Umbilical Cord Blood Derived Neutrophils Inhibit Proliferation Of Ovarian Cancer

Ovarian cancer is the most lethal type of gynecologic tumors,causing 140,000 death per year worldwild.The 5-year survival rate of patients diagnosed as stages III or IV is less than 25%.Only 15%of patients survived more than 10 years.Cytoreductive surgery and chemotherapy are the first-line treatment of ovarian cancer.Although aggressive advance has been made in surgery and chemotherapy,current therapeutic options are insufficient to improve long-term survival.Novel therapeutic approaches are needed to improve the prognosis of ovarian cancer patients.Immunotherapy including adoptive cell transfers,tumor vaccines,monoclonal antibodies has emerged as a therapeutic option with huge curative potential.Consolidation approaches such as immune checkpoint inhibitor PD-1,CTLA-4,adoptive T-cell therapy have given a high rate of upfront responses.These agents have been pursued in ovarian cancer but need more optimizations because of poorly response.Neutrophils,a substantial component of innate immune systems,have been reported to be involved in tumor suppression.Neutrophils induced tumor cell death in the presence of Ig G1 antitumor antibodies,which was referred to as antibody-dependent cellular cytotoxicity (ADCC).Neutrophil cytotoxicity could be further enhanced by Ig A antibodies or bispecific antibodies combining the agonistic activity of FcαRI targeting with Ig G1characteristics.Moreover,Neutrophils may lead to in situ generation of long-term antitumor immunity by releasing cytokines and chemokines,and/or via cross-talking with other immune cells such as dendritic cells(DCs).Infiltration of Neutrophils may also be used as a prognostic factor in tumors including ovarian cancer.These findings suggested that Neutrophils may be ideally poised to perform a comparable function in cancer therapy.The limited number and unstable status of autologous peripheral blood immune cells from tumor patients make it difficult to make off-the-shelf products for future treatment.To address these limitations,sources for immune cells from umbilical cord blood(UCB)have been tested for off-the-shelf availability.In the present study,we started with transcriptomic profiling of fresh isolation UCBN and peripheral blood-derived neutrophils(PBN)by RNA-sequencing.The transcriptomic analysis showed that the top upregulated genes in UCBN compared with PBN mainly related to neutrophils degranulation,neutrophils activation,cell killing,nuclear division,nuclear chromosome segregation and myeloid cell development.Accordingly,we found that UCBN showed high viability and enhanced tumor cell killing activity upon activation.Further transcriptomic combine proteomics analysis and experimental verifications screened the molecule to anti-tumor CCL3.Then,we verified that CCL3 may stimulated JAK2/STAT3 pathway to killing tumor cells.Part I:Cytokine activated umbilical cord blood-derived neutrophils inhibit the progression of ovarian cancer in vitroUmbilical cord blood(UCB)was collected from cesarean sections in lithium-heparin tubes(Greiner Bio-One 9m L LH,Austria)at Shanghai Tenth People’s Hospital after Informed consent was obtained from puerperas.Peripheral Blood(PB)collected in lithium-heparin collection tubes were obtained from adult volunteers.Density-gradient centrifugation was used to isolate Neutrophils.The identities and purity of neutrophils freshly isolated from human umbilical cord blood and peripheral blood were confirmed by flow cytometry analysis of CD11b and CD66b expression.CD11b~+cells and CD66b~+cells were over 90%in freshly isolated neutrophils from UCB and PBN.RNA-seq analysis was then performed.Differentially expressed genes(DEGs)and Gene ontology(GO)enrichment analyses showed that BPs including neutrophil degranulation,neutrophil activation,nuclear division,myeloid cell development;killing by a host of symbiont cells,killing of cells of other organisms were enriched in up-regulated DEG in UCBN.IFN-β,IFN-r,and LPS To evaluate the viability of UCBN and PBN,we assessed the expression of the activation markers CD11b(integrin alpha M)and CD66b (CEACAM8)by flow cytometry analysis.Consistent with our earlier observation,higher percentages of cells expressing CD11b and CD66b were observed with activated UCBN than with activated PBN at both time points.Next,we hoped to examine whether and how cytokine cocktail treatment may enhance the cytotoxicity of neutrophils.We subjected the ovarian cancer cells line OVCAR3 to treatment with activated UCBN,PBN,or their conditioned medium(CM)respectively and evaluated the viability of the cancer cells by CCK-8 assay.For UCBN,all three activated one manifested increased cytotoxicity when compared with non-activated states,but only two had enhanced killing activity when compared with cytokine cocktail treatment alone.For PBs,only a marginal increase in cytotoxicity was observed with activation but none of them had an enhanced killing effect when compared with cytokine cocktail treatment alone.More strikingly,CM from all the five activated UCBN had increased cytotoxicity when compared with non-activated ones and four out five had enhanced killing effects when compared with cytokine cocktail treatment alone,lending strong support to the earlier results and suggesting cytokine cocktail activation enhanced the ovarian cancer cell killing activity of UCBN.In contrast,again only marginal effects were seen for CM from PBN with activation when compared with those without activation and only two out of five had enhanced killing activity when compared with cocktail treatment alone.To further confirm the results above,we performed Ed U incorporation and Annexin V-PI staining assays.ovarian cancer cell proliferation was strongly inhibited by CM from activated UCBN.Taken together,our results strongly suggest that cytokine activated UCBN exerted strong proliferation-inhibitory and cytotoxic effects towards ovarian cancer cells likely through a secretory mechanism.Part II:Molecular mechanisms underlying the cytotoxic effect of activated UCBN on ovarian cancer cellsTo probe the mechanism underlying the anti-tumor function of activated UCBN,we performed the transcriptomic analysis with UCBN with and without cytokine cocktail activation.GO enrichment analysis of DEGs revealed that the top upregulated genes in activated UCBN were mainly enriched in immunity-related BPs such as response to interferon-gamma,positive regulation of cytokine production,response to lipopolysaccharide,cytokine secretion,peptide secretion,activation of the innate immune response,and cell killing.KEGG pathway analysis again confirmed that the top upregulated genes in activated UCBN mainly related to immunity-associated pathways such as NOD-like receptor signaling pathway,TNF signaling pathway,Cytokine-cytokine receptor interaction,NF-kappa B signaling pathway,Chemokine signaling pathway,RIG-I-like receptor signaling pathway,JAK-STAT signaling pathway,Proteasome MAPK signaling pathway,p53 signaling pathway,Notch signaling pathway.Then,we performed transcriptomic comparisons between activated UCBN and activated PBN since we have consistently seen an enhanced anti-tumor activity with UCBN when compared with PBN.Consistent with the phenotypic observations,GO term analysis showed that the top up-regulated DEGs in activated UCBN were enriched in neutrophil activation related BPs including neutrophil degranulation,neutrophil activation involved in immune response and cytokine biosynthetic process.To further probe the underlying anti-tumor molecule,we collected unactivated UCBN-CM(umbilical cord blood conditioned medium)and activated UCBN-CM to performed proteomics analysis.The results shown that GO enrichment analysis of BP revealed that the top upregulated peptides in activated UCBN were mainly enriched in immunity-related BPs such as---,which consistent with transcriptomic analysis.Nine molecules were screened after combined transcriptomic analysis with proteomics analysis:PI3,SERPING1,CXCL8,CSF1,ATRN,IL6,CCL3,CXCL9.Proteome profiler array were performed to further probe the underlying anti-tumor molecule.Protein assay analysis showed that cytokines such as I-TAC(CXCL11),MIP-3beta(CXCL19)were up-regulated in activated UCBN-CM,which were up-regulated in activated PBN-CM.And 8 molecules have no changes in activated PBN-CM,7 molecules were down regulated in activated PBN-CM,which were both up-regulated in activated UCBN-CM.These cytokines may be specificly involved in the inhibitory effect of activated UCBN.Finally,after unite these three different levels analysis,CCL3 may be specificly involved in the inhibitory effectAs CCL3 can inhibit OVCAR3 proliferation,then we further to prove the mechanism by RNA-seq.We found that CCL3 may activated JAK/STAT3 pathway to inhibit cancer cells proliferation.