EGFRvⅢ Colon Cancer Promotes Cetuximab Resistance Through Adenosine-mediated Immune Escape

BackgroundColorectal cancer is one of the most common malignancies in China,of which 4050%of the patients had distant metastasis(Metastatic Colorectal Cancer,mCRC)at their first diagnosis,leading to a poor overall prognosis.Epidermal growth factor(EGFR)-targeted therapy have become the cornerstone of the first-line treatment in mCRC nowadays,and cetuximab(Cet),an IgG1 monoclonal antibody,is the most iconic one due to its ability in significantly prolonging the survival of patients with advanced CRC.However,the therapeutic benefits of the mCRC patients are being severely compromised by the cetuximab resistance that develops during treatment.Several genetic alterations associated with cetuximab resistance have been identified,including RAS mutations(KRAS and NRAS),BRAF mutations,PI3KCA mutations as well as the amplification of HER2 and MET,and yet there still remained some patients that failed to be explained by any of these theories,indicating the underlying complexity of the mechanisms in cetuximab resistance and the greatly therapeutic potential in further exploring this phenomenon.Being the direct target of cetuximab,the mutation of EGFR itself has not been proved in previous studies to have any clear effect on the cetuximab sensitivity of CRC.Multiple novel EGFR mutations identified by several high throughput sequencing studies for CRC recently have suggested their closed connections with cetuximab resistance.EGFRvⅢ is a deletion mutant of extracellular domain of EGFR expressing in about 40%of high-grade gliomas and is associated with malignant transformation and poor clinical outcomes.Recent studies have discovered that about 10%of CRC patients carry EGFRvⅢ mutation,leading to worse prognosis and possible treatment resistance secondary to anti-EGFR therapy.Despite partially absence of extracellular segment,EGFRvⅢ still can combine with cetuximab,but the blocking effect is different from the wild type EGFR.Currently,the role of EGFRvⅢ mutations in the progression of drug resistance in colorectal cancer has yet to be explored,and further study of EGFRvⅢ mutations in CRC will be of great help in revealing its part in the evolution of drug resistance and providing novel ideas for population screening and individualized treatment decision making.Most anticancer drugs kill tumor cells in a non-immunogenic manner.However,in 2011,Garrido G et al.reported for the first time that anti-EGFR monoclonal antibody can induce the production of tumor specific cytotoxic T cells,suggesting that immunogenic apoptosis plays a key role in this mechanism.Another study published in 2016 in the journal of Nature further confirmed that both EGFR-targeting cetuximab and panimumab could induce Immunogenic cell death(ICD)in tumor cells.Together,the two studies reveal that immune factors play an important role in the effectiveness of cetuximab.Therefore,we focus on the effect of EGFRvⅢ mutation on the immune microenvironment of CRC when we analyze the mechanism of EGFRvⅢ induced cetuximab resistance,to explore the main mechanism of EGFRvⅢ tumor-induced immune microenvironment remodeling,which will lay an important foundation for finding effective drug targets and exploring the feasibility of targeted therapy combined with microenvironment remodeling strategy.Methods and ResultsIn this research,we first analyzed the proportion of EGFRvⅢ expression in colorectal cancer by immunohistochemical staining and the clinical characteristics of patients with EGFRvⅢ mutation.Next,we used EGFRvⅢ specific probes to detect circulating tumor cells in patients’ peripheral blood,TCGA database analysis,and animal studies to confirm that EGFRvⅢ colorectal cancer does not respond well to cetuximab therapy.However,in vitro sensitivity testing and subcutaneous tumor administration in nude mice,the difference in cetuximab response between EGFRwt and EGFRvⅢ tumor cells disappeared,suggsting that immune microenvironment may mediate EGFRvⅢ resistance to cetuximab.Therefore,we evaluated the immune microenvironment of EGFRWT and EGFRvⅢ patients and compared the expression levels of immune-related genes using GSVA-based immune infiltration score.It was found that EGFRvⅢ could induce the decrease of T cell infiltration and the inhibition of T cell function,which was confirmed by immunofluorescence staining of tumor tissue and flow analysis of tumor infiltration T cells.Furthermore,we purified human peripheral blood CD8+T cells and cultured them with tumor supernatant after removal of specific components or molecular weight filtration.The proliferation,activation and killing ability of EGFRvⅢ tumor were measured by Flow cytometry subsequently.The results showed that EGFRvⅢ tumor could promote the accumulation of adenosine in microenvironment to realize immune escape.At the mechanism level on the one hand,we used qRT-PCR,Western blot,flow cytometry,immunofluorescence,assay,in vitro phagocytosis,etc.to confirmed that cetuximab could induce stronger endoplasmic reticulum stress and ICD effect in EGFRvⅢ cells,promoting the release of adenosine precursor ATP.However,these EGFRvⅢ cells could not be successfully phagocytosed by DC cells.In addition,immunofluorescence staining and hippocampal energy metabolism experiments revealed that EGFRvⅢ might affect mitochondrial metabolism through mitochondrial translocation,thus modulating ER stress.On the other hand,by means of bioinformatics analysis and molecular biology experiments,we found that EGFRvⅢ cells can promote STAT3 phosphorylation and its entry into the nucleus to transcriptionally upregulates the expression of cell membrane adenosine metabolizing enzymes CD39 and CD73,which drives the release of ICD effect into the microenvironment to efficiently convert ATP into adenosine.Finally,we initially investigated the efficacy of cetuximab in combination with small molecular inhibitors of CD39 or CD73 to reverse immunosuppressive microenvironment in EGFRvⅢ CRC.ConclusionThis study found that the EGFRvⅢ mutation induces the formation of an adenosine mediated immune microenvironment that drives cetuximab resistance in CRC.On the mechanism level,EGFRvⅢ cells are stimulated by ICD and endoplasmic reticulum stress at higher level,which leads to ATP release on the one hand,and it upregulate the expression of adenosine metabolizing enzymes CD39 and CD73 through STAT3 pathway and promotes ATP conversion to immunosuppressive adenosine on the other hand.The combined strategy of targeted adenosine pathway and cetuximab improves the immune infiltration of EGFRvⅢ mutant colorectal cancer.