Therapeutic Effect Of Umbilical Cord Blood NK Cells Expanded By K562 Membrane Expressing IL-21 On Colorectal Cancer

Background and Aim:Colorectal cancer is among cancers with highest incidence globally and currently ranks second as the leading cause of cancer-related deaths worldwide.It remains an urgent need for novel strategies in the management of patients with advanced colorectal cancer.Adoptive transfer of allogeneic natural killer(NK)cells represent an attractive option in the treatment of patients with colorectal cancer.NK cell adoptive immunotherapy represents an important biological therapy for cancer.It kills cancer cells directly or indirectly in vivo to achieve the purpose of cancer treatment.How to obtain enough NK cells and how to maintain the activity of NK cells in vivo is a difficult problem in NK immunotherapy.Umbilical cord blood contains a high proportion of NK cells and is not susceptible to graft-versus-host disease.It is considered to be an ideal source of NK cells.Interleukin-21 is an important cytokine regulating the proliferation and function of NK cells.It promotes the proliferation of NK cells in vitro and up-regulates the activity of NK cells together with interleukin-2.In the present study,we explored the potential of a genetically engineered K562 cell line with membrane bound expression of interleukin-21(IL-21)for ex vivo generation of mature and functional NK cells from UCB.We next assessed the in vivo and in vitro efficacy of the expanded UCB-derived NK(eUCB-NK)cells against colorectal cancer cells and further determined whether combination with bevacizumab can synergize with this NK cell-based practice to better suppress colorectal cancer.Methods:The recombinant plasmid expressing IL-21 was constructed and lentivirus was packaged in 293 T cells.IL-21 was transfected into K562 cells by lentivirus,limited dilution method was then performed to subclone positive clones,designated "K562-mb21" cells.Cell surface expression of IL-21 on the genetically modified K562-mbIL21 cells was detected by flow cytometry.Monocytes nuclear cells(MNCs)were isolated from human umbilical cord blood(UCB)and co-cultured with K562-mb21 cells to expand NK cells,termed eUCB-NK cells.The purity of NK cells was measured using flow cytometry method.The expression of NK cells markers were determined before and after 13 days of cell expansion.The in vitro cytotoxic activities of eUCBNK cells were evaluated against various colorectal cancer cell lines.The expression of CD107 a and the secretion of several immunoactive cytokines were detected.In vivo,the HT29 and LoVo colorectal cancer xenografts were established and the anti-tumor activities of eUCB-NK cells were evaluated.We examined the tumors for the presence of NK cell infiltration with flow cytometry.Total RNA from HT29 and LoVo tumor specimens was extracted and quantitative PCR was performed to examine whether the antiangiogenic signals were associated with the level of NK cell infiltration.The angiogenesis inhibitor(bevacizumab)was added to LoVo tumor to evaluate the combination therapeutic effect Results:We found it feasible to efficiently generate a high dose of NK cells from UCBMNCs with stimulation from lethally irradiated K562-mb21 cells.NK cells could be selectively expanded to greater than 500-fold within 14 days of culture with purity approaching to 90%,whereas their counterpart T cells remained basically unchanged varying with time.Compared with IL-2 stimulated NK cells,the expression of almost all activating receptors were upregulated on eUCB-NK cells,among which NKG2 D and TRAIL that are strongly associated with NK cell activity had the greatest upregulation,indicating a highly functional active phenotype of these generated NK cells.Compared to the IL-2 stimulated NK cells,the eUCB-NK cells are highly toxic against diverse colorectal cancer targets as measured by the degranulation and cytotoxicity assay.Additionally,the eUCB-NK cells secreted higher levels of IFN-?,TNF-?,GM-CSF and CCL3 production.Significant anti-tumor activities of eUCB-NK cells were observed in HT29 xenografts but not in LoVo tumors.Flow cytometric results revealed that HT29 tumor xenografts,but not LoVo tumor xenografts have appreciable numbers of detectable NK cells in the sections of tumors.Contrary with HT29,a high level of tumor neoangiogenesis and insufficient NK cell infiltration was observed in LoVo tumors,indicating that insufficient NK cell infiltration in LoVo tumor xenografts might result from a high level of tumor neoangiogenesis.We applied bevacizumab in this study,finding that LoVo tumor growth was dramatically inhibited by eUCB-NK cells in combination with bevacizumab,while the eUCB-NK cells or bevacizumab alone has no such superior effect on tumor growth.Conclusions:(1)In this study,we successfully expanded NK cells from umbilical cord blood(UCB)with membrane-bound IL-21,termed eUCB-NK cells.eUCB-NK cells efficiently lysed colorectal cancer cell lines in vitro and secreted significantly higher levels of cytokines compared with IL-2 stimulated NK cells.(2)Adoptive transfer of these NK cells significantly inhibited the growth of some colorectal cancer xenografts,and the therapeutic effect was relevant to NK cell infiltration.Combination of bevacizumab can increase extravasation of adoptively transferred cells into the LoVo tumors and improve the therapeutic activity of eUCBNK cells.These results justified clinical translation of this UCB-derived NK cell-based therapeutics,either used alone or combined with bevacizumab,as a novel treatment option for patients with colorectal cancer.