| [Background and Aims]In recent years,the third-generation epidermal growth factor receptor(EGFR)tyrosine kinase inhibitors(TKI),represented by osimertinib,has brought benefits to survival and the quality of life to patients with advanced non-small cell lung cancer(NSCLC)who have been previous treated and acquired T790M drug-resistant mutation.However,most patients will inevitably have disease progression after second-line treatment with osimertinib,and the drug resistance mechanism of 30-40%patients is unknown.At present,the post-treatment of osimertinib resistance is mainly based on the clinical symptoms and disease progression mode.With the in-depth study of tumor micro environment(TME)in recent years,we realize that the occurrence and progress of tumor is a process of continuous interaction with the surrounding environment.Therefore,exploring and understanding the changes of these microenvironments plays a vital role in overcoming tumor drug resistance.Therefore,our research will explore the related changes of tumor microenvironment before and after the second-line treatment of osimertinib,find the potential intervention that can delay drug resistance,and explore its potential mechanism.[Methods]The first part of present research is mainly divided into four parts:in the first part,based on the results of RNA sequencing(RNA-seq),the difference of immune cell infiltration in the microenvironment before and after osimertinib resistance is inferred by CIBERSORT and xCell algorithm.Subsequently,we conducted dose climbing and continuous high-dose induction of osimertinib sensitive cell line H1975 in vitro to establish osimertinib resistant cell line H1975OR and further verify its drug-resistant phenotypes.Tumor cells,tumor cell derived exosomes and tumor cell conditioned medium were co-cultured with immune cells to observe the interaction between tumor cells and immune cells.The second part is clinical retrospective study,which have enrolled patients with EGFR mutant NSCLC who were resistant to osimertinib for second-line treatment.We compare their baseline information,analyzed the response rate,progression free survival(PFS)and overall survival(OS)of different treatment methods after drug resistance,and analyzed the prognostic factors of treatment methods after osimertinib resistance through Cox regression.In the third part,the humanized immune system was reconstructed by injecting human umbilical cord blood stem cells(HSC)into NOG-EXL mice with severe immune deficiency.In the xenograft tumor model of humanized immunized mice,osimertinib combined with anti-vascular therapy and single drug therapy were given respectively.The general growth,tumor size and weight changes of mice in each group were recorded,and the efficacy of combined treatment on drug-resistant tumors was observed.The transplanted tumor,peripheral blood and spleen of mice were harvested for flow cytometry to detect the difference of tumor infiltrating immune cells.In the fourth part,through the single-cell sequencing detection of fresh transplanted tumor samples of mice in the above treatment group,we explore the potential mechanism of osimertinib combined with anti-vascular therapy on the change of tumor microenvironment from the aspects of composition,function and intercellular communication analysis.[Results]RNA-seq results showed that the immune score and microenvironment score in drug-resistant tissues were significantly lower than those before treatment(P<0.001).After drug resistance,the infiltration of CD8+T cells decreased significantly(P<0.05),the proportion of M0 macrophages increased significantly(P<0.01),and the proportion of M2 macrophages increased.The induced H1975OR obtained the osimertinib resistant phenotype.The in vitro co-culture experiment of tumor cells and macrophages showed that the tumor cells derived exosomes could be absorbed by macrophages.The results of morphology and polarization markers showed that sensitive tumor cells could promote the polarization of macrophages to M1 type,and drug-resistant cells could promote the polarization of macrophages to M2 type,especially the stimulation effect of exosomes.The results of real-time cell dynamic monitoring showed that the inhibitory effect of macrophages stimulated by drug-resistant tumors on tumor proliferation was weakened,which was mainly mediated by exosomes.In the in vitro co-culture experiment of tumor cells and PBMC,flow cytometry showed that the proportion of late activated CD4+T cells decreased after stimulated by drug-resistant cell exosomes,and the proportion of depleted CD4+T cells increased after stimulated by drug-resistant cell conditioned medium.The results of real-time cell dynamic monitoring showed that PBMC from different sources showed tumor killing effect under two E:T ratios.When the E:T ratio was 10:1,PBMC had shorter response time and stronger killing effect on drug-resistant cells.A total of 111 advanced NSCLC patients were included in the retrospective analysis,who suffered disease progression after second-line osimertinib and re drug resistance,and post drug resistance treatment.The overall objective rate was 27.0%,the overall disease control rate was 86.5%,the median PFS was 6.3 months(95%Cl:5.4-7.2 months),and the median OS was 12.3 months(95%Cl:10.9-13.6 months).The median PFS of osimertinib combined with anti-vascular group was the longest 9.84 months(95%Cl:7.0-12.6 months),which was significantly better than that of chemotherapy group(P=0.012),osimertinib single drug group(P=0.003)and best supportive treatment group(P<0.001).The median OS of osimertinib combined with anti-vascular treatment group was the longest 16.79 months(95%Cl:14.97-18.61 months),which was significantly better than chemotherapy(P=0.026),chemotherapy combined with osimertinib(P=0.021),chemotherapy combined with immunotherapy(P=0.006),and the best supportive treatment(P=0.004).Cox univariate and multivariate analysis showed that PS score,biopsy site and treatment after drug resistance were all associated with the risk of disease progression,while no clinical features were associated with the risk of death.In vivo experiments,we successfully established the xenograft tumor model of humanized mice.The efficacy experiment showed that the growth inhibition effect of osimertinib on H1975OR was significantly weakened(P<0.05),and the inhibition effect of osimertinib combined with anlotinib on H1975OR was significantly stronger than that of osimertinib alone(P<0.05).The results of flow cytometry analysis of tumor tissues of mice in each treatment group showed that,compared with S-O group,the proportion of CD4+T cells activated in the middle and middle stage of drug-resistant tumors decreased significantly(P<0.05),the proportion of CD4+T cells expressing PD-1 also decreased significantly(P<0.05),and TGF-β expressing M2 immunosuppressive function.The proportion of macrophages increased significantly(P<0.001)in R-O group.Comparing the R-O+A and R-O group,the combined treatment could significantly increase the infiltration of CD4+T cells(P<0.05),significantly increase the proportion of CD4+T cells activated in the middle stage of CD25+and significantly increase the expression of PD-1 on CD8+T cells(P<0.05).The results of single cell test showed that there were fewer CD8+T cells in the drug-resistant tumor microenvironment,especially CD8 NK、CD8.TMERA、CD8.TEFF.In addition,drug-resistant tumors also had more TAM with M2 phenotype that secrete VEGFA,which can be mainly divided into two subtypes:IL1β.mo and CCL18.mo.Compared with osimertinib alone,osimertinib combined with anlotinib can significantly increase the number of T cells of CD8.TMERA、CD8.TEFF,and CD8.NK subtypes in transplant resistant tumors,while the number of immunosuppressive Treg cells decreased.In addition,the combination treatment group also reduced the number of CCL18.mo and IL-1β.mo and the expression of related functional genes.Further receptor-ligand communication analysis between tumor cells and macrophages showed that the interactions between them were significantly upregulated in the osimertinib-resistant cell than osimertinib-sensitive tumors,with upregulated ligand(macrophage)-receptor(tumor cell)pairs including ICAM-AREG,SPP1-CD44.Combined with the results of protein interaction network detected by exosome mass spectrometry analysis,we also noted a significant increase in tumor cell-derived exosomes like CD44,ICAM1.These results suggested that communication with macrophages can be upregulated via exosomes secreted from H1975OR cells.[Conclusions]In clinical specimens,the tumor tissue tends to be immunosuppressive after osimertinib resistance,which is mainly manifested in the decrease of T cell infiltration and the increase of macrophage polarization.In vitro co-culture suggests that this is related to the inhibition of T cell activation and the promotion of macrophage polarization towards M2 phenotype by drug-resistant cells.Osimertinib combined with anti-vascular therapy can improve PFS and OS in patients with second-line resistance to osimertinib.In vivo pharmacodynamic studies also confirmed that osimertinib combined with anti-vascular drug anlotinib can significantly inhibit tumor growth compared with osimertinib alone.In vivo experiments in humanized mice showed that the immunosuppressive phenotypes in drug-resistant transplanted tumors mainly demonstrated by the decrease of cytotoxic CD8+T cell infiltration,the decrease of the proportion of proliferating T cells,the increased infiltration of TAM.mo polarized to M2 phenotype,and increased VEGFA secretion of TAM.The above immunosuppressive phenotypes can be reversed after combined use of osimertinib and anti-vascular drugs,which may be achieved by inhibiting VEGFA-VEGFR pathway.The combined analysis of the tumor-derived exosome protein interaction network and the tumor cell-macrophages communication network suggests that macrophages can phagocytose tumor-derived exosomes and act back on tumor cells,possibly through the action of ICAM-AREG and SPP1-CD44 receptor ligands. |
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