Bacillus altitudinis AD13-4 Enhances Saline-Alkali Stress Tolerance of Alfalfa and Affects Composition of Rhizosphere Soil Microbial Community.

Saline and alkaline stresses limit plant growth and reduce crop yield. Soil salinization and alkalization seriously threaten the sustainable development of agriculture and the virtuous cycle of ecology. Biofertilizers made from plant growth-promoting rhizobacteria (PGPR) not only enhance plant growth and stress tolerance, but also are environmentally friendly and cost-effective. There have been many studies on the mechanisms underlying PGPRs enhancing plant salt resistance. However, there is limited knowledge about the interaction between PGPR and plants under alkaline-sodic stress. To clarify the mechanisms underlying PGPR's improvement of plants' tolerance to alkaline-sodic stress, we screened PGPR from the rhizosphere microorganisms of local plants growing in alkaline-sodic land and selected an efficient strain, Bacillus altitudinis AD13-4, as the research object. Our results indicate that the strain AD13-4 can produce various growth-promoting substances to regulate plant endogenous hormone levels, cell division and differentiation, photosynthesis, antioxidant capacity, etc. Transcriptome analysis revealed that the strain AD13-4 significantly affected metabolism and secondary metabolism, signal transduction, photosynthesis, redox processes, and plant-pathogen interactions. Under alkaline-sodic conditions, inoculation of the strain AD13-4 significantly improved plant biomass and the contents of metabolites (e.g., soluble proteins and sugars) as well as secondary metabolites (e.g., phenols, flavonoids, and terpenoids). The 16S rRNA gene sequencing results indicated that the strain AD13-4 significantly affected the abundance and composition of the rhizospheric microbiota and improved soil activities and physiochemical properties. Our study provides theoretical support for the optimization of saline-alkali-tolerant PGPR and valuable information for elucidating the mechanism of plant alkaline-sodic tolerance.

Harnessing microRNA-enriched extracellular vesicles for liquid biopsy.

Extracellular microRNAs (miRNAs) can be detected in body fluids and hold great potential as cancer biomarkers. Extracellular miRNAs are protected from degradation by binding various proteins and through their packaging into extracellular vesicles (EVs). There is evidence that the diagnostic performance of cancer-associated extracellular miRNAs can be improved by assaying EV-miRNA instead of total cell-free miRNA, but several challenges have hampered the advancement of EV-miRNA in liquid biopsy. Because almost all types of cells release EVs, cancer cell-derived EVs might constitute only a minor fraction of EVs in body fluids of cancer patients with low volume disease. Furthermore, a given cell type can release several subpopulations of EVs that vary in their cargo, and there is evidence that the majority of EVs contain low copy numbers of miRNAs. In this mini-review, we discuss the potential of several candidate EV membrane proteins such as CD147 to define cancer cell-derived EVs, and approaches by which subpopulations of miRNA-rich EVs in body fluids might be identified. By integrating these insights, we discuss strategies by which EVs that are both cancer cell-derived and miRNA-rich could be isolated to enhance the diagnostic performance of extracellular miRNAs.

Normal saline remodels the omentum and stimulates its receptivity for transcoelomic metastasis.

The omentum contains immune cell structures called milky spots that are niches for transcoelomic metastasis. It is difficult to remove the omentum completely, and there are no effective strategies to minimize the risk of colonization of preserved omental tissues by cancer cells that circulate in the peritoneal fluid. Normal saline is commonly administered into the peritoneal cavity for diagnostic and intraoperative lavage. Here we show that normal saline, when administered into the peritoneal cavity of mice, is prominently absorbed by the omentum, exfoliates its mesothelium, and induces expression of CX3CL1, the ligand for CX3CR1, within and surrounding the omental vasculature. Studies using CX3CR1-competent and CX3CR1-deficient mice showed that the predominant response in the omentum following saline administration is an accumulation of CX3CR1+ monocytes/macrophages that expand milky spots and promote neoangiogenesis within these niches. Moreover, saline administration promoted the implantation of cancer cells of ovarian and colorectal origin onto the omentum. By contrast, these deleterious effects were not observed following i.p. administration of lactated Ringer's solution. Our findings suggest that normal saline stimulates the receptivity of the omentum for cancer cells and that the risk of colonization can be minimized by using a biocompatible crystalloid for lavage procedures.

The glycoprotein CD147 defines miRNA-enriched extracellular vesicles that derive from cancer cells.

Extracellular vesicles (EVs) are ideal for liquid biopsy, but distinguishing cancer cell-derived EVs and subpopulations of biomarker-containing EVs in body fluids has been challenging. Here, we identified that the glycoproteins CD147 and CD98 define subpopulations of EVs that are distinct from classical tetraspanin+ EVs in their biogenesis. Notably, we identified that CD147+ EVs have substantially higher microRNA (miRNA) content than tetraspanin+ EVs and are selectively enriched in miRNA through the interaction of CD147 with heterogeneous nuclear ribonucleoprotein A2/B1. Studies using mouse xenograft models showed that CD147+ EVs predominantly derive from cancer cells, whereas the majority of tetraspanin+ EVs are not of cancer cell origin. Circulating CD147+ EVs, but not tetraspanin+ EVs, were significantly increased in prevalence in patients with ovarian and renal cancers as compared to healthy individuals and patients with benign conditions. Furthermore, we found that isolating miRNAs from body fluids by CD147 immunocapture increases the sensitivity of detecting cancer cell-specific miRNAs, and that circulating miRNAs isolated by CD147 immunocapture more closely reflect the tumor miRNA signature than circulating miRNAs isolated by conventional methods. Collectively, our findings reveal that CD147 defines miRNA-enriched, cancer cell-derived EVs, and that CD147 immunocapture could be an effective approach to isolate cancer-derived miRNAs for liquid biopsy.

In-vivo estimation of tissue electrical conductivities of a rabbit eye for precise simulation of electric field distributions during ocular iontophoresis.

Precise estimation of electrical conductivity of the eyes is important for the accurate analysis of electric field distributions in the eyes during ocular iontophoresis. In this study, we estimated the tissue electrical conductivities of a rabbit eye, which has been widely employed for neuro-ophthalmological experiments, through an in vivo experiment for the first time. Electrical potentials were measured at multiple locations on the skin, while weak currents were transmitted into the skin via two surface electrodes attached to the skin around the eye. A finite element model was constructed to calculate the electric potentials at the measurement locations. The conductivity values of different tissues were then estimated using an optimization procedure to minimize the difference between the measured and calculated electric potentials. The accuracy of the estimated tissue conductivity values of the rabbit eye was validated by comparing the measured and calculated electric potential values for different electrode montages. Further multi-physical analyses of iontophoretic drug delivery to the rabbit eye showed a significant influence of the conductivity profile on the resultant particle distribution. Overall, our results provide an important reference for the tissue electrical conductivity values of the rabbit eye, which could be further utilized for designing new medical devices for delivering electric fields to the eyes, such as transorbital and transscleral electrical stimulations.

Extracellular Vesicle Membrane-Associated Proteins: Emerging Roles in Tumor Angiogenesis and Anti-Angiogenesis Therapy Resistance.

The tumor vasculature is essential for tumor growth and metastasis, and is a prime target of several anti-cancer agents. Increasing evidence indicates that tumor angiogenesis is stimulated by extracellular vesicles (EVs) that are secreted or shed by cancer cells. These EVs encapsulate a variety of biomolecules with angiogenic properties, and have been largely thought to stimulate vessel formation by transferring this luminal cargo into endothelial cells. However, recent studies have revealed that EVs can also signal to recipient cells via proteins on the vesicular surface. This review discusses and integrates emerging insights into the diverse mechanisms by which proteins associate with the EV membrane, the biological functions of EV membrane-associated proteins in tumor angiogenesis, and the clinical significance of these proteins in anti-angiogenic therapy.

Comparative Solution Synthesis of Mn Doped (Na,K)NbO3 Thin Films.

(K0.5 Na0.5 )NbO3 (KNN) is a promising lead-free alternative for ferroelectric thin films such as Pb(Zr,Ti)O3 . One main drawback is its high leakage current density at high electric fields, which has been previously linked to alkali non-stoichiometry. This paper compares three acetate-based chemical solution synthesis and deposition methods for 0.5 mol % Mn-doped KNN film fabrication, using lower crystallization temperature processes in comparison to the sintering temperatures necessary for fabrication of KNN ceramics. This paper shows the crucial role of the A site homogenization step during solution synthesis in preserving alkali chemical homogeneity of Mn doped KNN films. Chemically homogeneous films show a uniform grain size of 80 nm and a leakage current density under 2.8×10-8  A cm-2 up to electric fields as high as 600 kV cm-1 , which is the highest breakdown strength reported for KNN thin films. Solution synthesis involving two-step pyrolysis resulted in films with dense, columnar microstructures, which are interesting for orientation control and enhancement of piezoelectric properties. This study reports detailed solution synthesis and deposition processes with good dielectric, ferroelectric and breakdown field properties. An optimized fabrication method that should couple low leakage current density with dense and oriented microstructures is proposed.

Interface Engineering in Hybrid Iodide CH3NH3PbI3 Perovskites Using Lewis Base and Graphene toward High-Performance Solar Cells.

Photovoltaic solar cells based on organic-inorganic hybrid halide perovskites have achieved a substantial breakthrough via advanced interface engineering. Reports have emphasized that combining the hybrid perovskites with the Lewis base and/or graphene can definitely improve the performance through surface trap passivation and band alignment alteration; the underlying mechanisms are not yet fully understood. Here, using density functional theory calculations, we show that upon the formation of CH3NH3PbI3 interfaces with three different Lewis base molecules and graphene, the binding strength with S-donors thiocarbamide and thioacetamide is higher than with O-donor dimethyl sulfoxide, while the interface dipole and work function reduction tend to increase from S-donors to O-donor. We provide evidences of deep trap state elimination in the S-donor perovskite interfaces through the analysis of defect formation on the CH3NH3PbI3(110) surface and of stability enhancement by estimation of activation barriers for vacancy-mediated iodine atom migrations. These theoretical predictions are in line with the experimental observation of performance enhancement in the perovskites prepared using thiocarbamide.

Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake.

Cancer-derived small extracellular vesicles (sEVs) induce stromal cells to become permissive for tumor growth. However, it is unclear whether this induction solely occurs through transfer of vesicular cargo into recipient cells. Here we show that cancer-derived sEVs can stimulate endothelial cell migration and tube formation independently of uptake. These responses were mediated by the 189 amino acid isoform of vascular endothelial growth factor (VEGF) on the surface of sEVs. Unlike other common VEGF isoforms, VEGF189 preferentially localized to sEVs through its high affinity for heparin. Interaction of VEGF189 with the surface of sEVs profoundly increased ligand half-life and reduced its recognition by the therapeutic VEGF antibody bevacizumab. sEV-associated VEGF (sEV-VEGF) stimulated tumor xenograft growth but was not neutralized by bevacizumab. Furthermore, high levels of sEV-VEGF were associated with disease progression in bevacizumab-treated cancer patients, raising the possibility that resistance to bevacizumab might stem in part from elevated levels of sEV-VEGF.

Neutrophils facilitate ovarian cancer premetastatic niche formation in the omentum.

Ovarian cancer preferentially metastasizes to the omentum, a fatty tissue characterized by immune structures called milky spots, but the cellular dynamics that direct this tropism are unknown. Here, we identified that neutrophil influx into the omentum is a prerequisite premetastatic step in orthotopic ovarian cancer models. Ovarian tumor-derived inflammatory factors stimulated neutrophils to mobilize and extrude chromatin webs called neutrophil extracellular traps (NETs). NETs were detected in the omentum of ovarian tumor-bearing mice before metastasis and of women with early-stage ovarian cancer. NETs, in turn, bound ovarian cancer cells and promoted metastasis. Omental metastasis was decreased in mice with neutrophil-specific deficiency of peptidylarginine deiminase 4 (PAD4), an enzyme that is essential for NET formation. Blockade of NET formation using a PAD4 pharmacologic inhibitor also decreased omental colonization. Our findings implicate NET formation in rendering the premetastatic omental niche conducive for implantation of ovarian cancer cells and raise the possibility that blockade of NET formation prevents omental metastasis.

Amino-Functionalized Mesoporous Silica Particles for Ocular Delivery of Brimonidine.

To treat glaucoma, conventional eye drops are often prescribed. However, the eye drops have limited effectiveness as a result of low drug bioavailability due to their rapid clearance from the preocular space. To resolve this, we proposed amino-functionalized mesoporous silica (AMS) particles as delivery carriers of the glaucoma drug, brimonidine. Because of the presence of mesopores, brimonidine (BMD) could be encapsulated in the AMS with a loading amount of 41.73 µg/mg (i.e., drug loading capacity of about 4.17%) to give the BMD-AMS, which could release the drug in a sustained manner over 8 h. BMD-AMS was also shown to be mucoadhesive due to the presence of both hydroxyl and amino groups in the surface, allowing for formation of hydrogen bonds and an ionic complex with the mucin, respectively. Therefore, when topically administered to rabbit eyes in vivo, BMD-AMS could reside in the preocular space for up to 12 h because of its adherence to the mucous layer. To assess in vivo efficacy, we examined the variance in intraocular pressure (IOP) and brimonidine concentration in the aqueous humor (AH) after applying BMD-AMS to the eye, which was compared with that induced by Alphagan P, the marketed brimonidine eye drops. For BMD-AMS, the duration in the decrease in IOP and the area under the drug concentration in the AH-time curve (AUC) were 12 h and 2.68 µg·h/mL, respectively, which were about twice as large as those obtained with Alphagan P; this finding indicated enhanced ocular bioavailability of brimonidine with BMD-AMS.

Direct Synthesis of Carbon Sheathed Tungsten Oxide Nanoparticles via Self-Assembly Route for High Performance Electrochemical Charge Storage Electrode.

Using a stabilizing agent-assisted co-assembly method, a novel nanocomposite of mesoporous carbon embedded with uniform tungsten oxide nanorods is obtained, which is converted into carbon-sheathed tungsten oxide nanoparticles by delicate calcination and further reduction. Through optimization of tungsten content, it is found that highly crystalline tungsten oxide nanoparticles are uniformly coated with an ultra-thin carbon layer. When applied into electrochemical charge-storage electrodes for supercapacitor and lithium-ion battery, an excellent average capacitance (129 F g−1, above 400 F cm−3), higher rate performance and significantly advanced cycle stability are observed. These improved charge storage properties are attributed to improved electrical conductivity and enhanced structural stability, which is induced by uniform carbon coating on partially reduced tungsten oxide nanoparticles.

Radiosensitizing Pancreatic Cancer Xenografts by an Implantable Micro-Oxygen Generator.

Over the past decades, little progress has been made to improve the extremely low survival rates in pancreatic cancer patients. Extreme hypoxia observed in pancreatic tumors contributes to the aggressive and metastatic characteristics of this tumor and can reduce the effectiveness of conventional radiation therapy and chemotherapy. In an attempt to reduce hypoxia-induced obstacles to effective radiation treatment, we used a novel device, the implantable micro-oxygen generator (IMOG), for in situ tumor oxygenation. After subcutaneous implantation of human pancreatic xenograft tumors in athymic rats, the IMOG was wirelessly powered by ultrasonic waves, producing 30 µA of direct current (at 2.5 V), which was then utilized to electrolyze water and produce oxygen within the tumor. Significant oxygen production by the IMOG was observed and corroborated using the NeoFox oxygen sensor dynamically. To test the radiosensitization effect of the newly generated oxygen, the human pancreatic xenograft tumors were subcutaneously implanted in nude mice with either a functional or inactivated IMOG device. The tumors in the mice were then exposed to ultrasonic power for 10 min, followed by a single fraction of 5 Gy radiation, and tumor growth was monitored thereafter. The 5 Gy irradiated tumors containing the functional IMOG exhibited tumor growth inhibition equivalent to that of 7 Gy irradiated tumors that did not contain an IMOG. Our study confirmed that an activated IMOG is able to produce sufficient oxygen to radiosensitize pancreatic tumors, enhancing response to single-dose radiation therapy.

The homeoprotein DLX4 stimulates NF-κB activation and CD44-mediated tumor-mesothelial cell interactions in ovarian cancer.

Ovarian cancers often highly express inflammatory cytokines and form implants throughout the peritoneal cavity. However, the mechanisms that drive inflammatory signaling and peritoneal metastasis of ovarian cancer are poorly understood. We previously identified that high expression of DLX4, a transcription factor encoded by a homeobox gene, is associated with reduced survival of ovarian cancer patients. In this study, we identified that DLX4 stimulates attachment of ovarian tumor cells to peritoneal mesothelial cells in vitro and increases the numbers of peritoneal implants in xenograft models. DLX4 induced expression of the cell surface molecule CD44 in ovarian tumor cells, and inhibition of CD44 abrogated the ability of DLX4 to stimulate tumor-mesothelial cell interactions. The induction of CD44 by DLX4 was attributed to increased activity of NF-κB that was stimulated by the inflammatory cytokine IL-1ß, a transcriptional target of DLX4. The stimulatory effects of DLX4 on CD44 levels and tumor-mesothelial cell interactions were abrogated when IL-1ß or NF-κB was inhibited in tumor cells. Furthermore, DLX4 expression levels strongly correlated with NF-κB activation and disease stage in clinical specimens of ovarian cancer. Collectively, these findings indicate that DLX4 induces CD44 by stimulating IL-1ß-mediated NF-κB activity, thereby promoting peritoneal metastasis of ovarian cancer.

The homeoprotein DLX4 controls inducible nitric oxide synthase-mediated angiogenesis in ovarian cancer.

BACKGROUND: Homeobox genes encode transcription factors that control patterning of virtually all organ systems including the vasculature. Tumor angiogenesis is stimulated by several homeobox genes that are overexpressed in tumor cells, but the mechanisms of these genes are poorly understood. In this study, we investigated the mechanisms by which DLX4, a homeobox gene that is associated with increased tumor microvessel density, stimulates ovarian tumor angiogenesis. METHODS: Expression of DLX4 and nitric oxide synthases was analyzed in publicly available transcriptional profiles of ovarian cancer clinical specimens. Levels of inducible nitric oxide synthase (iNOS) were evaluated by quantitative RT-PCR, flow cytometry and nitric oxide assays using ovarian cancer cell lines in which DLX4 was overexpressed or knocked down. Signal Transducer and Activator of Transcription 1 (STAT1) expression and activity were evaluated by luciferase reporter assays, immunofluorescence staining, Western blot and immunoprecipitation. Endothelial cell growth and tumor angiogenesis were evaluated in in vitro assays and xenograft models. RESULTS: We identified that DLX4 induces expression of iNOS, an enzyme that stimulates angiogenesis by generating nitric oxide. Analysis of datasets of two independent patient cohorts revealed that high DLX4 expression in ovarian cancer is strongly associated with elevated expression of iNOS but not of other nitric oxide synthases. Studies using STAT1-expressing and STAT1-deficient cells revealed that DLX4 interacts with STAT1 and induces iNOS expression in part by stimulating STAT1 activity. Expression of DLX4 in ovarian cancer cells stimulated endothelial cell growth in vitro and increased microvessel density in xenograft models, and these stimulatory effects of DLX4 were abrogated when its induction of iNOS was inhibited. CONCLUSION: These findings indicate that DLX4 promotes ovarian tumor angiogenesis in part by stimulating iNOS expression.

HOXA9 promotes homotypic and heterotypic cell interactions that facilitate ovarian cancer dissemination via its induction of P-cadherin.

BACKGROUND: Epithelial ovarian cancer (EOC) is a lethal disease that frequently involves the peritoneal cavity. Dissemination of EOC is a multi-step process in which exfoliated tumor cells survive in the peritoneal fluid as multi-cellular aggregates and then form invasive implants on peritoneal surfaces. The mechanisms that control this process are poorly understood. We previously identified that high expression of the developmental patterning gene HOXA9 is associated with poor survival in EOC patients. In this study, we investigated the significance and mechanisms of HOXA9 in controlling aggregation and implantation of floating EOC cells. METHODS: HOXA9 was inhibited by shRNAs or expressed in EOC cells that were propagated in suspension cultures and in the peritoneal cavity of mice. Cell death was assayed by flow cytometry and ELISA. Cell aggregation, attachment and migration were evaluated by microscopy, transwell chamber assays and histopathologic analysis. DNA-binding of HOXA9 and its effect on expression of the cell adhesion molecule P-cadherin were assayed by chromatin immunoprecipitation, quantitative RT-PCR and Western blot. HOXA9 and P-cadherin expression was evaluated in publicly available datasets of EOC clinical specimens. RESULTS: We identified that HOXA9 promotes aggregation and inhibits anoikis in floating EOC cells in vitro and in xenograft models. HOXA9 also stimulated the ability of EOC cells to attach to peritoneal cells and to migrate. HOXA9 bound the promoter of the CDH3 gene that encodes P-cadherin, induced CDH3 expression in EOC cells, and was associated with increased CDH3 expression in clinical specimens of EOC. Inhibiting P-cadherin in EOC cells that expressed HOXA9 abrogated the stimulatory effects of HOXA9 on cell aggregation, implantation and migration. Conversely, these stimulatory effects of HOXA9 were restored when P-cadherin was reconstituted in EOC cells in which HOXA9 was inhibited. CONCLUSION: These findings indicate that HOXA9 contributes to poor outcomes in EOC in part by promoting intraperitoneal dissemination via its induction of P-cadherin.

Therapeutic strategies for targeting the ovarian tumor stroma.

Epithelial ovarian cancer is the most lethal type of gynecologic malignancy. Sixty percent of women who are diagnosed with ovarian cancer present with advanced-stage disease that involves the peritoneal cavity and these patients have a 5-year survival rate of less than 30%. For more than two decades, tumor-debulking surgery followed by platinum-taxane combination chemotherapy has remained the conventional first-line treatment of ovarian cancer. Although the initial response rate is 70%-80%, most patients with advanced-stage ovarian cancer eventually relapse and succumb to recurrent chemoresistant disease. A number of molecular aberrations that drive tumor progression have been identified in ovarian cancer cells and intensive efforts have focused on developing therapeutic agents that target these aberrations. However, increasing evidence indicates that reciprocal interactions between tumor cells and various types of stromal cells also play important roles in driving ovarian tumor progression and that these stromal cells represent attractive therapeutic targets. Unlike tumor cells, stromal cells within the tumor microenvironment are in general genetically stable and are therefore less likely to become resistant to therapy. This concise review discusses the biological significance of the cross-talk between ovarian cancer cells and three major types of stromal cells (endothelial cells, fibroblasts, macrophages) and the development of new-generation therapies that target the ovarian tumor microenvironment.

Monitoring the effects of anti-angiogenesis on the radiation sensitivity of pancreatic cancer xenografts using dynamic contrast-enhanced computed tomography.

PURPOSE: To image the intratumor vascular physiological status of pancreatic tumors xenografts and their response to anti-angiogenic therapy using dynamic contrast-enhanced computed tomography (DCE-CT), and to identify parameters of vascular physiology associated with tumor x-ray sensitivity after anti-angiogenic therapy. METHODS AND MATERIALS: Nude mice bearing human BxPC-3 pancreatic tumor xenografts were treated with 5 Gy of radiation therapy (RT), either a low dose (40 mg/kg) or a high dose (150 mg/kg) of DC101, the anti-VEGF receptor-2 anti-angiogenesis antibody, or with combination of low or high dose DC101 and 5 Gy RT (DC101-plus-RT). DCE-CT scans were longitudinally acquired over a 3-week period post-DC101 treatment. Parametric maps of tumor perfusion and fractional plasma volume (Fp) were calculated and their averaged values and histogram distributions evaluated and compared to controls, from which a more homogeneous physiological window was observed 1-week post-DC101. Mice receiving a combination of DC101-plus-RT(5 Gy) were imaged baseline before receiving DC101 and 1 week after DC101 (before RT). Changes in perfusion and Fp were compared with alternation in tumor growth delay for RT and DC101-plus-RT (5 Gy)-treated tumors. RESULTS: Pretreatment with low or high doses of DC101 before RT significantly delayed tumor growth by an average 7.9 days compared to RT alone (P ≤ .01). The increase in tumor growth delay for the DC101-plus-RT-treated tumors was strongly associated with changes in tumor perfusion (ΔP>-15%) compared to RT treated tumors alone (P=.01). In addition, further analysis revealed a trend linking the tumor's increased growth delay to its tumor volume-to-DC101 dose ratio. CONCLUSIONS: DCE-CT is capable of monitoring changes in intratumor physiological parameter of tumor perfusion in response to anti-angiogenic therapy of a pancreatic human tumor xenograft that was associated with enhanced radiation response.

P-cadherin promotes ovarian cancer dissemination through tumor cell aggregation and tumor-peritoneum interactions.

UNLABELLED: More than 60% of patients who are diagnosed with epithelial ovarian cancer (EOC) present with extensive peritoneal carcinomatosis. EOC cells typically disseminate by shedding into the peritoneal fluid in which they survive as multicellular aggregates and then implant onto peritoneal surfaces. However, the mechanism that facilitates aggregation and implantation of EOC cells is poorly understood. The cell adhesion molecule P-cadherin has been reported to be induced during early progression of EOC and to promote tumor cell migration. In this study, P-cadherin not only promoted migration of EOC cells, but also facilitated the assembly of floating EOC cells into multicellular aggregates and inhibited anoikis in vitro. Furthermore, inhibiting P-cadherin by short hairpin RNAs (shRNA) or a neutralizing antibody prevented EOC cells from attaching to peritoneal mesothelial cells in vitro. In mouse intraperitoneal xenograft models of EOC, inhibition of P-cadherin decreased the aggregation and survival of floating tumor cells in ascites and reduced the number of tumor implants on peritoneal surfaces. These findings indicate that P-cadherin promotes intraperitoneal dissemination of EOC by facilitating tumor cell aggregation and tumor-peritoneum interactions in addition to promoting tumor cell migration. IMPLICATIONS: Inhibiting P-cadherin blocks multiple key steps of EOC progression and has therapeutic potential.

Expression of the homeobox gene HOXA9 in ovarian cancer induces peritoneal macrophages to acquire an M2 tumor-promoting phenotype.

Tumor-associated macrophages (TAMs) exhibit an M2 macrophage phenotype that suppresses anti-tumor immune responses and often correlates with poor outcomes in patients with cancer. Patients with ovarian cancer frequently present with peritoneal carcinomatosis, but the mechanisms that induce naïve peritoneal macrophages into TAMs are poorly understood. In this study, we found an increased abundance of TAMs in mouse i.p. xenograft models of ovarian cancer that expressed HOXA9, a homeobox gene that is associated with poor prognosis in patients with ovarian cancer. HOXA9 expression in ovarian cancer cells stimulated chemotaxis of peritoneal macrophages and induced macrophages to acquire TAM-like features. These features included induction of the M2 markers, CD163 and CD206, and the immunosuppressive cytokines, IL-10 and chemokine ligand 17, and down-regulation of the immunostimulatory cytokine, IL-12. HOXA9-mediated induction of TAMs was primarily due to the combinatorial effects of HOXA9-induced, tumor-derived transforming growth factor-ß2 and chemokine ligand 2 levels. High HOXA9 expression in clinical specimens of ovarian cancer was strongly associated with increased abundance of TAMs and intratumoral T-regulatory cells and decreased abundance of CD8(+) tumor-infiltrating lymphocytes. Levels of immunosuppressive cytokines were also elevated in ascites fluid of patients with tumors that highly expressed HOXA9. HOXA9 may, therefore, stimulate ovarian cancer progression by promoting an immunosuppressive microenvironment via paracrine effects on peritoneal macrophages.