IFN-γ Withdrawal After Immunotherapy Potentiates B16 Melanoma Invasion And Metastasis By Intensifying Tumor Integrin αvβ3 Signaling

Progress in molecular and cellular immunology during the past two decades hasadvanced our understanding of tumor-host interactions and opened extraordinaryopportunities for the development of antitumor immunotherapy. So far, there aremany available approaches designed to modulate the immune system for a betterefficacy in tumor immunotherapy. Although the exciting efficacy has been obtained byusing these approaches, the complete tumor eradication occurs infrequently. Tumorrecurrence and metastasis are frequently the final and fatal step in the progression ofsolid malignancies in patients after immunotherapy. It has been found that a variety ofmolecular alterations occur in tumors so that they become more progressive and betterequipped to evade or inhibit host defenses. However, it is still unclear whichfactor(s) is responsible for the inhibition of tumor metastasis during immunotherapyand what happens if such factor(s) is downregulated after immunotherapy.Interferonγ(IFN-γ) is an important cytokine produced by the activatedlymphocytes and contributes to antitumor activity via several mechanisms, includingphenotypic or functional modification of neoplastic cells, rendering them moreamenable to immune recognition and attack via Fas-dependent and Fas-independentpathways. Although IFN-γ, has also been found to promote tumor immune evasionby downregulating cellular level of an endogenous tumor antigen and induce thenegative immune regulation by upregulating B7-H1 expression, the increase ofIFN-γlevel is still crucial for the therapeutic efficacy of different immunotherapeuticapproaches. However, despite the extensive studies on the positive and negative effectsof IFN-γon tumor growth, little is known about the influence of IFN-γon the metastatic behavior of tumor cells.In this study, we found that the metastatic potential of tumor cells was increasedafter the termination of immunotherapy. Underlying mechanisms involve the increasedactivity of integrin.Theαvβ3 signaling pathway and the increased responses of tumorcells to ECM molecules after the removal of IFN-γ. This pitfall of immunotherapytermination could be remedied by the administration of a recombinant polypeptide offibronectin, which counteracts the increasedαvβ3 signaling of tumor cells. Thesefindings suggest that the termination of immunotherapy, especially the drop of IFN-γ,may increase the risk of tumor metastasis and that targetingαvβ3 signaling.Thisexperiments are subdivided into four parts.Part 1: To explore the invasive behavior of tumor cells after immunotherapy.Although the exciting efficacy has been obtained by using tumor immunotherapy. Thecomplete tumor eradication occurs infrequently. Plasmid p4-1BBL was injected intomuscle of mice. The expressed 4-1BBL and sPD-1 were identified by RT-PCR andWestern blot Gene therapy with p4-1BBL/psPD-1 inhibits the tumor growth but theresidual tumor exhibits an enhanced capability of invasive growth. The donor mice(n=8 per group) were inoculated by subcutaneous injection of B16 cells, and genetherapy with p4-1BBL/psPD-1 was performed. Small pieces of tumor tissues afterimmunotherapy were transplanted to recipient mice. The transplanted tumors weremeasured and other tumor nodes in the liver of recipient mice were counted. The resultshowed that the transplanted tumors from p4-1BBL/psPD-1-treated mice grew fasterthan those from control mice, concomitant with the appearance of more satellitenodules around the main tumor.Part 2: To explore which factor(s) is responsible for the enhanced of the residualtumor metastasis after immunotherapy and what happens if such factor(s) is changed inmicroenvironment. Interferonγ(IFN-γ) is an important cytokine produced by theactivated lymphocytes and contributes to antitumor activity. IFN-γexpression in tumormicroenvironment was 10 fold lower several days after the termination of P4-1BBLtransfaction. Gene therapy with pIFN-γinhibits the tumor growth but the residualtumor exhibits an enhanced capability of invasive growth. The donor mice (n=8 pergroup) were inoculated by subcutaneous injection of B16 cells, and gene therapy with pIFN-γwas performed. Small pieces of tumor tissues after immunotherapy weretransplanted to recipient mice. The transplanted tumors were measured and othertumor nodes in the liver of recipient mice were counted. The result showed that thetransplanted tumors from pIFN-γ-treated mice grew faster than those from controlmice, concomitant with the appearance of more satellite nodules around the maintumor. B16 cells and H22 cells were treated with IFN-γ(20 ng/ml) in vitro for 48 h,and then injected into the liver of C57BL/6 and BALB/c mice (n=8 per group)respectively. The mice were sacrificed d14 later. The result showed that the injectedinto the liver tumors from IFN-γtreatment mice grew faster than those from controlmice, concomitant with the appearance of more satellite nodules around the maintumor.Part 3: This study was designed to investigate the tumor cells biologicalphenotype after the treatment of the IFN-γand to analyze the possible mechanism.Decrease or removal of IFN-γresulted in the increase of tumor cell proliferation.Similarly, and the cells were more resistant to MMC-induced apoptosis. The resultsshowed that the expression of c-Myc, Bcl-2, and Bcl-xL was down-regulated and thatof p21^WAF1, p27^Kip1, and Bax was up-regulated in the presence of IFN-γ, but theexpression pattern of these genes was totally reversed after the decrease or removal ofIFN-γ. And the activation of ERK and JNK in tumor cells was intensified in therecovered tumor cells. Adhesion assay showed that the continuous presence of IFN-γslightly suppressed the adhesive ability of tumor cells to fibrinogen, fibronectin andlaminin, whereas the adhesion of tumor cells to these molecules was strongly increased24 h after the removal of IFN-γ. The increased adhesive ability of tumor cells lastedfor several days after the removal of IFN-γ. After the injection of CSFE-labeled B16cells into mice via tail vein, the fluorescent spots in lung tissues, both 5 h and 24 hafter tumor cell injection, were significantly increased in the treatment/recover group.The results showed that the metastatic tumor nodes in lung were significantlyincreased and the survival of mice was shortened if B16 cells were pretreated withIFN-γbefore inoculation.Part 4: To elucidate the molecular mechanism involved in the increased invasivegrowth and metastasis of tumor cells after IFN-γ. The results showed that the activation of FAK in tumor cells was intensified in the recovered tumor cells,indicating thatαvβ3 signalling pathway is indeed intensified. The expression of cdc2gene, a down-stream gene ofαvβ3 signalling pathway, was up-regulated in thepresence of matrigel, and further up-regulated in the recovered cells. The productionand activation of MMP-2 and MMP-9, and the polymerization of actin in response toECM molecules were also increased in the recovered B16 cells.Part 5: This study was designed to explore possible method of therapy that theresidual tumor metastasis were enhanced after immunotherapy. The CH50 mRNAs of12 h, 24 h, 48 h and 72 h on hepatic cells of the mouse after in vivo transfection ofpCH510 by i.v. injection were detected by RT-PCR. 72 h after in vivotransfection of pCH510 by i.v. injection, the expressed CH50 in serum or livertissue was detected by Western blot. The results showed that the in vitropretreatment with IFN-γsignificantly increased the lung metastasis of tumor cells,which was abolished by in vivo expressed CH50. The in vitro pretreatment of tumorcells with IFN-γtogether with CH50 also suppressed the tumor metastasis to lung sothat the in vivo expressed CH50 effectively inhibited the lung metastasis of tumor cells.We then investigated the effect of CH50 in immunotherapy/transplantation model.The treatment with in vivo expression of CH50 either during immunotherapy (in donormice) or after immunotherapy (in recipient mice) significantly inhibited the invasivegrowth of transplanted tumor.