Courtney Jarvis

PEDF increases the tumoricidal activity of macrophages towards prostate cancer cells in vitro

Background Although inflammation and prostate cancer (PCa) have been linked, the molecular interactions between macrophages and PCa cells are poorly explored. Pigment Epithelium-Derived Factor (PEDF) is an anti-angiogenic and anti-tumor factor. We previously showed that PEDF induces macrophages recruitment in vitro, correlates with macrophages density in human prostate, and stimulates macrophages polarization towards the classically activated pathway. Here, we demonstrate that PEDF modulates the interaction between macrophages and PCa cells through a bidirectional signalling leading to tumor cell apoptosis and phagocytosis. Methods RAW 264.7 and THP-1 cells, and BMDMs were grown in vitro as mono- or co-cultures with PC3 or CL1 tumor cells. The effects of PEDF and its derived P18 peptide were measured on macrophages differentiation, migration, and superoxide production, and tumor cell apoptosis and phagocytosis. PEDF receptors (ATP5B, PNPLA2, and LRP6) and CD47 mRNA and protein expression were quantified in macrophages and tumor cells by quantitative RT-PCR, western blot, immunofluorescence and flow cytometry. Results We found that PEDF induced the migration of macrophages towards tumor 3D spheroids and 2D cultures. In co-culture, PEDF increased PCa cells phagocytosis through an indirect apoptosis-dependent mechanism. Moreover, PEDF stimulated the production of superoxide by macrophages. Conditioned media from macrophages exposed to PEDF induced tumor cells apoptosis in contrast to control conditioned media suggesting that ROS may be involved in tumor cells apoptosis. ATP5B and PNPLA2 PEDF receptors on macrophages and CD47 on tumor cells were respectively up- and down-regulated by PEDF. As PEDF, blocking CD47 induced phagocytosis. Inhibiting ATP5B reduced phagocytosis. Inversely, PNPLA2 inhibition blocks differentiation but maintains phagocytosis. CD47-induced phagocytosis was partially reverted by ATP5B inhibition suggesting a complementary action. Similar effects were observed with P18 PEDF-derived peptide. Conclusions These data established that modulating the molecular interactions between macrophages and PCa cells using PEDF may be a promising strategy for PCa treatment.

Organoselenium Polymer Inhibits Biofilm Formation in Polypropylene Contact Lens Case Material.

Objectives: Contact lens-acquired bacterial infections are a serious problem. Of the reported cases, inadequate cleaning of the lens case was the most common cause of lens contamination. Organoselenium has been shown to inhibit bacterial attachment to different polymer materials. This study evaluates the ability of an organoselenium monomer, incorporated into the polymer of a polypropylene contact lens case coupon, to block the formation of biofilms in a lens case. Methods: The bacteria tested were Pseudomonas aeruginosa, Staphylococcus aureus, Stenotrophomonas maltophilia, and Serratia marcescens. For this study, the bacteria were allowed to grow overnight, in trypticase soy broth media, in the presence of the selenium-containing polymer or the same polymer without organoselenium. The material was studied by both colony-forming unit determination and by confocal laser scanning microscopy. Results: The results showed that the organoselenium polymer versus the control polymer resulted in the following effect on biofilm formation: (1) a reduction in P. aeruginosa of 7.3 logs (100%); (2) a reduction in S. aureus of 7.3 logs (100%); (3) a reduction in S. maltophilia of 7.5 logs (100%); and (4) a reduction in S. marcescens reduction of 3.3 logs (99.9%). To test the stability of the organoselenium polypropylene contact lens coupon, the coupon was soaked in PBS for eight weeks at room temperature. It was found that when these soaked coupons were tested against S. aureus, complete inhibition (8.1 logs) was obtained. Because organoselenium cannot leach from the polymer, this would imply that the organoselenium polypropylene contact lens case coupon would be inhibitory toward bacterial biofilm for the life of the case. Conclusion: The organoselenium polypropylene contact lens case coupon shows the ability to inhibit biofilm formation. The use of organoselenium copolymer should play an important role in protecting against contact lens case-acquired infection.

A Novel Organo-Selenium Bandage that Inhibits Biofilm Development in a Wound by Gram-Positive and Gram-Negative Wound Pathogens

Biofilm formation in wounds is a serious problem which inhibits proper wound healing. One possible contributor to biofilm formation in a wound is the bacteria growing within the overlying bandage. To test this mechanism, we used bandages that contained a coating of organo-selenium that was covalently attached to the bandage. We tested the ability of this coating to kill bacteria on the bandage and in the underlying tissue. The bandage material was tested with both lab strains and clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa and Staphylococcus epidermidis. It was found that the organo-selenium coated bandage showed inhibition, of biofilm formation on the bandage in vitro (7–8 logs), with all the different bacteria tested, at selenium concentrations in the coating of less than 1.0%. These coatings were found to remain stable for over one month in aqueous solution, 15 min in boiling water, and over 6 years at room temperature. The bandages were also tested on a mouse wound model where the bacteria were injected between the bandage and the wound. Not only did the selenium bandage inhibit biofilm formation in the bandage, but it also inhibited biofilm formation in the wound tissue. Since selenium does not leave the bandage, this would appear to support the idea that a major player in wound biofilm formation is bacteria which grows in the overlying bandage.

Cabazitaxel regimens inhibit the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with various efficacy and toxicity

Taxanes chemotherapies represent the major therapeutic alternative for symptomatic mCRPC. While docetaxel is the most commonly prescribed Taxane for mCRPC; cabazitaxel has been approved for patients unresponsive to docetaxel. Still mCRPC remains incurable and patients often experience severe side effects. Recently, the FIRSTANA trial first demonstrated the absence of superiority in overall survival between cabazitaxel and docetaxel in mCRPC patients. Inversely, different toxicity were reported suggesting that cabazitaxel may provide a first line treatment option for some patients urging for a deeper characterization of cabazitaxel mechanisms of action as well as a re‐evaluation of cabazitaxel conventional dose and schedule. In this study, our goal was therefore to evaluate the anti‐tumor efficacy of various cabazitaxel regimens delivered as monotherapy or in combination with PEDF, a known anti‐angiogenic and anti‐neoplastic agent.

Organo-Selenium Coatings Inhibit Gram-Negative and Gram-Positive Bacterial Attachment to Ophthalmic Scleral Buckle Material.

Purpose Biofilm formation is a problem for solid and sponge-type scleral buckles. This can lead to complications that require removal of the buckle, and result in vision loss due to related ocular morbidity, primarily infection, or recurrent retinal detachment. We investigate the ability of a covalent organo-selenium coating to inhibit biofilm formation on a scleral buckle. Methods Sponge and solid Labtican brand scleral buckles were coated with organo-selenium coupled to a silyation reagent. Staphylococcus aureus biofilm formation was monitored by a standard colony-forming unit assay and the confocal laser scanning microscopy, while Pseudomonas aeruginosa biofilm formation was examined by scanning electron microscopy. Stability studies were done, by soaking in phosphate buffer saline (PBS) at room temperature for 2 months. Toxicity against human corneal epithelial cell was examined by growing the cells in the presence of organo-selenium–coated scleral buckles. Results The organo-selenium coating inhibited biofilm formation by gram-negative and gram-positive bacteria. The buckle coatings also were shown to be fully active after soaking in PBS for 2 months. The organo-selenium coatings had no effect on the viability of human corneal epithelial cells. Conclusions Organo-selenium can be used to covalently coat a scleral buckle, which is stable and inhibits biofilm formation for gram-negative and gram-positive bacteria. The organo-selenium buckle coating was stable and nontoxic to cell culture. Translational Relevance This technology provides a means to inhibit bacterial attachment to devices attached to the eye, without damage to ocular cells.

Assessment of phagocytic activity in live macrophages-tumor cells co-cultures by Confocal and Nomarski Microscopy

Abstract Macrophages have been recognized as the main inflammatory component of the tumor microenvironment. Although often considered as beneficial for tumor growth and disease progression, tumor-associated macrophages have also been shown to be detrimental to the tumor depending on the tumor microenvironment. Therefore, understanding the molecular interactions between macrophages and tumor cells in relation to macrophages functional activities such as phagocytosis is critical for a better comprehension of their tumor-modulating action. Still, the characterization of these molecular mechanisms in vivo remains complicated due to the extraordinary complexity of the tumor microenvironment and the broad range of tumor-associated macrophage functions. Thus, there is an increasing demand for in vitro methodologies to study the role of cell–cell interactions in the tumor microenvironment. In the present study, we have developed live co-cultures of macrophages and human prostate tumor cells to assess the phagocytic activity of macrophages using a combination of Confocal and Nomarski Microscopy. Using this model, we have emphasized that this is a sensitive, measurable, and highly reproducible functional assay. We have also highlighted that this assay can be applied to multiple cancer cell types and used as a selection tool for a variety of different types of phagocytosis agonists. Finally, combining with other studies such as gain/loss of function or signaling studies remains possible. A better understanding of the interactions between tumor cells and macrophages may lead to the identification of new therapeutic targets against cancer.

Abstract 288: Low-dose cabazitaxel inhibits the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with greater efficacy than docetaxel

Despite recently approved novel agents, taxane-based chemotherapy remains the major therapeutic strategy for metastatic castration-resistant prostate cancer (mCRPC). Still mCRPC continues to be incurable. Furthermore, patients often experience severe side effects, prompting a re-evaluation of conventional regimen. Past studies have demonstrated promise for Low-Dose Metronomic (LDM) chemotherapy; schedule defined as the frequent administration of low doses of chemotherapeutic drugs with no prolonged drug-free breaks. Yet relative activities of LDM taxanes and combinations with known anti-neoplasic agents have to be investigated. Using CRPC cell lines (PC3, Du145 and the LNCaP-derivative CL1), we have shown that cabazitaxel-treated cells have a significantly lower EC50 compared to docetaxel, with Du145 cells presenting the greatest differences. In cell cycle analysis, both low-dose taxanes increased the sub-G1 cells population. However, the sub-G1 increase was significantly greater in cabazitaxel- than in docetaxel-treated Du145 cells, but not in PC3 and CL1. Accordingly, plasma membrane Annexin V elevation occurred in Du145 cells at lower doses of cabazitaxel than docetaxel validating a higher efficacy due to increased apoptosis. As other possible cell death mechanisms, autophagy and necrosis were investigated. Beclin1 expression levels remain unchanged in all three cell lines. In contrast, necrosis was stimulated in all the taxanes-treated cell lines in a dose dependent manner. In vivo, LDM cabazitaxel was significantly more efficient in delaying tumor growth than docetaxel. This effect was markedly increased when cabazitaxel was combined with the angio-inhibitor and anti-tumor Pigment Epithelium-Derived Factor (PEDF). Other results showed that PEDF and LDM chemotherapy combination induces more phagocytosis of CRPC cells when compared to single treatments. In conclusion, our data demonstrate a higher efficacy of cabazitaxel on CRPC both in vitro and in vivo, and suggest that LDM taxane chemotherapy/PEDF combination could be used as a novel therapeutic strategy for CRPC. Citation Format: Courtney L. Jarvis, Thomas Nelius, Dalia Martinez-Marin, Srirupa Cheerla, Stephanie Filleur. Low-dose cabazitaxel inhibits the growth of prostate cancer cells and enhances the anti-tumor properties of PEDF with greater efficacy than docetaxel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 288.

MP66-04 CABAZITAXEL INHIBITS THE PROLIFERATION OF HUMAN CASTRATION-REFRACTORY PROSTATE CANCER CELLS IN VITRO AND ENHANCES THE ANTI-TUMOR PROPERTIES OF THE ANGIO-INHIBITORY PIGMENT EPITHELIUM-DERIVED FACTOR IN VIVO WITH A GREATER EFFICACY THAN DOCETAXEL

therapeutic response. The goal of this study was to evaluate and genetically characterize exosome derived RNA (exoRNA) isolated from blood of metastatic CRPC patients. METHODS: Whole blood samples from 18 consented clinically annotated mCRPC patients and 1 normal control were collected. Exosomes were isolated with ultracentrifugation and exoRNA extracted. Following library prep, paired-end sequencing was performed using Illumina Hi-Seq 2000. A bioinformatics pipeline was used for data prepossessing including alignment, duplicate removal, normalization and variant calling. Visualization and differential analyses were performed with SNP & Variation Suite v8.x. RESULTS: In exoRNA there is evidence of extensive chromosomal rearrangement resulting in a myriad of gene fusions and isoforms. Through preliminary analyses we identified 39 genes commonly expressed in the exosomes of these mCRPC patients. These include PDPK1, USP9X, MAGI2, HMGA2 and PTGFR all of which have been previously expressed in prostate cancer tissue. A diverse variety of lcnRNA, ncRNA and miRNA were also identified in circulating exosomes. Validation of these alterations and additional analyses evaluating translocations and splice variants, PCR validation, and/or direct tumor based nucleic acid assays is ongoing. CONCLUSIONS: These preliminary analyses of circulating exoRNA have identified gene expression signatures of several prostate cancer associated transcripts. Ultimately, the identification of PCa associated transcripts in plasma derived exosomes provides evidence that exosomes and exosomal cargo may serve as biomarker in CRPC patients. Exosomes and exoRNA may provide otherwise unattainable insight into tumor evolution and disease progression. Additional studies evaluating the clinical relevance and prognostic value of exosomal RNA will be critical for biomarker development.

Effect of cabazitaxel on the growth of human castration-refractory prostate cancer cells in vitro compared to docetaxel and on the anti-tumor properties of the angio-inhibitor PEDF in vivo.

205 Background: Docetaxel/DTX and cabazitaxel/CBZ have shown promise in the treatment of metastatic Castration-Refractory Prostate Cancer/mCPRC however, comparative studies are missing. Toxicities of these drugs are significant, urging the need to modify taxane regimens. Recently, low-dose metronomic/LDM treatments using conventional chemotherapeutic drugs have shown benefits in CPRC in improving the effect of anti-angiogenic agents. Previously, we have demonstrated that LDM-DTX in combination with PEDF curbs significantly CRPC growth, limits metastases formation and prolongs survival in vivo. In this study, we intended to compare the cytotoxic effect of CBZ and DTX on CRPC cells in vitro and CL1 tumors in vivo. Methods: PC3, DU145 cell lines were from ATCC.CL1 cells were obtained from androgen-deprived LNCaP cells. Cell proliferation was assessed by crystal violet staining and cell cycle analyses. In vitro cytotoxicity assays were performed on CL1 cells/RAW264.7 macrophages co-cultures treated with PEDF ...