Antitumor Strategies Based On TLR3 Agonist Poly (I:C) And The Underling Mechanism

The malignant tumor is regarded as the emperor of all maladies and it has become the biggest killer that threatens human life and health.Despite enormous progress in targeted therapies have demonstrated some curative effects,there are still a substantial number of patients cannot benefit from current treatments.Because almost all tumor patients will develop drug resistance after treatment,and metastasis is one of the main causes of tumor death,it is imperative to develop more efficacious approaches to overcome the drug resistance and metastasis of cancer.TLR3 is a Toll-like family receptor that mediates antiviral responses by recognizing double-strand RNA(dsRNA).TLR3 is expressed not only in immune cells but also in non-immune cells,and many tumor cells also express TLR3,which is closely related to the treatment and prognosis of clinical tumors.Based on the significance of expression in tumor cells and the properties of activating immune system,TLR3 has become a target for tumor therapy.The TLR3 agonist poly(I:C)(PIC)is a synthetic dsRNA analog that has been found to induce expression of type I and pro-inflammatory cytokines by activating professional antigen-presenting dendritic cells,which in turn activate specific T-cell immune responses and exert antitumor immunity.PIC can also directly inhibit tumor growth by inhibiting cell proliferation or inducing cell apoptosis.In addition,it has been also found to play a crucial role in reprogramming tumor-associated macrophages(TAMs).TAMs are macrophages infiltrating into tumor tissues with a high degree of plasticity.Current studies suggest that TAMs play a dual role in the development of tumors and a rational steering of the macrophage plasticity may alter the immunosuppressed microenvironment and strengthen the organic immunity to inhibit tumors.Thus applying TLR agonists to reprogram TAMs from immunosuppressed phenotype to immunestimulated phenotype is a very promising anti-tumor strategy.Previous studies have shown that macrophages activated by PIC singly are inefficient to fight tumors.In particular,the susceptibility of PIC to nuclease degradation leads to insufficient immune-boosting,which has hindered its clinical application.Therefore,it is extremely desirable to combine PIC with other macrophage activators or to develop an efficient delivery system to address these issues.Ferumoxytol(FMT)is an iron oxide nanoparticle composed of iron oxide core and dextran shell.As a commonly used nano-carrier,FMT also integrates the crucial essence of both diagnostic and therapeutic functions by improving gene transfection and system delivery.In a latest research,FMT is reported to possess an appealing feature of anti-tumor therapeutic potential by promoting the production of pro-inflammatory cytokines and reactive oxygen species in macrophages to kill tumor cells.Therefore,the untapped potential of macrophages activated by the combination of FMT and PIC or nano-particles composited by FMT surface functionalized with PIC provides impetus to discover whether it's an efficient strategy to against tumor.In this study,the treatment effect of PIC combined with paclitaxel(PTX)on PTX-resistant colon cancer was analyzed;a combination of PIC and FMT for the inhibition of subcutaneous melanoma was investigated;FMT-NH2-PIC composite nanomaterials(FP-NPs)was constructed and the therapeutic effects of FP-NPs on pulmonary metastases in mice with the underling mechanism were explored.The results showed that PIC could enhance the chemotherapy effect of PTX on PTX-resistant colon cancer through the signal axis of TLR3-UNC93B1-IFN-?;PIC combined with FMT synergistically promoted macrophage activation and inhibited melanoma progression in mice;FP-NPs alleviated lung metastases in mice by steering macrophage to M1 phenotype through NF-?B signaling pathway.Therefore,these findings provide novel strategies which have potential value for overcoming drug resistance and metastasis.The detailed results of the study are as follows.1.TLR3 agonist PIC reinforces the potency of cytotoxic chemotherapy via TLR3-UNC93B1-IFN-? signaling axis in PTX-resistant colon cancerResults of cell viability showed that PIC specifically and significantly inhibited cell viability of PTX-resistant cell line HCT-8/PTX.It is well known that type I IFN can mediate anti-tumor effects by inducing cell apoptosis or retarding cell proliferation.Further results showed that PIC significantly promoted the gene and protein expression of IFN-? in HCT-8/PTX,and exogenous addition of IFN-? also dose-dependently inhibited the cell viability of HCT-8/PTX.These results suggested that PIC inhibited the viability of HCT-8/PTX cells via upregulation of IFN-?.It is generally believed that PIC initiates signaling by activating the antiviral pattern recognition receptors TLR3,RIG-I,and MDA5.The results of western blotting(WB)displayed that stimulation of PIC barely altered the expression level of these three receptors in PTX-sensitive cell lines HCT116 and S W620,whereas it significantly up-regulated the expression level of MDA-5 and RIG-1 in HCT-8/PTX and its parental cell line HCT-8.It may be cleaved upon activation,WB assay did not detect the expression changes of TLR3.However,results of flow cytometry(FCM)showed that TLR3 was expressed both in cytoplasm and cell surface in HCT-8/PTX and its parental cell line,and the expression level of it was as well increased upon PIC stimulation.Collectively,though PIC stimulation could significantly upregulate the expression of MDA-5 and RIG-1 in HCT-8 cells,it hardly affected the gene transcription of IFN-P and cell viability.These results suggested that TLR3 may be involved in the secretion of IFN-? and thus downregulating the cell viability of HCT-8/PTX cells.Results of TLR3 overexpression and interference further confirmed that it was through TLR3-NF-?B signaling pathway that PIC reinforced the secretion of IFN-P and thus inhibited the cell viability of HCT-8/PTX.However,it was intriguing that despite PIC as well up-regulated the expression level of TLR3,it did not cause the upregulation of IFN-? and down-regulation of cell viability in HCT-8 cells.As a chaperone protein,UNC93B1 promotes the secretion of IFN-? for initiating signal transduction by transporting TLR3 from endoplasmic reticulum to endosome.Next,the alteration of UNC93B1 after PIC stimulation in HCT-8/PTX and its parental cell HCT-8 was examined.As results indicated that PIC specifically up-regulated the expression level of UNC93B1 in HCT-8/PTX,and TLR3 overexpression further enhanced its expression.Moreover,it was found that upon silence of UNC93B1,PIC further up-regulated the IFN-? expression level in HCT-8/PTX,accompanied by a decreased cell viability.Interference with TLR3 yielded a similar result.Therefore,it implied that TLR3 and UNC93B1 should be on the same signal axis.Besides,data indicated that the gene expression level of MDA-5 and RIG-1 was significantly up-regulated upon PIC stimulation in control group,whereas only the gene expression of RIG-1 appeared an increased trend in cells interfered with UNC93B1 and TLR3,indicating that it may be RIG-1 that promote the production of IFN-?.Taken together,PIC preferentially promoted the production of IFN-? for decreased cell viability in HCT-8/PTX cells through TLR3-UNC93B1 signal axis.To examine whether PIC could enhance the cytotoxicity of PTX,the combination of them on cell viability of HCT-8/PTX was detected.Results showed that compared to sole treatment,the combination of PIC and PTX at different doses significantly reduced the cell viability of HCT-8/PTX.Analysis of synergy index indicated that there exhibited a strong synergy(CI=0.1-0.3)between PIC and PTX.In addition,results of qRT-PCR displayed that PTX alone did not affect the expression of TLR3,UNC93B1 and IFN-?.Whereas when it was combined with PIC,they synergistically promoted the upregulation of these genes.Moreover,PIC combined with PTX significantly inhibited or even eliminated the xenograft HCT-8/PTX colon tumors in vivo while PTX itself only slightly reduced the tumor burden.2.Anti-tumor macrophage activated by FMT combined with the TLR3 agonist PIC promotes melanoma regressionSince both PIC and FMT could effectively activate macrophages,herein we tested the hypothesis that combination of them would exert a more efficient anti-tumor effect.Results of CCK-8 showed that selected doses of PIC,FMT and PIC combined with FMT did not affect tumor cell viability.Whereas the results of co-culture experiments showed that the combined treatment of PIC and FMT synergistically inhibited tumor cell growth via biasing macrophages towards a tumoricidal phenotype with upregulation of TNF-a,iNOS and NO.This phenomenon was confirmed in both RAW 264.7 cell line and mouse primary macrophages.Results of indirect co-culture experiments indicated that the prohibited growth of B16F10 incubated with PIC and PIC combined with FMT was partially mediated by direct cell contact.As professional phagocytes,the phagocytic function of macrophages plays a crucial role in clearing pathogenic microorganisms,apoptotic cells and tumor cells.Given the prohibited growth of B16F10 incubated with PIC and FMT/PIC was partially mediated by direct cell contact,it was of great interest to observe whether their addition could motivate the capacity of macrophage to engulf tumor cells.As expected,there was a more statistically elevated in percentage of phagocytosis in the group administrated with combined PIC and FMT than either PIC or FMT alone.It is well known that NOX2-derived ROS plays an important role in the phagocytosis of macrophages.Results of FCM showed that FMT and PIC solely treatment only slightly promoted the production of ROS,while the combination of them dramatically enhanced the production level of ROS.In addition,the ROS induced phagocytic capacity was distinctly blunt with the addition of ROS scavenger NAC.WB experiments showed that PIC combined with FMT synergistically promoted NOX2 expression,indicating that it was NOX2-derived ROS that predicted to boost the phagocytosis.As a product of cellular oxidative stress,ROS is prone to damage tumor cell proliferation.The results of FCM showed that cell growth suppression was partly recovered by injection with NAC in the co-stimulated group,which meant that ROS was as well involved in macrophage activation-mediated tumor cell growth inhibition.Moreover,in vivo studies showed that PIC combined with FMT significantly promoted melanoma regression and prolonged mouse survival by enhancing the pro-inflammatory macrophages infiltration and destroying the blood vessels formation in tumor tissue.3.FP-NPs inhibits lung metastases by promoting Ml macrophage polarization through NF-?B signaling pathwayEncouraged by the combined therapeutic effect of FMT and PIC on subcutaneous tumors,the therapeutic potential of them for pulmonary metastasis was further evaluated.To better realize the immune stimulatory function and reduce systemic toxicity of PIC,FMT modified with a group of amino was composited with PIC to construct FMT-NH2-PIC nanomaterials(FP-NPs).The bio-distribution analysis of FMT-NH2 and FP-NPs fluorescently labeled with near-infrared molecules NIR797 was detected using the IVIS Spectrum In Vivo Imaging System.In vitro results showed that the FP-NPs could be efficiently internalized by macrophages and ex vivo imaging of the dissected tissues indicated that they were more prone to be accumulated in the lung than FMT-NH2.Most importantly,FP-NPs were more powerful in reducing pulmonary metastases of B16F10 and 4T1 in mice and also effectively relieved the body weight loss in 4T1 mice caused by lung metastases than PIC combined with FMT-NH2.Images of laser scanning confocal microscopy showed that FP-NPs significantly increased the proportion of M1/M2 macrophages in the lungs of mice.In addition,in vitro experimental results showed that FP-NPs itself did barely affect the vitality of tumor cells,but significantly inhibited the proliferation of tumor cells in the co-culture system.Further,we found that FP-NPs promoted macrophage polarization to M1 phenotype through NF-?B signaling pathway.In summary,the different anti-tumor strategies based on TLR3 agonist PIC:PIC combined with chemotherapy drugs PTX,PIC combined with FMT,and FP-NPs nano-composites significantly inhibited the progression of PTX-resistant colon cancer,solid melanoma,and lung metastases respectively,demonstrating that PIC,has a broad spectrum of anti-tumor efficacy.In addition,as the FMT has been approved by FDA for the treatment of hypoferric anemia and PIC used in this study has already been widely used in clinical practice,and the effectiveness and safety of FP-NPs for treatment of lung metastases were also confirmed by our experiments in vivo.This will provide a novel therapeutic reference for clinical treatment of tumors.