The Effect Of Systemic CpG ODN Combined With TGF-β2 Antisense Oligonucleotide On The Growth Of Subcutaneous Solid Tumor And Formation Of Tumor Microenvironment

Tumor immunotherapy has been a popular immunotherapy for tumor treatment in the current stage.However,it displays tremendous discrepancy in the therapeutic effect on cancer patients,which showed lower objective response rate.Two main limitations are dampening the effect of tumor immunotherapy:1.There were few lymphocytes which could play cytotoxic effect on tumor cells in the tumor tissues;2.The lymphocyte in the tumor tissues could not be activated efficiently.Therefore,the solutions for the two main limitations can make the immunotherapy achieve better results.Immune activators were used to increase the lymphocytes infiltrated in tumors,such as TLR3 agonists,TLR7 agonists and TLR9 agonist CpG ODN etc.could increase the lymphocytes in tumors very well.However,the antitumor effect of CpG ODN monotherapy displayed not ideal,which may be related to the insufficient activation of lymphocytes in tumors.Several studies have shown that amount of immunosuppressive cytokines existed in the tumor microenvironment,such as TGF-β2.TGF-β2 produced by the tumor cells and the activated immune cells not only can block the lymphocytes infiltrate into tumor but inhibit the activation of lymphocytes in tumors.In some clinical trials,tumors such as glioma,melanoma and non-small cell lung cancer can express and secret abundant TGF-β2.As an immunosuppressive cytokine,TGF-β2 can directly inhibit the activation of CD8+T cells and NK cells,and also inhibit the maturation and activation of DCs.Therefore,it seems extremely important to inhibit TGF-β2 in tumor tissues.TIO3 is a self-designed TGF-β2 antisense oligonucleotide which has been proved to show the ability of inhibiting the expression of TGF-β2 specifically.We inferred that CpG ODN as an immune activator combined with TI03 as an inhibitor of immunosuppressive cytokine might increase and activated the lymphocytes in tumor tissues to achieve an antitumor effect.CpG ODN increases lymphocytes in tumors,and TIO3 inhibits TGF-β2 produced by tumor cells and activated immune cells at mean time,which may promote the increase and activation of lymphocytes in tumor tissues.The combinatorial therapy might achieve better anti-tumor effect from two aspects of enhancing immune response and inhibiting immune inhibitory factors.However,it is not clear whether the tumor microenvironment already formed in solid tumors can be reshaped.Nevertheless,it is worth exploring to inhibit the tumor growth by creating the lymphocyte-enriched and activated tumor microenvironment in the early stage of tumor microenvironment formation.To explore whether early administration of CpG ODN combined with TIO3 can promote the formation of lymphocyte-enriched and activated tumor microenvironment in tumor-bearing mice,increase the infiltration and activation of lymphocytes in tumors,thus inhibit tumor growth and prolong the survival of tumor-bearing mice.In this study,LLC subcutaneous tumor-bearing mouse model was used to observe the effects of CpG ODN combined with TIO3 on the formation of tumor microenvironment and the survival of mice and through early systemic administration.1.The antitumor effect of CpG ODN combined with TIO3To investigate whether CpG ODN combined with TIO3 can inhibit subcutaneous tumor growth in tumor-bearing mice,LLC and B16 tumor-bearing mice were administered intraperitoneally with CpG ODN combined with TIO3 on 1,3,5 and 7 day,and the tumor volume and survival of tumor-bearing mice were detected.Meanwhile,CpG ODN and TIO3 monotherapies were used as experimental controls,and PBS was used as blank control.We found that CpG ODN combined with TIO3 could inhibit the LLC tumor growth significantly.The average tumor volume of mice in CpG ODN+TIO3 group was half of that in CpG ODN and TIO3 monotherapy groups,and only 1/4 of that in PBS group.Two mice in CpG ODN+TIO3 group had complete tumor regression.The survival of mice in CpG ODN+TIO3 was prolonged obviously.On day 44 after tumor cell inoculation,all tumor-bearing mice in CpG ODN+TIO3 group survived,while all mice in PBS group died.On day 54 after tumor cell inoculation,half of the mice in CpG ODN+TIO3 group survived,while all mice in the CpG ODN and TIO3 monotherapy groups died.Up to day 80 after inoculation,2 mice in CpG ODN+TIO3 group were still alive.These results indicated that CpG ODN combined with TIO3 can significantly inhibit LLC tumor growth and prolong the survival of tumor-bearing mice,which might induce a specific antitumor response.To generalize the utility of CpG ODN combined with TIO3 in antitumor application,we processed the same experiment by using B16 melanoma tumor-bearing mouse model.We found that CpG ODN combined with TIO3 could prolong the survival of tumor-bearing mice significantly.On day 34 after tumor cell inoculation,all tumorbearing mice in CpG ODN combined with TIO3 group survived,while all tumorbearing mice in PBS group died.On day 45 after tumor cell inoculation,half of the mice in CpG ODN+TIO3 group survived,while all mice in the CpG ODN and TIO3 monotherapy groups died.In conclusion,CpG ODN combined with TIO3 can significantly inhibit the subcutaneous tumor growth and prolong the survival of tumorbearing mice,also induce specific antitumor effects.2.The effect of CpG ODN combined with TIO3 on systemic immune response of subcutaneous tumor bearing miceTo investigate the regulatory effect of intraperitoneal administration of CpG ODN combined with TIO3 on systemic immune response in subcutaneous tumor-bearing mice,the peripheral blood,spleens and draining lymph nodes of treated tumor-bearing mice were harvested to interrogate the IFN-γ and TLR9 expression by flow cytometry and qPCR on day 8 and 13 after tumor inoculation.We found that no matter CpG ODN alone or combined with TIO3 could raise the expression of IFN-γ and TLR9 with no discrepancy,while TIO3 monotherapy showed no effect on systemic immune response.The IFN-γ expression and TLR9 expression of systemic tissues in CpG ODN+TIO3 or CpG ODN group is 3 times and 2 times of those in PBS group,respectively.CpG ODN combined with or without TIO3 could upregulate the lymphocyte count to 2 times of that in PBS group and down-regulate the Neutrophil-to-Lymphocyte ratio to 1/4 of that in PBS group.In conclusion,CpG ODN,whether combined with TIO3 or not,can increase the lymphocyte count in peripheral blood,and significantly activate peripheral blood lymphocytes,splenocytes and DLN cells.However,the antitumor effect of CpG ODN combined with TIO3 is much stronger than that of CpG ODN monotherapy.Therefore,we inferred that CpG ODN combined with TIO3 might work more effectively in activating immune response in tumor tissues.3.The effect of CpG ODN combined with TIO3 on the formation of tumor microenvironment of tumor-bearing miceTo explore the possible mechanism of antitumor effect by combining CpG ODN and TIO3,we interrogated TGF-β2,IFN-γ,the counts and activation of lymphocytes and DCs in the tumor tissues of treated tumor-bearing mice on day 13 after tumor cell inoculation.Then,we further interrogated the activation of lymphocytes and DCs,and paid attention to whether DCs played an indispensable role in initiating antitumor response by establishing a co-culture system of tumor cells and splenocytes in vitro.We also traced the fluorescence conjugated TIO3 to determine whether TIO3 administrated systemically could enter tumor tissues and cells.We found that CpG ODN combined with TIO3 could downregulate the expression of TGF-β2 to less than 1/2 of that in PBS group,while up-regulate the expression of IFN-γ to more than 3 times that in PBS group in the tumor tissues of tumor-bearing mice.CpG ODN combined with TIO3 could increase the lymphocytes,CD8+T cells and NK cells were 2 and 3 times higher than those in PBS group,respectively,and the expression of IFN-γ in lymphocytes was 5 times higher than that in PBS group.The frequency of DCs in CpG ODN+TIO3 group was 4 times of that in PBS group,and the CD86 expression on DCs was double that in PBS group.Although TIO3 could downregulate the expression of TGF-β2,and CpG ODN could also increase the expression of IFN-γ to twice that of PBS group,the ability of increasing and activating lymphocytes in tumor tissues was far less than that of the combination of TIO3 and CpG ODN.These results indicated that CpG ODN combined with TIO3 can inhibit the expression and secretion of immunosuppressive factors,increase the expression of proinflammatory factors,and activate CD8+T cells and NK cells in tumor tissues,depending on both CpG ODN and TIO3.We found that the effects of CpG ODN+TIO3 in the in vitro co-culture system were similar to those in mouse tumor tissues.CpG ODN combined with TIO3 could inhibit TGF-β2 and other immunosuppressive factors,also upregulate the expression of IFN-γ and other proinflammatory factors.The expression of IFN-γ in lymphocytes in CpG ODN+TIO3 group,including CD8+T cells and NK cells,was more than 2 times higher than that in PBS group,while no difference occurred on CD4+T cells.CpG ODN combined with TIO3 could also activate DCs in co-culture system.However,CpG ODN combined with TIO3 failed to upregulate the expression of IFN-γ in CD8+T cells and NK cells in the co-culture system without DCs,and showed no difference in effect of tumor eliminating compared with PBS group.These results indicated that CpG ODN combined with TIO3 in the co-culture system could inhibit the expression and secretion of immune inhibitory factors,increase the expression of proinflammatory factors,also activate CD8+T cells and NK cells.It is worth noting that the activation of CD8+T cells and NK cells depends on the activation of DCs.Fluorescence conjugated TIO3 could enter tumor tissues 24 hours after intraperitoneal injection in vivo,and also enter tumor cells in vitro,indicating that TIO3 could enter tumor tissues and tumor cells to play an important role in inhibiting TGFβ2.In conclusion,the anti-tumor effect of CpG ODN combined with TIO3 may be due to the entry of CpG ODN and TIO3 into tumors.While CpG ODN activates immune response,TIO3 inhibits immune inhibitory factors to assist to enhance the immune response,and activates CD8+T cells and NK cells by activating DCs.Thus promoting the formation of lymphocyte enriched and activated tumor microenvironment.In summary,this study demonstrated that systemic application of CpG ODN combined with TIO3 in the early stage of tumor formation can inhibit the tumor growth and prolong the survival of tumor-bearing mice.CpG ODN combined with or without TIO3 both can activate systemic immune response,but the systemic immune response activated by CpG ODN alone does not match its antitumor effect.CpG ODN combined with TIO3 induces stronger immune activation effect in the tumor microenvironment,mainly manifested as that CpG ODN activates the immune response,TIO3 inhibits immune inhibitory factors to assist to enhance the immune response meanwhile,and promotes the increase and activation of lymphocytes in tumor tissues,so as to achieve better anti-tumor effect.In this study,the treatment at the early stage of tumor formation can simulate the immunotherapy in post-surgery tumor patients to a certain extent.The use of immune activating ODN combined with the ODN that blocks the immune suppressive factors can affect the formation of tumor microenvironment,which may provide a novel idea for the treatment to prevent tumor recurrence in post-surgery patients.