HC-NPs "Re-educate" TAMs And Exhibit Anti-tumor Effect Via STAT3Inhibition

Objective To investigate the effect of hydrazinocurcuminnanoparticles （HC-NPs）“re-education” of tumor associated macrophages（TAMs） and anti-tumor both in vitro and in vivo，and to prove whether itsmechanisms is related to inhibiting the STAT3signal pathway.Methods We firstly co-cultured mouse monocyte/macrophage cell lineRAW264.7（M1phenotype） with mouse breast cancer cell line4T1toobserve whether the macrophages could be “educated” to M2phenotype ofTAMs．Cell surface antigens and intra-cellular cytokines expression ofRAW264.7cells were detected with flow cytometry（FCM）．p-STAT3，MMP9，MMP2，VEGFexpressions were tested by western blot．Then wedivided E-RAW264.7cells into PBS control group，NPs control group andHC-NPs treatment group．Cell cytomorphology were observed by Liustaining, and cell proliferation was demonstrated by MTT assay．STAT3and its downstream proteins expression were tested by western blot, andthe cell surface antigens and intra-cellular cytokines expression ofRAW264.7cells were detected with FCM． The effect of HC-NPs“re-educate” E-RAW264.7cells were observed，and it showed that HC-NPs could reverse phenotype of E-RAW264.7cells from M2to M1．We co-cultured “re-educate” RAW264.7with4T1，divided them intoPBS control group，E-RAW264.7control group，E-RAW264.7cellspre-treated by NPs group （NPs-E-RAW264.7） and E-RAW264.7cellspre-treated by HC-NPs group （HC-NPs-E-RAW264.7）．Counted mortalityof4T1cells through Trypan Blue staining, and FCM was performed todetermine the cell cycle and cells apoptosis. The migration and invasion wasmeasured by Transwell assay; expression of STAT3protein and itsdownstream proteins related to proliferation，apoptosis and invasion weremeasured by western blot．Co-transplanted4T1tumor cells and E-RAW264.7cells into fat padwhich near to the right hind of6-8-week-old female mice.Then gave5IVinjection of drug at3days intervals during day10to day25aftertransplantation，and mice were divided into PBS control group，NPs controlgroup，HC-NPs treatment group and HC treatment group．Measured volumeof tumor after transplantation continuously until mice sacrifice．After micesacrifice on day30，weigh the weight of tumor，detected proliferation（Ki-67positive cells）and angiogenesis（CD31positive micrangium）byimmunohistochemistry （IHC）; tumor cell apoptosis was measured byTUNEL staining，counted numbers of pulmonary metastasis of tumor cellsafter HEstaining．Observe survivaltime of mice untilday60，detected thesurvival rate． Results FCM demonstrated RAW264.7cells performed M2phenotype(IL-10^high，IL-12^low，TGF-β^high) after co-cultured with4T1cells，western blotshowed p-STAT3， MMP2， MMP9and VEGF expressions alsoincreased．After treated with HC-NPs，E-RAW264.7cells indicated themorphology change by Liu staining, and FCM demonstrated the phenotypeof E-RAW264.7cells had been reversed(IL-10^low，TGF-β^low，IL-12^high)，along with decreased expressions of p-STAT3protein and its downstreamMMP2，MMP9and VEGF proteins．Besides, E-RAW264.7cells pre-treatedby HC-NPs （HC-NPs-E-R AW264.7）didn’t affect the motality andapoptosis of4T1cells，and however, they resulted in decreased numbersin the S phase of4T1cells compared with control groups，and inhibited theabilities of migrationand invasionof4T1cells．Some of proteins expressionrelated to the biological functions also were decreasd．In vivo，tumor growth and tumor weight in HC-NPs treatment groupwere decreased compared with control groups．Survival curve indicatedHC-NPs group（80%） prolonged survival time of mice compared with HCgroup（40%）．IHC demonstrated Ki-67positive tumor cells and CD31positive micrangium in HC-NPs treatment group decreased remarkably.TUNEL staining showed more tumor cells apoptosis in HC-NPs treatmentgroup compared with control groups．HC-NPs also dramaticly reducedpulmonary metastasis by counting tumor cells numbers on lung tissue slice.Conclusion Inhibition of STAT3signal pathway in TAMs by HC-NPs was able to reverse the phenotype of macrophages from M2toM1，and macrophages demonstrated effect of inhibiting4T1tumor cellsproliferation，migration，invasion and angiogenesis after co-cultured with4T1mouse breast cancer cells in vitro．Moreover， HC-NPs exhibitedremarkable anti-tumor effect，prolonged mice survival time and reduceddrug toxicity compared with HC in vivo．These findings confirmed thatHC-NPs was more capable for clinical application．...