A Developed NK-92MI Cell Line with Siglec-7neg Phenotype Exhibits High and Sustainable Cytotoxicity against Leukemia Cells

Abstract

Altered sialic acid processing that leads to upregulation of cell surface sialylation is recognized as a key change in malignant tissue glycosylation. This cancer-associated hypersialylation directly impacts the signaling interactions between tumor cells and their surrounding microenvironment, especially the interactions mediated by immune cell surface sialic acid-binding immunoglobulin-like lectins (Siglecs) to relay inhibitory signals for cytotoxicity. First, we obtained a Siglec-7neg NK-92MI cell line, NK-92MI-S7N, by separating a group of Siglec-7neg cell population from an eight-month-long-term NK-92MI in vitro culture by fluorescence-activated cell sorting (FACS). The effect of Siglec-7 loss on NK-92MI-S7N cells was characterized by the cell morphology, proliferation, and cytotoxic activity via FACS, MTS assay, cytotoxic assay, and natural killer (NK) degranulation assay. We found the expression levels of Siglec-7 in NK-92MI were negatively correlated with NK cytotoxicity against leukemia cells. This NK-92MI-S7N cell not only shared very similar phenotypes with its parental cells but also possessed a high and sustainable killing activity. Furthermore, this Siglec-7neg NK line was unexpectedly capable of eliminating a NK-92MI-resistant leukemia cell, THP-1, through enhancing the effector-target interaction. In this study, a NK cell line with high and sustainable cytotoxicity was established and this cell may provide a potential application in NK-based treatment for leukemia patients.

DNA methylation‐mediated Siglec‐7 regulation in natural killer cells via two 5′ promoter CpG sites

“…Besides DNMT inhibitor, treatment with HDAC inhibitor, including suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA), also suppresses NK cytotoxicity 49 . In this study, we showed the surface Siglec‐7 of peripheral NK cells can be upregulated by epigenetic therapeutic drugs, and whether this change in Siglec‐7 expression may potentially impact NK cytotoxic functions remains to be investigated, as our cell model showed NK cytotoxicity could be greatly affected in the presence of a high level of Siglec‐7 expression 35 …”

“…Briefly, we found that NK‐92MI cells lost significant cytotoxicity capability after long‐term in vitro culture, and the resulting cell population exhibited a dominant Siglec‐7 + expressing phenotype (designated as NK‐92MI‐S). More interestingly, from this NK‐92MI‐S cell population, we were able to isolate a Siglec‐7 − sub‐population (NK‐92MI‐S7N), which possessed high cytotoxicity to kill a leukemia cell line that even parental NK‐92MI cells cannot 35 . Moreover, our result suggested that the expression level of cell surface Siglec‐7 is regulated at the transcription step as the cell surface expression was positively correlated with the transcript level in these three cell lines, in which NK‐92MI‐S possessed the highest Siglec‐7 expression, followed by parental NK‐92MI, and then NK‐92MI‐S7N 35 …”

“…1b). 35 We addressed this goal using two approaches: focusing on screening transactivators and examining epigenetics. With RNA‐seq results and putative binding site data, we narrowed down FoxO1 as a candidate, but later data suggested the change of FoxO1 in expression was unlikely to function as a key Siglec‐7 regulator for expression because there was no correlation in the expression levels between Fox01 and Siglec‐7 (Fig.…”

“…Human NK cell line, NK‐92MI, was purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan), and the NK‐92MI‐S and ‐S7N cells were established in our laboratory as described previously 35 . NK cells were grown in α‐MEM medium, supplemented with 12·5% fetal bovine serum (FBS), 12·5% horse serum, 2 m m l ‐glutamine, 2 m m inositol, 0·1 m m 2‐mercaptoethanol, 0·02 m m folic acid, 50 units/ml penicillin and 50 μg/ml streptomycin in a humidified incubator with 5% CO 2 at 37°.…”

“…Currently, the regulatory mechanism for Siglec‐7 is still unclear in detail. By our previous study, we found that cell surface Siglec‐7 amount was positively correlated to the transcript level, suggesting the main Siglec‐7 regulatory step is via transcription 35 . In this study, we aimed to characterize the regulatory mechanism for Siglec‐7 activation in NK cells by investigating the involvement of transcription factor(s), DNA methylation and histone modification.…”