Immunomodulatory Effects And Mechanisms Of Recombinant TRAIL In The Tumor Microenvironment

Tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL)is a member of the TNF superfamily.TRAIL can selectively induce apoptosis in tumor cells by binding to a receptor(TRAILR)with little systemic toxicity.As a result,TRAIL has rapidly attracted interest in the search for ideal tumor therapeutic agents.Several anti-tumour drug candidates targeting TRAIL-TRAILR have been developed,and several of these recombinant soluble TRAIL protein drugs have entered clinical trials.Unfortunately,significant anti-tumor activity has not been shown in clinical trials,leading to failure,including drug resistance,off-target toxicity,and short half-life.To address these drawbacks,researchers have bioengineered recombinant TRAIL to obtain a more stable trimeric form,used nanomaterials for targeted delivery,and used vectors to express TRAIL genes for gene therapy,but have not yet further advanced the clinical application of TRAIL.In addition to TRAIL’s ability to specifically induce apoptosis in tumor cells,studies of inflammatory and autoimmune models have shown that TRAIL can affect immune cells in many different ways.While TRAIL was initially found to have an important role in tumor defense by natural killer(NK)cells or cytotoxic T cells,the additional effects of TRAIL on regulatory T cells(Tregs),effector T cells,neutrophils,and antigen-presenting cells have also become a focus of attention.Although several studies have reported that recombinant TRAIL is able to affect the tumor immune microenvironment by inducing apoptosis of immune cells such as Tregs,its potential regulatory role and mechanism in the tumor microenvironment(TME)are still unclear.Investigating the immunomodulatory role and mechanism of recombinant TRAIL in the TME may provide an important perspective to understand the clinical resistance of recombinant TRAIL and help to explore more effective tumor therapeutic strategies.Due to the structural differences between human and murine TRAILR,the affinity of human TRAIL for murine TRAILR is weaker,in order to be able to better evaluate the regulatory role and mechanism of recombinant TRAIL in the tumor immune microenvironment in immune totipotent mice,this thesis firstly constructed and purified murine soluble TRAIL(sm TRAIL)to obtain murine TRAIL for use in the murine system for the Evaluation.In vitro,murine breast cancer cell 4T1 was identified as a sensitive cell line of sm TRAIL,and colorectal cancer cell CT26 and melanoma cell B16 were identified as non-sensitive cell lines of sm TRAIL by cell killing assay.Tumor models were established in immunotolerant mice with the above three cells,and three doses of 0.5 mg/kg,2 mg/kg,and 8 mg/kg sm TRAIL were selected for treatment with reference to the therapeutic concentrations in the TRAIL clinical trials.However,it is worth noting that the tumor therapeutic effect did not show a dose-dependence,with the 2 mg/kg sm TRAIL treatment group showing the best tumor therapeutic effect in both sensitive and non-sensitive cell lines,while the 8mg/kg sm TRAIL group had a poorer therapeutic effect or even showed a tumor growth-promoting effect.The detection of caspases in tumor tissues by immunohistochemistry showed that different doses of sm TRAIL were able to induce apoptosis in 4T1 cells,but the ability to induce apoptosis in CT26 and B16 cells was weaker,which suggests that there are factors other than apoptosis(e.g.,immune regulation)that affect the anti-tumor effects of sm TRAIL in vivo in the course of tumor treatment.Next,we chose sm TRAIL-sensitive cells 4T1 and non-sensitive cells CT26 as research models and analyzed immune cells in mouse tumor tissues by flow cytometry,and the assay revealed that:2 mg/kg sm TRAIL activated innate immune cells and CD8~+T cells;whereas,8 mg/kg sm TRAIL inhibited NK cell and CD8~+T cell activation and significantly increased the number of M2-like macrophages infiltrated.At the same time,we performed transcriptome sequencing and single-cell RNA sequencing of tumors from 4T1 and CT26 mouse tumor models,respectively,and the conclusions obtained from the sequencing were consistent with the results of the flow assay,which further confirms that different doses of sm TRAIL have different regulatory effects on the intratumoral immune cells,which in turn affects the tumor growth.We also examined the expression of cytokines in the two tumor models at the protein level and the transcriptional level,and the results showed that 2 mg/kg sm TRAIL promoted the expression of IL-12 and IFN-γ,and 8 mg/kg sm TRAIL increased the expression of IL-10 and VEGF.Combined with the results of immune cell infiltration and activation,it can be seen that 2 mg/kg sm TRAIL treatment facilitates immune activation and achieves tumor killing,while the 8 mg/kg sm TRAIL group promotes the generation of an immunosuppressive microenvironment,which counteracts the apoptotic effect of sm TRAIL on tumor cells.Subsequently,we focused on exploring the mechanism of immunosuppression triggered by 8 mg/kg sm TRAIL treatment.Firstly,the inhibitory effect of sm TRAIL on NK cells was verified by in vivo and in vitro experiments,which revealed that NK cells were crucial in tumor therapy with sm TRAIL,but the inhibition of NK cell activation was not caused by the direct action of sm TRAIL on NK cells.Subsequently,we explored the effects of sm TRAIL on tumor cells,and the results showed that sm TRAIL could induce CCL2 secretion from tumor cells through the TRAIL-TRAILR axis,and blocking the TRAIL-TRAILR axis could effectively control intratumoral CCL2 secretion and inhibit the recruitment of tumor-associated macrophages(TAMs),which in turn led to significant inhibition of NK cell activation in the 8 mg/kg sm TRAIL group significantly inhibited tumor growth.Results such as single-cell RNA sequencing also indicated that 8 mg/kg sm TRAIL treatment promoted the recruitment of M2-like macrophages.In addition,we found that sm TRAIL also exerts a direct effect on macrophages to promote the polarisation of M2-like macrophages.sm TRAIL binding to TRAILR on the surface of macrophages resulted in increased expression of intracellular Fox O1,which binds to the IL-10promoter and promotes the secretion of IL-10.The mechanism of immunosuppression triggered by 8 mg/kg sm TRAIL treatment was summarised:sm TRAIL binding to TRAILR of tumor cells induced tumor cells to secrete CCL2,which recruited CCR2~+TAMs;meanwhile,sm TRAIL can also act directly on TAMs to promote the polarization of M2-like TAMs as well as the secretion of IL-10,which further promotes the formation of an immunosuppressive microenvironment,thus deepening the suppression of the inhibitory activation and function of NK cells and CD8~+T cells in the tumor microenvironment and the formation of an immunosuppressive microenvironment.Finally,we evaluated the anti-tumor effect and immunomodulatory effects of soluble human TRAIL(sh TRAIL)in a humanized mouse model of the immune system(HU-HSC-NPG.GM3),which showed that both 2 mg/kg and 8 mg/kg sh TRAIL significantly inhibited the growth of human colorectal cancer cells HCT116,whereas 8 mg/kg sh TRAIL caused an increase in the number of intratumoral Tregs and M2-like macrophages,resulting in no better therapeutic effect compared with the2 mg/kg sh TRAIL-treated group.sh TRAIL’s modulatory effect in TME was similar to that of sm TRAIL,which produced immunosuppression by increasing intratumoral M2-like macrophages,and in order to lift this immunosuppression We combined sh TRAIL with the macrophage-targeting drug trabectedin.The combination therapy group was able to significantly enhance the infiltration of lymphocytes and activated CD8~+T cells,significantly reduce the number of intratumoral Tregs and M2-like macrophages,remodel the tumor immune microenvironment,enhance the intratumoral anti-tumor immunity,and improve the anti-tumor effect of sh TRAIL.In summary,this thesis comprehensively evaluated the anti-tumor effect of sm TRAIL and its effects on various intratumoural immune cells in different mouse tumor models.sm TRAIL was found to have a dose-related immunomodulatory effect in a mouse tumor model with normal immune function,and its immunomodulatory mechanism in the tumor microenvironment was explored.Meanwhile,sh TRAIL was used in the humanized mouse tumor model to verify its anti-tumor effect and immunomodulatory effect.It further amplified the anti-tumor effect and activated the body’s anti-tumor immunity by combining it with chemotherapeutic agents.The investigation of the immune regulation mechanism of recombinant TRAIL in the tumor microenvironment provides an important foundation and basis for the further clinical application of TRAIL and has a certain guiding significance for the optimization of the combination treatment strategy of TRAIL with chemotherapeutic and immunotherapeutic drugs.