Cetuximab Enhanced The Cytotoxic Activity Of Immune Cells During Treatment Of Colorectal Cancer

Backgrouds:Colorectal cancer(CRC)carries a high mortality worldwide.Surgery is first-line treatment for CRC but up to 30-40%of patients relapse within the first few years after initial surgery.Also,it has been reported that ?60%CRC patients will go on to suffer from tumor metastasis.Studies have indicated a high prevalence of mortality,recurrence and metastasis of CRC to be associated with immunocompromised patients.One epidemiologic survey showed low activity of natural killer(NK)cells to be associated with an increased risk of cancer,and that infiltration of CD8+T cells and NK cells are closely related to the clinical outcome of CRC.One study demonstrated that NK cells can kill human tumor cells isolated immediately from colon carcinomas.Moreover,NK cells can serve as effective immunotherapy for patients with metastatic CRC.For example,the adoptive transfer of NK cells to patients is an emerging method to treat the metastasis of CRC.Cetuximab is a chimeric human-mouse anti-epidermal growth factor receptor(EGFR)immunoglobulin(Ig)G1monoclonal antibody.It has been used to inhibit the proliferation,invasion,and metastasis of tumor cells.Cetuximab was approved recently by the US Food and Drug Administration for first-line treatment of advanced CRC.By crosslinking CD16a(FcyR?a),cetuximab mediates the crosstalk between NK cells and dendritic cells(DCs),which is important for the induction of antitumor cellular immunity.Cetuximab-induced activation of NK cells can upregulate expression of the costimulatory receptor CD137(4-1BB),which is closely related to the efficiency of NK cells,and subsequently promotes T cell-based immunity in vitro.Importantly,if DCs and NK cells are removed from peripheral blood,the antitumor activity of cetuximab is attenuated significantly.It has been shown that the number of CD8+T cells is reduced significantly in patients with metastatic CRC compared with healthy individuals.Some in vitro and mouse-model experiments have investigated the role of cetuximab in CRC treatment,but few studies have tried to determine the mechanism of action(MoA)of cetuximab during CRC treatment by analyzing the lymphocyte subsets of CRC patients.Mice-model experiments have suggested that cetuximab may target tumor cells by antibody-dependent cell-mediated cytotoxicity(ADCC),and that cetuximab can intensify the ADCC antitumor activity of adoptive NK cells towards CRC with increased expression of the EGFR.Several studies have provided some information on how cetuximab acts on CRC in vitro,but the MoA of cetuximab during CRC treatment is not known due to a lack of in vivo studies.Cetuximab is used widely for the treatment of metastatic CRC,but its MoA is not known.Thus,we analyzed the immune characteristics of the peripheral blood of CRC patients.We found that NK cells and CD8+T cells have important roles in cetuximab treatment.These findings provide further understanding of the MoA of cetuximab during CRC treatment.Methods:Collect and analyze the clinical data of 63 CRC patients and healthy control.Isolate peripheral blood mononuclear cells for studying the proportion and function by in vivo and vitro experiments.Then test the number and the proportion of NK and CD8+T cells and their ability to kill tumor cells through flow cytometry,Detect the levels of CRC-related cytokines through enzyme-linked immune response(ELISA)and enzyme immunoassay.Results:Part1 The results showed that compared with healthy controls,the number of CD3+ T,CD8+ T,and natural killer(NK)cells was increased significantly and T-regulatory cells reduced gradually after cetuximab treatment.There was no significant difference in CD4+T cells compared with healthy controls.The number of CD3+CD56+ NKT(natural killer)like cells was not significantly different from the healthy group.Whereas,Tregs(Treg cells)has immunosuppressive effects.It was found that Tregs in CRC patients had a significantly lower inhibitory effect than healthy controls;In addition,there was no significant change in the BDCA1 co-stimulatory molecules on DC surface of CRC patients;There was also no significantly different in iNKT(invariant natural killer T)cells between CRC patients and healthy group.invariant NKT.It is further proved that NK cells and CD8+T are important immune cells,they are associated with CD 107a and CD 137 molecules with degranulation and cytotoxic functions.The level of CD 107? and CD 137 expression on NK and CD8+T cells was increased significantly after 4 weeks of cetuximab therapy.In vitro cetuximab treatment markedly increased expression of CD 137 and CD 107a on NK and CD8+ T cells.Cetuximab treatment promoted the cytotoxic activity of NK and CD8+ T cells against tumor cells.It is also found that plasma levels of cytokines IL-6 and IL-10 in peripheral blood of CRC patients decreased;IFN-ylevels increased.Part2 We divided CRC patients into effective and inactive groups according to the clinical symptoms and imaging diagnosis after treatment.We found that IL-33 levels in the peripheral blood of Cetuximab-effective group were increased for 2 weeks.The level of OPN was significantly decreased;The levels of IL-33 and OPN in the peripheral blood of the patients in the ineffective group had no significant changes before and after treatment;In addition,the IL-10 levels in the plasma of the patients in the effective treatment group were significantly lower than no treatment.There was no significant difference in IL-10 levels in the inactive group.There was no change in the levels of IL-4 and TNF-a before and after treatment.The levels of IL-33,IL-10 and OPN were significantly lower in the culture supernatant of peripheral blood mononuclear cells in patients with effective treatment.We speculate that IL-33 may play an anti-tumor role in regulating the expression of IL-10 and OPN in peripheral blood in the early stages of colorectal cancer.Conclusions:Cetuximab treatment promotes activation of the immune response but alleviates immunosuppression:this might be the underlying anti-CRC effect of cetuximab.In the process of treatment of CRC,CD8+ T and NK cells play an important role in the mechanism is closely related with cetuximab;At the same time,in vitro,CD8+T cells and NK cells play an important role in anti-tumor activity.IL-33 inflammatory factors are closely related to the early onset of colorectal cancer,and may regulate tumor-related cytokines through the two key factors of OPN and IL-10.