Effect And Mechanism Of Particulate Ganoderma Lucidum Spore β-glucan In Remodeling Tumor Immunosuppressive Microenvironment As An Adjuvant To Gemcitabine Chemotherapy

TME plays an important role in mediating the escape of tumor cells during chemotherapy.The myelosuppressive side effect of chemotherapy depletes the effector T cells in TME,and the host’s protective anti-tumor immunity is suppressed by the non-malignant leukocyte lineage,which makes TME bias towards an immunosuppressive state and significantly affects the long-term efficacy.With the deepening understanding of the complex mechanisms involved in TME,it is important to develop adjuvant chemotherapy strategies targeting immunosuppressive cells in TME.Fungal β-glucans have been shown to regulate TME adjuvant chemotherapy,but the specific mechanism is still unknown.Ganoderma lucidum spore powder capsule which was approved and marketed by the National Medical Products Administration(NMPA)has been used to reduce the side effects during chemotherapy and improve patient quality of life.Firstly,crude cell wall β-1,3-glucan from Ganoderma lucidum spore powder was extracted by high temperature alkaline reflux and then purified by Sevage method to deproteinization,30%H2O2 to decolorization and dialysis method to deionization.Next,Zeta potential and scanning electron microscopy(SEM)showed that PGSG is a spherical particle with a porous surface and a particle size of 2-6 μm;Fourier transform infrared spectroscopy(FTIR)analysis proved that PGSG is a β-1,3-D-glucan;Elemental analysis showed that the content of C,H and N in PGSG was 39.28%,6.23%and 4.07%respectively;Finally,X-ray diffraction(XRD)showed that PGSG was amorphous and thermogravimetric analysis(TGA)suggested that the structure of PGSG only changed when the temperature exceeded 200℃.Therefore,PGSG is a kind of stable particle β-1,3-D-glucan.Secondly,we tested the immunomodulatory activities of PGSG on macrophages and BMDC in vitro.PGSG up-regulates the expression of CD40,CD86,CD80 and MHC-II on the surface of macrophages and BMDC,which suggested PGSG promotes the maturation and activation of macrophages and BMDC;At the same time,PGSG enhanced the ability of antigen presenting cells to phagocytosis of DTAF-PGSG,and the phagocytosis of neutral red in macrophages was concentration dependent.Finally,we found that PGSG increased the release of inflammatory cytokines IL-6 and TNF-α by macrophages and BMDC.This suggests that PGSG has good abilities to regulate innate immunity.Thirdly,we verified the anti-tumor effects of PGSG combined with GEM on breast cancer and Lewis lung cancer in vivo experiments.We found that repeated GEM chemotherapy can indeed delay the development of tumors,but it caused the accumulation of MDSC,M2 and Treg in tumor and spleen.Our results proved that PGSG combined with GEM can further delay tumor development and reduce the side effects of weight loss caused by GEM.In tumor and spleen,PGSG reduced the ratio of MDSC(CD11b+Gr-1+)and Treg(CD4+Foxp3+),promoted the conversion of M2(F4/80+CD206+)to M1(F4/80+MHC-Ⅱ+),and enhanced Thl(CD4+IFN-γ+)and CTL(CD8+IFN-γ+)immune response.Finally,we prepared GEM-treated tumor cell conditioned medium to simulate TME to observe the effect of PGSG on the differentiation of bone marrow-derived cells into MDSC.The content of cytokine GM-CSF in our conditioned medium is significantly increased,and PGSG can decrease the distribution of MDSC whether induced by cytokine or conditioned medium.Therefore,PGSG can target MDSC and regulate TME in vivo and in vitro.