with taxane-based conventional chemotherapy could be effective for the treatment of ovarian carcinomas, regardless of VEGF status.

Role of the endothelin axis in astrocyte- and endothelial cell-mediated chemoprotection of cancer cells.

BACKGROUND: Recent evidence suggests that astrocytes protect cancer cells from chemotherapy by stimulating upregulation of anti-apoptotic genes in those cells. We investigated the possibility that activation of the endothelin axis orchestrates survival gene expression and chemoprotection in MDA-MB-231 breast cancer cells and H226 lung cancer cells. METHODS: Cancer cells, murine astrocytes, and murine fibroblasts were grown in isolation, and expression of endothelin (ET) peptides and ET receptors (ETAR and ETBR) compared with expression on cancer cells and astrocytes (or cancer cells and fibroblasts) that were co-incubated for 48 hours. Type-specific endothelin receptor antagonists were used to evaluate the contribution of ETAR and ETBR to astrocyte-induced activation of the protein kinase B (AKT)/mitogen-activated protein kinase (MAPK) signal transduction pathways, anti-apoptotic gene expression, and chemoprotection of cancer cells. We also investigated the chemoprotective potential of brain endothelial cells and microglial cells. RESULTS: Gap junction signaling between MDA-MB-231 cancer cells and astrocytes stimulates upregulation of interleukin 6 (IL-6) and IL-8 expression in cancer cells, which increases ET-1 production from astrocytes and ET receptor expression on cancer cells. ET-1 signals for activation of AKT/MAPK and upregulation of survival proteins that protect cancer cells from taxol. Brain endothelial cell-mediated chemoprotection of cancer cells also involves endothelin signaling. Dual antagonism of ETAR and ETBR is required to abolish astrocyte- and endothelial cell-mediated chemoprotection. CONCLUSIONS: Bidirectional signaling between astrocytes and cancer cells involves upregulation and activation of the endothelin axis, which protects cancer cells from cytotoxicity induced by chemotherapeutic drugs.

DNA-damage-induced nuclear export of precursor microRNAs is regulated by the ATM-AKT pathway.

Expression of microRNAs (miRNAs) involves transcription of miRNA genes and maturation of the primary transcripts. Recent studies have shown that posttranscriptional processing of primary and precursor miRNAs is induced after DNA damage through regulatory RNA-binding proteins in the Drosha and Dicer complexes, such as DDX5 and KSRP. However, little is known about the regulation of nuclear export of pre-miRNAs in the DNA-damage response, a critical step in miRNA maturation. Here, we show that nuclear export of pre-miRNAs is accelerated after DNA damage in an ATM-dependent manner. The ATM-activated AKT kinase phosphorylates Nup153, a key component of the nucleopore, leading to enhanced interaction between Nup153 and Exportin-5 (XPO5) and increased nuclear export of pre-miRNAs. These findings define an important role of DNA-damage signaling in miRNA transport and maturation.

Biologic effects of dopamine on tumor vasculature in ovarian carcinoma.

Chronic sympathetic nervous system activation results in increased angiogenesis and tumor growth in orthotopic mouse models of ovarian carcinoma. However, the mechanistic effects of such activation on the tumor vasculature are not well understood. Dopamine (DA), an inhibitory catecholamine, regulates the functions of normal and abnormal blood vessels. Here, we examined whether DA, an inhibitory catecholamine, could block the effects of chronic stress on tumor vasculature and tumor growth. Exogenous administration of DA not only decreased tumor microvessel density but also increased pericyte coverage of tumor vessels following daily restraint stress in mice. Daily restraint stress resulted in significantly increased tumor growth in the SKOV3ip1 and HeyA8 ovarian cancer models. DA treatment blocked stress-mediated increases in tumor growth and increased pericyte coverage of tumor endothelial cells. Whereas the antiangiogenic effect of DA is mediated by dopamine receptor 2 (DR2), our data indicate that DA, through DR1, stimulates vessel stabilization by increasing pericyte recruitment to tumor endothelial cells. DA significantly stimulated migration of mouse 10T1/2 pericyte-like cells in vitro and increased cyclic adenosine mono-phosphate (cAMP) levels in these cells. Moreover, DA or the DR1 agonist SKF 82958 increased platinum concentration in SKOV3ip1 tumor xenografts following cisplatin administration. In conclusion, DA stabilizes tumor blood vessels through activation of pericyte cAMP-protein kinase A signaling pathway by DR1. These findings could have implications for blocking the stimulatory effects of chronic stress on tumor growth.

Coronary microvascular pericytes are the cellular target of sunitinib malate-induced cardiotoxicity.

Sunitinib malate is a multitargeted receptor tyrosine kinase inhibitor used in the treatment of human malignancies. A substantial number of sunitinib-treated patients develop cardiac dysfunction, but the mechanism of sunitinib-induced cardiotoxicity is poorly understood. We show that mice treated with sunitinib develop cardiac and coronary microvascular dysfunction and exhibit an impaired cardiac response to stress. The physiological changes caused by treatment with sunitinib are accompanied by a substantial depletion of coronary microvascular pericytes. Pericytes are a cell type that is dependent on intact platelet-derived growth factor receptor (PDGFR) signaling but whose role in the heart is poorly defined. Sunitinib-induced pericyte depletion and coronary microvascular dysfunction are recapitulated by CP-673451, a structurally distinct PDGFR inhibitor, confirming the role of PDGFR in pericyte survival. Thalidomide, an anticancer agent that is known to exert beneficial effects on pericyte survival and function, prevents sunitinib-induced pericyte cell death in vitro and prevents sunitinib-induced cardiotoxicity in vivo in a mouse model. Our findings suggest that pericytes are the primary cellular target of sunitinib-induced cardiotoxicity and reveal the pericyte as a cell type of concern in the regulation of coronary microvascular function. Furthermore, our data provide preliminary evidence that thalidomide may prevent cardiotoxicity in sunitinib-treated cancer patients.

ATP11B mediates platinum resistance in ovarian cancer.

Platinum compounds display clinical activity against a wide variety of solid tumors; however, resistance to these agents is a major limitation in cancer therapy. Reduced platinum uptake and increased platinum export are examples of resistance mechanisms that limit the extent of DNA damage. Here, we report the discovery and characterization of the role of ATP11B, a P-type ATPase membrane protein, in cisplatin resistance. We found that ATP11B expression was correlated with higher tumor grade in human ovarian cancer samples and with cisplatin resistance in human ovarian cancer cell lines. ATP11B gene silencing restored the sensitivity of ovarian cancer cell lines to cisplatin in vitro. Combined therapy of cisplatin and ATP11B-targeted siRNA significantly decreased cancer growth in mice bearing ovarian tumors derived from cisplatin-sensitive and -resistant cells. In vitro mechanistic studies on cellular platinum content and cisplatin efflux kinetics indicated that ATP11B enhances the export of cisplatin from cells. The colocalization of ATP11B with fluorescent cisplatin and with vesicular trafficking proteins, such as syntaxin-6 (STX6) and vesicular-associated membrane protein 4 (VAMP4), strongly suggests that ATP11B contributes to secretory vesicular transport of cisplatin from Golgi to plasma membrane. In conclusion, inhibition of ATP11B expression could serve as a therapeutic strategy to overcome cisplatin resistance.

The biology of brain metastasis.

BACKGROUND: It is estimated that at least 200 000 cases of brain metastases occur each year in the US, which is 10 times the number of patients diagnosed with primary brain tumors. Brain metastasis is associated with poor prognosis, neurological deterioration, diminished quality of life, and extremely short survival. Favorable interactions between tumor cells and cerebral microvascular endothelial cells encourage tumor growth in the central nervous system, while tumor cell interactions with astrocytes protect brain metastases from the cytotoxic effects of chemotherapy. CONTENT: We review the pathogenesis of brain metastasis and emphasize the contributions of microvascular endothelial cells and astrocytes to disease progression and therapeutic resistance. Animal models used to study brain metastasis are also discussed. SUMMARY: Brain metastasis has many unmet clinical needs. There are few clinically relevant tumor models and no targeted therapies specific for brain metastases, and the mean survival for untreated patients is 5 weeks. Improved clinical outcomes are dependent on an enhanced understanding of the metastasis-initiating population of cells and the identification of microenvironmental factors that encourage disease progression in the central nervous system.

Biological roles of the Delta family Notch ligand Dll4 in tumor and endothelial cells in ovarian cancer.

Emerging evidence suggests that the Notch/Delta-like ligand 4 (Dll4) pathway may offer important new targets for antiangiogenesis approaches. In this study, we investigated the clinical and biological significance of Dll4 in ovarian cancer. Dll4 was overexpressed in 72% of tumors examined in which it was an independent predictor of poor survival. Patients with tumors responding to anti-VEGF therapy had lower levels of Dll4 than patients with stable or progressive disease. Under hypoxic conditions, VEGF increased Dll4 expression in the tumor vasculature. Immobilized Dll4 also downregulated VEGFR2 expression in endothelial cells directly through methylation of the VEGFR2 promoter. RNAi-mediated silencing of Dll4 in ovarian tumor cells and tumor-associated endothelial cells inhibited cell growth and angiogenesis, accompanied by induction of hypoxia in the tumor microenvironment. Combining Dll4-targeted siRNA with bevacizumab resulted in greater inhibition of tumor growth, compared with control or treatment with bevacizumab alone. Together, our findings establish that Dll4 plays a functionally important role in both the tumor and endothelial compartments of ovarian cancer and that targeting Dll4 in combination with anti-VEGF treatment might improve outcomes of ovarian cancer treatment.

Selection of brain metastasis-initiating breast cancer cells determined by growth on hard agar.

An approach that facilitates rapid isolation and characterization of tumor cells with enhanced metastatic potential is highly desirable. Here, we demonstrate that plating GI-101A human breast cancer cells on hard (0.9%) agar selects for the subpopulation of metastasis-initiating cells. The agar-selected cells, designated GI-AGR, were homogeneous for CD44(+) and CD133(+) and five times more invasive than the parental GI-101A cells. Moreover, mice injected with GI-AGR cells had significantly more experimental brain metastases and shorter overall survival than did mice injected with GI-101A cells. Comparative gene expression analysis revealed that GI-AGR cells were markedly distinct from the parental cells but shared an overlapping pattern of gene expression with the GI-101A subline GI-BRN, which was generated by repeated in vivo recycling of GI-101A cells in an experimental brain metastasis model. Data mining on 216 genes shared between GI-AGR and GI-BRN breast cancer cells suggested that the molecular phenotype of these cells is consistent with that of cancer stem cells and the aggressive basal subtype of breast cancer. Collectively, these results demonstrate that analysis of cell growth in a hard agar assay is a powerful tool for selecting metastasis-initiating cells in a heterogeneous population of breast cancer cells, and that such selected cells have properties similar to those of tumor cells that are selected based on their potential to form metastases in mice.

Astrocytes upregulate survival genes in tumor cells and induce protection from chemotherapy.

In the United States, more than 40% of cancer patients develop brain metastasis. The median survival for untreated patients is 1 to 2 months, which may be extended to 6 months with conventional radiotherapy and chemotherapy. The growth and survival of metastasis depend on the interaction of tumor cells with host factors in the organ microenvironment. Brain metastases are surrounded and infiltrated by activated astrocytes and are highly resistant to chemotherapy. We report here that coculture of human breast cancer cells or lung cancer cells with murine astrocytes (but not murine fibroblasts) led to the up-regulation of survival genes, including GSTA5, BCL2L1, and TWIST1, in the tumor cells. The degree of up-regulation directly correlated with increased resistance to all tested chemotherapeutic agents. We further show that the up-regulation of the survival genes and consequent resistance are dependent on the direct contact between the astrocytes and tumor cells through gap junctions and are therefore transient. Knocking down these genes with specific small interfering RNA rendered the tumor cells sensitive to chemotherapeutic agents. These data clearly demonstrate that host cells in the microenvironment influence the biologic behavior of tumor cells and reinforce the contention that the organ microenvironment must be taken into consideration during the design of therapy.

The seed and soil hypothesis revisited--the role of tumor-stroma interactions in metastasis to different organs.

The fact that certain tumors exhibit a predilection for metastasis to specific organs has been recognized for well over a century now. An extensive body of clinical data and experimental research has confirmed Stephen Paget's original "seed and soil" hypothesis that proposed the organ-preference patterns of tumor metastasis are the product of favorable interactions between metastatic tumor cells (the "seed") and their organ microenvironment (the "soil"). Indeed, many of the first-line therapeutic regimens, currently in use for the treatment of human cancer are designed to target cancer cells (such as chemotherapy) and also to modulate the tumor microenvironment (such as antiangiogenic therapy). While some types of tumors are capable of forming metastases in virtually every organ in the body, the most frequent target organs of metastasis are bone, brain, liver and the lung. In this review, we discuss how tumor-stromal interactions influence metastasis in each of these organs.

Upregulated stromal EGFR and vascular remodeling in mouse xenograft models of angiogenesis inhibitor-resistant human lung adenocarcinoma.

Angiogenesis is critical for tumor growth and metastasis, and several inhibitors of angiogenesis are currently in clinical use for the treatment of cancer. However, not all patients benefit from antiangiogenic therapy, and those tumors that initially respond to treatment ultimately become resistant. The mechanisms underlying this, and the relative contributions of tumor cells and stroma to resistance, are not completely understood. Here, using species-specific profiling of mouse xenograft models of human lung adenocarcinoma, we have shown that gene expression changes associated with acquired resistance to the VEGF inhibitor bevacizumab occurred predominantly in stromal and not tumor cells. In particular, components of the EGFR and FGFR pathways were upregulated in stroma, but not in tumor cells. Increased activated EGFR was detected on pericytes of xenografts that acquired resistance and on endothelium of tumors with relative primary resistance. Acquired resistance was associated with a pattern of pericyte-covered, normalized revascularization, whereas tortuous, uncovered vessels were observed in relative primary resistance. Importantly, dual targeting of the VEGF and EGFR pathways reduced pericyte coverage and increased progression-free survival. These findings demonstrated that alterations in tumor stromal pathways, including the EGFR and FGFR pathways, are associated with, and may contribute to, resistance to VEGF inhibitors and that targeting these pathways may improve therapeutic efficacy. Understanding stromal signaling may be critical for developing biomarkers for angiogenesis inhibitors and improving combination regimens.

Consistent interactions between tumor cell IL-6 and macrophage TNF-α enhance the growth of human prostate cancer cells in the bone of nude mouse.

To test the hypothesis that tumor-associated macrophages (TAMs) enhance the growth and metastasis of human prostate cancer in the bone, we evaluated the effects of decreasing interleukin-6 (IL-6) production by tumor cells and TAMs in a mouse model of bone metastasis. Human PC-3MM2 cells that produce IL-6 were transfected with lentivirus containing IL-6 small hairpin RNA (shRNA) or nonspecific RNA and injected into the tibias of nude mice treated intraperitoneally every 5days for 5weeks with phosphate-buffered saline (PBS), liposomes containing PBS, or liposomes containing clodronate (to decrease the number of macrophages). Transfection of PC-3MM2 cells with IL-6 shRNA significantly decreased cellular expression of IL-6 and the number of TAMs and osteoclasts in bone tumors, which correlated with significant decreases in tumor size, bone lysis, and incidence of lymph node metastasis. Treatment of mice with clodronate liposomes significantly decreased the number of TAMs and osteoclasts in the bone tumors, the expression of IL-6 in the PC3-MM2 cells, and the production of tumor necrosis factor (TNF)-α by TAMs. These findings correlated with a significant decrease in tumor size, bone lysis, and lymph node metastasis. Knocking down IL-6 in tumor cells and decreasing TAMs was associated with the lowest incidences of bone tumors and lymph node metastasis. These results suggest that TAMs enhance the growth of prostate cancer cells in the bone.