ATRA Treatment Enhanced The Adhesion Behaviors Of Flowing HL60 Cells In A Mechano-chemical Dependent Manner

All-trans retinoic acid(ATRA)can differentiate acute promyelocytic leukemia(APL)cells towards granulocytes.At the same time,using ATRA can also trigger a severe complication-differentiation syndrome(DS).This study aims to deeply understand and reveal the still unclear scientific problem of the occurrence mechanism of APL DS and its mechanical-chemical regulation mechanism.Firstly,we used ATRA to differentiate HL60 cells for 0-120 h.By detecting the cell growth rate,observing the change of nuclear morphology,counting the nuclear-cytoplasmic ratio,detecting the cell differentiation rate,etc.,we wanted to examine whether the cells occurred differentiation.Next,we used flow cytometry to investigate the expression levels of three main adhesion molecules of HL60 cells involved in the adhesion cascade,PSGL-1(CD162),LFA-1(CD11a/CD18,αLβ2),and Mac-1(CD11b/CD18,αMβ2),then compared them with neutrophils extracted from the healthy volunteers.Then,using the parallel plate flow chamber,the adhesion behaviors of HL60 cells under flow conditions before and after ATRA differentiation were observed and compared with neutrophils.Here we mainly focused on the adhesion behaviors of fast rolling,slow rolling,firm adhesion,and crawling.Finally,we constructed a cytoskeletal tension sensor based on ICAM-1 to investigate the changes in cytoskeletal tension signal exerted on ICAM-1 by HL60 cells before and after differentiation and compared them with neutrophils.We found that ATRA treatment indeed induced the differentiation of HL60 cells toward granulocytes,which showed that the proliferation rate of HL60 cells decreased after ATRA differentiation,and this decrease was ATRA concentration and ATRA treating time-dependent;ATRA differentiation induced the nuclei of HL60 cells towards polymorphic nuclei;The nucleocytoplasmic ratio of HL60 cells decreased toward neutrophils after differentiation;the differentiation rate of HL60 cells increased with the increase of ATRA incubation time and concentration.The flow cytometry results showed that with the increase of ATRA differentiation time,the expression of CD162,CD11a,and CD11b on HL60 cells did differentiate toward neutrophils.However,there were still some differences,and the expression of these three molecules was regulated by the treatment time and by the concentration of ATRA.The parallel plate flow chamber experiment results showed that the adhesion behavior of ATRA-differentiated HL60 cells was indeed enhanced.In the fast rolling mediated by P-selectin,the proportion of firm adhesion was higher than that of the neutrophil group.In contrast,the proportion of rolling cells was close to that of the neutrophil group,and the slow rolling was mediated by the P-selectin/ICAM-1.Further analysis of the cell rolling velocity showed that the rolling velocity of ATRA-120 h-HL60 cells was lower than that of neutrophils under shear stress from 0.1 to 0.5 dyn/cm~2.Further analysis of the firm adhesion cells’stay time showed that the stay time of ATRA-120 h-HL60 cells was longer than that of neutrophils under shear stress conditions from 0.1 to 0.5 dyn/cm~2.The results of the crawling experiment showed that the crawling velocity and crawling Euclidean distance(ED)of ATRA-120 h-HL60 cells was lower than that of neutrophils under resting conditions under shear stress conditions of 1dyn/cm~2.In addition,ATRA-120 h-HL60 cells had a higher proportion of up-flow crawling cells than neutrophils under 1 dyn/cm~2 shear stress.Then ICAM-1-based cytoskeleton tension sensor was successfully constructed and used to detect the force change of HL60 cytoskeleton tension force acting on ICAM-1 before and after ATRA differentiation.At the same time,compared with neutrophils,we found that ATRA differentiation leads to the change of tension signals of ICAM-1 on HL60 cells,from a scattered and sparse tension force signal of pre-differentiation to an aggregated and dense tension force signal of post-differentiation;Both of them are different from the scattered and dynamic ICAM-1 tension force signal of neutrophils.The occurrence of APL DS is likely due to the enhanced adhesion behavior of APL cells under the flow field in fast-rolling,slow-rolling,and firm adhesion after ATRA differentiation.The enhanced cytoskeletal tension signal of the HL60 cytoskeleton acting on its ligand molecule ICAM-1 after ATRA differentiation may also be one of the reasons for APL DS.The changes in the adhesion behavior of APL cells after ATRA differentiation may inspire the clinical treatment of APL DS.In addition,the differences in the adhesion behavior of cells after ATRA differentiation may provide new thinking for the clinical identification of the occurrence of APL DS.