S-1 eliminates MDSCs and enhances the efficacy of PD-1 blockade via regulation of tumor-derived Bv8 and S100A8 in thoracic tumor.

Myeloid-derived suppressor cells (MDSCs) have been known to play a pivotal role in the induction of immune tolerance, which limits the benefits of immune checkpoint inhibitors (ICIs). Recent studies revealed that several chemotherapeutic agents decreased tumor-infiltrating MDSCs. Therefore, combination therapy with cytotoxic chemotherapeutic agents and ICIs was approved for first-line treatment for lung cancer. However, the impact of chemotherapeutic agents on MDSCs and an optimal partner of ICIs has not been fully investigated in thoracic tumors, including lung cancer and malignant pleural mesothelioma. In the present study, we found that treatment with 5-FU and its oral formulation, S-1, suppressed tumor progression and inhibited the accumulation of MDSCs in thoracic tumor-bearing mice. Tumor-infiltrating T cells and dendritic cells were significantly expanded in S-1-treated mice. 5-FU suppressed the ability of tumor cells to recruit MDSCs, while it did not suppress the survival and differentiation of mouse MDSCs in vitro. We also revealed that 5-FU or S-1 significantly downregulated the expression of tumor-derived Bv8 and S100A8. The knockdown of Bv8 or S100A8 in tumor cells suppressed tumor growth and MDSC recruitment in vivo. Furthermore, in comparison with pemetrexed, administration of S-1 improved the synergistic therapeutic efficacy of anti-PD-1 antibodies with or without carboplatin. Our findings revealed a novel mechanism wherein S-1 primed a favorable tumor microenvironment to provide the rationale for combination therapy with S-1 and ICIs as the optimal therapy for thoracic cancer.

Neutralization of Bombina variegata peptide 8 suppresses retinal neovascularization in two different murine models: The oxygen-induced retinopathy model and the rhodopsin promoter/VEGF transgenic mouse model.

Bombina variegata 8 (Bv8), also known as prokineticin-2 (PK-2), is a potent pro-angiogenic factor. However, its role in retinal neovascularization (RNV) remains unknown. In this study, we explored the role of Bv8 in the pathogenesis of RNV. We found that the expression of Bv8 was significantly increased in two different models of retinal neovascularization: the oxygen-induced retinopathy (OIR) mouse model and the rhodopsin promoter (rho)/VEGF transgenic mouse model. Neutralization of Bv8 by intravitreal injections of its antibody, not only inhibited retinal and subretinal neovascularization but also decreased the mRNA and protein levels of several pro-angiogenic factors. Our in vitro assay showed that recombinant human Bv8 (RhBv8) protein promoted human retinal microvascular endothelial cells (HRECs) tube-formation, cell proliferation and vascular endothelial growth factor receptor 1 (VEGFR1) and receptor 2 (VEGFR2) expression. Our findings suggest that Bv8 could be used as a novel target for the treatment of RNV-related ocular diseases.

Method development to quantify Bv8 expression in circulating CD11b+ cells in patients with neovascular age-related macular degeneration (nvAMD) exhibiting Anti-VEGF refractoriness.

A subset of neovascular age-related macular degeneration (nvAMD) subjects appears to be refractory to the effects of anti-VEGF treatment and require frequent intravitreal injections. Prokineticin-2 (Bv8) expression in CD11b(+) cells has been linked to anti-VEGF response. We have developed a reproducible method to quantify gene expression in circulating CD11b + cells. Utilizing this method we tested the hypothesis that high Bv8 expression in circulating CD11b(+) cells is associated with anti-VEGF refractoriness in nvAMD patients. Two groups of nvAMD subjects undergoing treatment with anti-VEGF agents were recruited and classified as refractory or non-refractory to anti-VEGF treatment (n = 33 for each group). Two blood draws were obtained from each subject 1-9 months apart. Peripheral blood mononuclear cells (PBMCs) were isolated and CD11b(+) cells were purified via magnetic bead separation. RNA was purified, and relative expression of Bv8 among the subjects was compared via quantitative PCR analysis. Utilizing this approach no significant difference was detected in the mean LogRQ values between the first and second blood draws (t-test, p = 0.826) indicating low intra-patient variability and demonstrating good reproducibility of the assay. There was no significant difference in Bv8 expression between nvAMD subjects classified as refractory versus non-refractory. We were unable to find a correlation between Bv8 expression in CD11b + cells and anti-VEGF refractoriness in human nvAMD subjects. Relatively high expression in Bv8 in these subjects did not correlate with clinical treatment history, as measured by the frequency of injections. Utilizing this well characterized technique, studies are underway to examine alternative gene expression profiles in various circulating cell populations that may contribute to anti-VEGF refractoriness.

EG-VEGF, BV8, and their receptor expression in human bronchi and their modification in cystic fibrosis: Impact of CFTR mutation (delF508).

Enhanced lung angiogenesis has been reported in cystic fibrosis (CF). Recently, two highly homologous ligands, endocrine gland vascular endothelial growth factor (EG-VEGF) and mammalian Bv8, have been described as new angiogenic factors. Both ligands bind and activate two closely related G protein-coupled receptors, the prokineticin receptor (PROKR) 1 and 2. Yet, the expression, regulation, and potential role of EG-VEGF, BV8, and their receptors in normal and CF lung are still unknown. The expression of the receptors and their ligands was examined using molecular, biochemical, and immunocytochemistry analyses in lungs obtained from CF patients vs. control and in normal and CF bronchial epithelial cells. Cystic fibrosis transmembrane conductance regulator (CFTR) activity was evaluated in relation to both ligands, and concentrations of EG-VEGF were measured by ELISA. At the mRNA level, EG-VEGF, BV8, and PROKR2 gene expression was, respectively, approximately five, four, and two times higher in CF lungs compared with the controls. At the cellular level, both the ligands and their receptors showed elevated expressions in the CF condition. Similar results were observed at the protein level. The EG-VEGF secretion was apical and was approximately two times higher in CF compared with the normal epithelial cells. This secretion was increased following the inhibition of CFTR chloride channel activity. More importantly, EG-VEGF and BV8 increased the intracellular concentration of Ca(2+) and cAMP and stimulated CFTR-chloride channel activity. Altogether, these data suggest local roles for epithelial BV8 and EG-VEGF in the CF airway peribronchial vascular remodeling and highlighted the role of CFTR activity in both ligand biosynthesis and secretion.

G-protein-coupled receptor agonist BV8/prokineticin-2 and STAT3 protein form a feed-forward loop in both normal and malignant myeloid cells.

BACKGROUND: Signaling pathways underlying BV8-mediated oncogenesis remain unknown. RESULTS: BV8-STAT3 forms a feed-forward loop in both normal and malignant myeloid cells and promotes tumor growth. CONCLUSION: JAK2/STAT3 signaling plays critical roles in BV8-mediated myeloid cell-dependent oncogenesis. SIGNIFICANCE: This study identifies a novel role of BV8-STAT3 signaling in mediating cross-talk between tumor microenvironment and tumor cells. An important role of BV8 in mobilization of myeloid cells and myeloid cell-dependent angiogenesis has been established. Recently, it has also been shown that granulocyte colony-stimulating factor (G-CSF)-induced BV8 expression is STAT3 dependent in CD11b(+)Gr1(+) myeloid cells. However, the BV8 downstream signaling pathway(s) intrinsic to myeloid cells crucial for angiogenesis, and potentially also for development of cancers of myeloid origin, remains largely unknown. Here we show that BV8 activates STAT3, which is critical for regulating genes important for both tumor cell proliferation/survival and tumor angiogenesis, in both normal and malignant myeloid cells. Further, BV8-induced STAT3 activation requires Janus-activated kinase 2 (JAK2) activity as shown by both genetic and pharmacologic inhibition. Knocking down BV8 in human myeloid leukemia cells inhibits STAT3 activity and expression of STAT3 downstream angiogenic and pro-proliferation/survival genes, leading to a decrease in tumor cell viability. BV8 shRNA expressing leukemia cells exhibit reduced STAT3 activity and tumor growth in vivo. Taken together, we have delineated a signaling pathway downstream of BV8 that plays critical roles in both the tumor microenvironment and malignant myeloid cells for angiogenesis and tumor cell proliferation/survival.

Bv8 mediates myeloid cell migration and enhances malignancy of colorectal cancer.

Colorectal cancer (CRC) is the third most predominant malignancy in the world. Although the importance of immune system in cancer development has been well established, the underlying mechanisms remain to be investigated further. Here we studied a novel protein prokineticin 2 (Prok2, also known as Bv8) as a key pro-tumoral factor in CRC progression in in vitro and ex vivo settings. Human colorectal tumor tissues, myeloid cell lines (U937 cells and HL60 cells) and colorectal cancer cell line (Caco-2 cells) were used for various studies. Myeloid cell infiltration (especially neutrophils) and Bv8 accumulation were detected in human colorectal tumor tissue with immunostaining. The chemotactic effects of Bv8 on myeloid cells were presented in the transwell assay and chemotaxis assy. Cultured CRC cells treated with myeloid cells or Bv8 produced reactive oxygen species (ROS) and vascular endothelial growth factor (VEGF). Furthermore, ROS and VEGF acted as pro-angiogenesis buffer in myeloid cell-infiltrated CRC microenvironment. Moreover, myeloid cells or Bv8 enhanced energy consumption of glycolysis ATP and mitochondria ATP of CRC cells. Interestingly, myeloid cells increased CRC cell viability, but CRC cells decreased the viability of myeloid cells. ERK signalling pathway in CRC cells was activated in the presence of Bv8 or co-cultured myeloid cells. In conclusion, our data indicated the vital roles of Bv8 in myeloid cell infiltration and CRC development, suggesting that Bv8 may be a potential therapeutic target for colorectal cancer-related immunotherapy.

Bv8 Blockade Sensitizes Anti-PD1 Therapy Resistant Tumors.

Myeloid-derived suppressor cells (MDSCs) are known to promote tumor growth in part by their immunosuppressive activities and their angiogenesis support. It has been shown that Bv8 blockade inhibits the recruitment of MDSCs to tumors, thereby delaying tumor relapse associated with resistance to antiangiogenic therapy. However, the impact of Bv8 blockade on tumors resistant to the new immunotherapy drugs based on the blockade of immune checkpoints has not been investigated. Here, we demonstrate that granulocytic-MDSCs (G-MDSCs) are enriched in anti-PD1 resistant tumors. Importantly, resistance to anti-PD1 monotherapy is reversed upon switching to a combined regimen comprised of anti-Bv8 and anti-PD1 antibodies. This effect is associated with a decreased level of G-MDSCs and enrichment of active cytotoxic T cells in tumors. The blockade of anti-Bv8 has shown efficacy also in hyperprogressive phenotype of anti-PD1-treated tumors. In vitro, anti-Bv8 antibodies directly inhibit MDSC-mediated immunosuppression, as evidenced by enhanced tumor cell killing activity of cytotoxic T cells. Lastly, we show that anti-Bv8-treated MDSCs secrete proteins associated with effector immune cell function and T cell activity. Overall, we demonstrate that Bv8 blockade inhibits the immunosuppressive function of MDSCs, thereby enhancing anti-tumor activity of cytotoxic T cells and sensitizing anti-PD1 resistant tumors. Our findings suggest that combining Bv8 blockade with anti-PD1 therapy can be used as a strategy for overcoming therapy resistance.

Bv8-Like Toxin from the Frog Venom of Amolops jingdongensis Promotes Wound Healing via the Interleukin-1 Signaling Pathway.

Prokineticins are highly conserved small peptides family expressed in all vertebrates, which contain a wide spectrum of functions. In this study, a prokineticin homolog (Bv8-AJ) isolated from the venom of frog Amolops jingdongensis was fully characterized. Bv8-AJ accelerated full-thickness wounds healing of mice model by promoting the initiation and the termination of inflammatory phase. Moreover, Bv8-AJ exerted strong proliferative effect on fibroblasts and keratinocytes isolated from newborn mice by activating interleukin (IL)-1 production. Our findings indicate that Bv8 is a potent wound healing regulator and may reveal the mechanism of rapid wound-healing in amphibian skins.

Targeted Next-Generation Sequencing Identification of Mutations in Disease Resistance Gene Analogs (RGAs) in Wild and Cultivated Beets.

Resistance gene analogs (RGAs) were searched bioinformatically in the sugar beet (Beta vulgaris L.) genome as potential candidates for improving resistance against different diseases. In the present study, Ion Torrent sequencing technology was used to identify mutations in 21 RGAs. The DNA samples of ninety-six individuals from six sea beets (Beta vulgaris L. subsp. maritima) and six sugar beet pollinators (eight individuals each) were used for the discovery of single-nucleotide polymorphisms (SNPs). Target amplicons of about 200 bp in length were designed with the Ion AmpliSeq Designer system in order to cover the DNA sequences of the RGAs. The number of SNPs ranged from 0 in four individuals to 278 in the pollinator R740 (which is resistant to rhizomania infection). Among different groups of beets, cytoplasmic male sterile lines had the highest number of SNPs (132) whereas the lowest number of SNPs belonged to O-types (95). The principal coordinates analysis (PCoA) showed that the polymorphisms inside the gene Bv8_184910_pkon (including the CCCTCC sequence) can effectively differentiate wild from cultivated beets, pointing at a possible mutation associated to rhizomania resistance that originated directly from cultivated beets. This is unlike other resistance sources that are introgressed from wild beets. This gene belongs to the receptor-like kinase (RLK) class of RGAs, and is associated to a hypothetical protein. In conclusion, this first report of using Ion Torrent sequencing technology in beet germplasm suggests that the identified sequence CCCTCC can be used in marker-assisted programs to differentiate wild from domestic beets and to identify other unknown disease resistance genes in beet.

Modulators of pain: Bv8 and prokineticins.

Bv8 is a small protein secreted by frog skin. Mammalian homologues of Bv8, the prokineticins PK1 and PK2, and their G-protein coupled receptors PKR1 and PKR2 have been identified and linked to several biological effects. Bv8 elicits a dose-dependent reduction in nociceptive threshold to thermal and mechanical stimuli applied to the skin of tail and paw of rats and mice and increases the sensitivity to nociceptive mediators as capsaicin and prostaglandins. The receptors for Bv8/PKs are present in a fraction of peptidergic population of C-fibre neurons, and in a fraction of A myelinated-fibre neurons. In mouse and rat dorsal root ganglia, PKR-expressing neurons also express TRPV1 and the activation of PKRs sensitises TPRV1 to the action of capsaicin. Mice lacking PKR1 gene exhibit impaired Bv8-induced hyperalgesia, develop deficient responses to noxious heat, capsaicin and protons and show reduced thermal and mechanical hypersensitivity to paw inflammation, indicating a requirement for PKR1 signalling associated with activation and sensitisation of primary afferent fibres. PKs are highly expressed by neutrophils and other inflammatory cells and must be considered as new pronociceptive mediators in inflammatory tissues. Bv8-like hyperalgesic activity was demonstrated in extracts of rat inflammatory granulocytes. Bv8 stimulate macrophage and T lymphocyte to differentiate between an inflammatory and Th1 profile indicating that Bv8/PK proteins play a role in immuno-inflammatory responses. Blockade of PKRs may represent a novel therapeutic strategy in acute and inflammatory pain conditions.

Involvement of Prokineticin 2 and Prokineticin Receptor 1 in Lipopolysaccharide-Induced Testitis in Rats.

Prokineticin 2, a newly discovered proinflammatory peptide, has been amply evidenced to be involved in the occurrence and progress of local and systematical inflammation. Although the presence of Prokineticn 2 in mammal testis has been documented clearly, research targeting the involvement of prokineticin 2 in testicular pathology, especially testitis, is rather scarce. Employing a lipopolysaccharide-induced testitis rat model, we for the first time demonstrated the expression and upregulation of prokineticin 2 in orchitis at several levels. Our effort also addressed the differential expression patterns of prokineticin 2 and interleukin-1ß, a key inflammation indicator, during testitis suggesting Prokineticn 2 serves more than a proinflammatory factor in the context of testitis. Given one of the cognate receptors of prokineticin 2, prokineticin receptor 1 (PKR1) was also significantly upregulated in orchitis as discussed in the current study, it is very likely that PK2/PKR1 signaling contribute to the development of inflammation-related testicular diseases.

Prokineticins are neuroprotective in models of cerebral ischemia and ischemic tolerance in vitro.

Bv8/prokineticin 2 (PK2) is a member of a bioactive family of peptides that regulate multiple functions in the CNS including hyperalgesia, neurogenesis, neuronal survival and inflammation. Recent studies have associated PK2 and prokineticin receptors (PKR) with human diseases, but because their role in neuropathology is still debated we examined whether prokineticins exert a protective or deleterious role in models of cerebral ischemia and ischemic tolerance in vitro. In order to mimic cerebral ischemia, we exposed primary murine cortical cell cultures or rat organotypic hippocampal slices to appropriate periods of oxygen-glucose deprivation (OGD), which leads to neuronal damage 24 h later. Ischemic tolerance was induced by exposing hippocampal slices to a preconditioning subtoxic pharmacological stimulus (3 µM NMDA for 1 h) 24 h before the exposure to OGD. Bv8 (10-100 nM) attenuated OGD injury in cortical cultures and hippocampal slices, and the effect was prevented by the PKR antagonist PC7. The development of OGD tolerance was associated with an increase in the expression of PK2, PKR1 and PKR2 mRNA and proteins and was prevented by addition of the antagonist PC7 into the medium during preconditioning. Both Bv8 at protective concentrations and the NMDA preconditioning stimulus promoted the phosphorylation of ERK1/2 and Akt. These findings indicate that the prokineticin system can be up-regulated by a defensive preconditioning subtoxic NMDA stimulus and that PK2 may act as an endogenous neuroprotective factor through the activation of the ERK1/2 and Akt transduction pathways.

Location, location, location: functional and phenotypic heterogeneity between tumor-infiltrating and non-infiltrating myeloid-derived suppressor cells.

An increasing number of studies is focusing on the role of myeloid-derived suppressor cells (MDSCs) in the suppression of antitumor immune responses. Although the main site of action for MDSCs is most likely the tumor microenvironment, the study of these cells has been largely restricted to MDSCs derived from peripheral lymphoid organs. Only in a minority of studies MDSCs isolated from the tumor microenvironment have been characterized. This review will give an overview of the data available on the phenotypical and functional differences between tumor-derived MDSCs and MDSCs isolated from the spleen of tumor-bearing mice or from the peripheral blood of cancer patients.

Role of BV8 and its antibody in angiogenesis / 上海交通大学学报（医学版）

BV8, a lately defined secreted protein, is proved to play an important role in the angiogenesis process of endocrine, cardiovascular system, kidney and other organs. Moreover, it contributes to the CD11b+Gr1+ myeloid cell-dependent tumor neo-vascularization. Meanwhile, anti-BV8 antibody therapy has been shown to be against solid tumors via inhibition of angiogenesis in animal models. This review systematically introduced the current knowledge on BV8 from structure to function, from mechanism to its role in diseases. Consequently, this review will probably provide new ideas and direction to the new target of the current anti-vascular therapy for cancer and to the future investigation.

Role of BV8 and its antibody in angiogenesis / 上海交通大学学报（医学版）

BV8,a lately defined secreted protein,is proved to play an important role in the angiogenesis process of endocrine,cardiovascular system,kidney and other organs.Moreover,it contributes to the CD1 lb+Grl+ myeloid cell-dependent tumor neo-vascularization.Meanwhile,anti-BV8 antibody therapy has been shown to be against solid tumors via inhibition of angiogenesis in animal models.This review systematically introduced the current knowledge on BV8 from structure to function,from mechanism to its role in diseases.Consequently,this review will probably provide new ideas and direction to the new target of the current anti-vascular therapy for cancer and to the future investigation.