Major surgery-induced expansion of myeloid-derived suppressor cells promotes breast cancer cell bone metastasis though PD-1
Authors: Dong Linlin , Chen Hong , Jiang Fen , Zheng Xiaoyu 
Published: February 2021

Abstract
Background

Bones metastasis is a serious clinical complication and increases morbidity and mortality. Surgery remains a main therapy of most solid tumors, but itself may accelerate tumor metastasis. Myeloid-derived suppressor cells (MDSCs) have been reported to be contribute to tumor growth. Little is known about the presence and function of surgery-induced MDSCs during bones metastasis.
Methods

In the present study, implantation of 4T-1 breast cancer into the mouse was used as a bone metastasis model. We treated 4T1 bearing mice with surgical procedures and mice were divided into Control group, Surgery group and Surgery-hemorrhage group randomly. The femur and tibia fragments from three groups were isolated and bone resorption was identified by hematoxylin-eosin (HE) staining seven days after surgery. The tumor volumes, metastasis and invasion among three groups were also assessed. We detected the expression of ALP and TRAP to quantify the activities of osteoblasts and osteoclasts respectively. The percentage of MDSCs in the bones and spleens among three groups were detected though flow cytometry analysis. We analyzed the programmed cell death-1 (PD-1) and programmed cell death-Ligand 1 (PD-L1) expression on the MDSCs isolated from mice by western blot. We also detected inducible nitric oxide synthase (iNOS), indoleamine2, 3-dioxygenase (IDO) and arginase-1 (Arg-1) expression in MDSCs by Real time-polymerase chain reaction (RT-PCR). In vitro, we isolated MDSCs in bone myeloid cells (BMCs) of surgical mice and co-cultured with 4T-1 cells. The effects of MDSCs on tumor cell proliferation migration and invasion were further assessed. We also compared the migration, invasion and proliferation of 4T-1 cells co-cultured with MDSCs pretreatment with anti-mouse PD-1 or isotype antibodies.
Results

In the present study, we found more severe bone resorption and destruction in Surgery-hemorrhage group (p < 0.05). We did not find that there was difference about the tumor volumes among three groups. The total percentages of tumor metastasis and the invasion were more larger and deeper in surgery-hemorrhage mice. We found that mice accepted major surgery showed more serious bone lesions and increased activity of osteoblasts and osteoclasts (p < 0.05). Meanwhile, we also observed a striking increase of MDSCs in the bones (p < 0.01) and spleens obtained from surgical mice (p < 0.05). MDSCs derived from surgical mice at 72 hr after surgery expressed high levels of PD-1 (p < 0.05), IDO (p < 0.05), Arg-1 (p < 0.05) and iNOS (p < 0.01). The protein level of PD-L1 did not show difference among three groups (p > 0.05). In addition, the data showed that the proliferation rate were significantly higher when breast cancer cells co-cultued with MDSCs compared with these co-cultued with MDSC-depleted BMCs (p < 0.05) in vitro. The migration (p < 0.05) and invasion ability (p < 0.05 at 48h and p < 0.01 at 72h, respectively) were significantly increased in MDSCs-treated 4T-1 cells. Furthermore, blockade of PD-1 weakened MDSC-mediated promotion of 4T-1 cells migration (p < 0.05), invasion (p < 0.05) and proliferation (p < 0.05 at 24h, p < 0.01 at 48h and 72h, respectively).
Conclusions

Taken together, our results suggest that surgery-induced MDSCs showed tumor-promotive ability and elimination or reduction of MDSCs may significantly delay and limit bone metastasis.