Endoplasmic Reticulum Stress In Prostate Cancer Cells Promotes Macrophage M2 Polarization Via STAT3 Signaling Pathway

Objectives:A prostate cancer ERS model was established,and exosomes released from ERS and non-ERS prostate cancer cells were co-cultured with macrophages to determine whether macrophages undergo M2 polarization by determining changes in the expression of inflammatory factors in macrophages,and to further explore the mechanisms through which exosomes regulate the immune phenotype of macrophages.Methods:（1）The prostate cancer cells were stimulated with different concentrations of thapsigargin for 24 hours and with the same concentration of thapsigargin for different times,and the changes in GRP78 protein expression were detected using Western Blot to determine the optimal stimulation concentration and optimal stimulation time.（2）Prostate cancer cells were stimulated with 3 μM thapsigargin for 24 h.Cells and supernatants were collected from the thapsigargin stimulation groupand normal culture group,and the supernatants were used to extract exosomes using the Exo Quick-TC kit.Exosomes were observed using transmission electron microscopy（TEM）,proteins of the cultured cells were extracted,and the expression of exosomal marker proteins TSG101,CD63 and Calnexin protein were detected by Western Blot.（3）Exosomes were labeled with PKH67 membrane fluorescent dye,and then the labeled exosomes were co-cultured with macrophages for 12 hours and placed under a laser confocal microscope to observe the phagocytosis of exosomes by macrophages.（4）Exo-TG and Exo-Con were stimulated in macrophages with different concentrations of exosomes for 24 hours and different times using the same concentration of exosomes,and the optimal stimulation concentration and optimal stimulation time were determined using Western Blot to detect changes in PD-L1 protein expression.（5）Exo-TG and Exo-Con were co-cultured with PMA-induced macrophages at a concentration of 30 μg/ml for 24 h.The supernatants and cells were collected separately,and the changes in the expression of macrophage inflammatory factors IL-1β,IL-6,IL-10 and TNF-α were detected using chemiluminescence assay.（6）Exo-TG and Exo-Con were co-cultured with macrophages after PMA induction at a concentration of 30 μg/ml for 24 h.Cells were collected and proteins were extracted,and the expression of macrophage phosphorylated proteins p-STAT3 and STAT3 was detected by Western Blot.Results:（1）TG could increase GRP78 protein expression in prostate cancer cells concentration-dependently and time-dependently,but the expression of endoplasmic reticulum stress protein GRP78 reached its maximum when TG concentration reached 3μM,while GRP78 protein expression was not significantly elevated at 12 versus 24 hours of stimulation（P=0.051）.（2）Transmission electron microscopy revealed that the exosomes were 30-100 nm in diameter,relatively homogeneous in size,round or circular bilayer structured vesicles,in accordance with the relevant reports.Western Blot detected that the exosomes expressed exosomal marker proteins CD63 and TSG101,but not Calnexin,and compared with the Exo-Con group,Exo-TG carried more of CD63 and TSG101 compared with the Exo-Con group.（3）Laser confocal microscopy showed that exosomes labeled with PKH67 were able to be phagocytosed by macrophages.（4）Exosomes can increase PD-L1 protein expression in macrophages in a concentration-dependent and time-dependent manner,and the maximum PD-L1 protein expression was reached when the stimulation concentration reached 30 μg/ml and the stimulation time was 24 hours.（5）Chemiluminescence results showed that macrophages co-cultured with exosomes secreted large amounts of IL-1β,IL-10 and TNF-α cellular inflammatory factors,while the expression of IL-6 did not change significantly.（6）Western Blot results showed that macrophages co-cultured with exosomes had increased p-STAT3 protein expression,while STAT3 protein expression was not significantly increased and the p-STAT3/STAT3 ratio was significantly increased.Conclusion:（1）Thapsigargin can induce endoplasmic reticulum stress in prostate cancer cells in a concentration-dependent and time-dependent manner.（2）Exosomes of prostate cancer cell origin are capable of being taken up by macrophages.（3）ERS prostate cancer exosomes were able to induce a change in the M2 phenotype of macrophages,secreting large amounts of inflammatory factors IL1β,IL-10 and TNF-α,as well as being able to increase the expression of PD-L1 protein in macrophages.（4）Exosomes from prostate cancer cells in ERS may cause M2-type polarization in macrophages via the STAT3 signaling pathway.