Study On Therapeutic Biomarkers Of Targeted Therapy And Immunotherapy In Non-small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related death.With the development of molecular targeted therapy and immunotherapy,the prognosis and survival of non-small cell lung cancer(NSCLC)patients have been significantly improved.However,some patients remain insensitive to current therapeutic drugs or develop resistance after treatment,leading to disease progression.Therefore,identifying therapeutic markers for targeted therapy and immunotherapy in NSCLC will contribute to the risk stratification of patients and monitoring of drug resistance,provide molecular targets for studies of drug resistance,and guide the follow-up treatment for drug-resistant patients.This thesis consists of two parts:The first part focuses on EML4-ALK positive NSCLC patients.Based on Luminex liquid suspension chip and data-independent acquisition-mass spectrometry technology,we screened and validated prognostic protein markers in baseline and longitudinal plasma samples.Additionally,we elucidated the molecular mechanisms of CCL20-induced resistance and its reversal by anlotinib in cell and animal experiments.The second part focuses on NSCLC patients treated with immunotherapy.Through integrated analysis of bulk and single-cell genomics,transcriptomics,and proteomics profiles,we constructed a cancer-associated fibroblasts-related prognostic model and validated the prognostic efficacy of FBLIM1 in a clinical cohort treated with immunotherapy.Part 1:Study on drug resistance and prognostic markers of targeted therapy for EML4-ALK positive non-small cell lung cancerChapter 1:Role of chemokine-mediated angiogenesis in resistance towards crizotinib and its reversal by anlotinib in EML4-ALK positive NSCLCThe identification of early plasma biomarkers for clinical outcomes and drug resistance has key importance for risk stratification in anaplastic lymphoma kinase(ALK)positive advanced NSCLC patients.Moreover,it remains unclear whether the antiangiogenic drug anlotinib can reverse the resistance of ALK-tyrosine kinase inhibitor(ALK-TKI)crizotinib,and no research has explored the effect of anlotinib combined with crizotinib on ALK-positive NSCLC.In this study,76 baseline and longitudinal plasma samples from 61 ALK-positive NSCLC patients receiving crizotinib treatment were analyzed by Luminex liquid suspension chip for 40 chemokines.RNA sequence(RNA-seq)was used to identify differentially expressed genes(DEGs)between H3122 and H3122-crizotinib resistant(H3122CR)cells.Tube formation assay was performed to investigate the effect of chemokines on angiogenesis.And H3122CR-derived xenograft model was constructed to validate the efficacy and safety of anlotinib combined with crizotinib in vivo.Baseline and progression plasma samples detection suggested that CCL20 played a crucial role in monitoring and predicting the clinical response of crizotinib(hazard ratio for progression-free survival:2.27(1.13-4.58);for overall survival:2.7(1.23-5.8)).RNAseq results for H3122 and H3122CR cells showed that high expression of chemokines and angiogenesis pathways were involved in crizotinib resistance.Subsequently,in vitro experiments indicated that CCL20 may induce crizotinib resistance by activation of angiogenesis via JAK2/STAT3-CCL20-VEGFA/IL6 axis.We further found that antiangiogenic TKI anlotinib could reverse crizotinib resistance by inhibiting chemokinesinduced angiogenesis,and anlotinib combined with crizotinib has a better antitumor effect than monotherapy in vitro&in vivo.Overall,CCL20-mediated angiogenesis is involved in crizotinib resistance and could be overcome by using anlotinib in EML4-ALK positive NSCLC.The combination of anlotinib and crizotinib is a promising strategy for patients resistant to ALK-TKIs.Chapter 2:Longitudinal plasma proteomic profiling of EML4-ALK positive lung cancer receiving ALK-TKIs therapyThe introduction of ALK-TKIs has demonstrated remarkable therapeutic effect in ALK-positive NSCLC.Identifying robust prognostic and drug-resistance biomarkers can enhance our understanding of the mechanism underlying drug resistance and guide the next-line therapies for patients.In this study,we profiled 737 plasma proteins from 159 pre-treatment and ontreatment plasma samples of 63 ALK-positive NSCLC patients using data-independent acquisition-mass spectrometry(DIA-MS).Consensus clustering algorithm was used to identify subtypes with distinct biological features.A plasma-based prognostic model was constructed using LASSO-Cox method.We performed Mfuzz analysis to classify the patterns of longitudinal changes in plasma proteins during treatment.52 treatment-naive plasma samples from another independent ALK-TKI treatment cohort were collected to validate the potential prognostic markers using ELISA.We identified three subtypes of ALK-positive NSCLC with distinct biological features and clinical efficacy.Patients in subgroup 1 exhibited activated humoral immunity and inflammatory responses,increased expression of positive acute-phase response proteins,and the worst prognosis.Then we constructed and verified a prognostic model that predicts the efficacy of ALK-TKI therapy using the expression levels of five plasma proteins(SERPINA4,ATRN,APOA4,TF,and MYOC)at baseline.Next,we explored the longitudinal changes in plasma protein expression during treatment and identified four distinct change patterns(Clusters 1-4).The longitudinal changes of acute-phase proteins during treatment can reflect the treatment status and tumor progression of patients.Finally,we validated the prognostic efficacy of baseline plasma CRP,SAA1,AHSG,SERPINA4,and TF in another independent NSCLC cohort undergoing ALK-TKI treatment.This study contributes to the search for prognostic and drug-resistance biomarkers in plasma samples for ALK-TKI therapy and provides new insights into the mechanism of drug resistance and the selection of follow-up treatment.Part 2:Integrative analyses of bulk and single-cell RNA-seq identified cancerassociated fibroblasts-related signature as a prognostic factor for immunotherapy in NSCLCAn emerging view regarding cancer-associated fibroblast(CAF)is that it plays a critical role in tumorigenesis and immunosuppression in the tumor microenvironment(TME),but the clinical significance and biological functions of CAFs in NSCLC are still poorly explored.Here,we aimed to identify the CAF-related signature for NSCLC through integrative analyses of bulk and single-cell genomics,transcriptomics,and proteomics profiling.Using CAF marker genes identified in weighted gene co-expression network analysis(WGCNA),we constructed and validated a CAF-based risk model that stratifies patients into two prognostic groups from four independent NSCLC cohorts.The high-score group exhibits a higher abundance of CAFs,decreased immune cell infiltration,increased epithelial-mesenchymal transition(EMT),activated transforming growth factor beta(TGFβ)signaling,and a limited survival rate compared with the low-score group.Considering the immunosuppressive feature in the high-score group,we speculated an inferior clinical response for immunotherapy in these patients,and this association was successfully verified in two NSCLC cohorts treated with immune checkpoint blockades(ICBs).Furthermore,single-cell RNA sequence datasets were used to clarify the molecular mechanisms underlying the aggressive and immunosuppressive phenotype in the highscore group.We found one of the genes in the risk model,filamin binding LIM protein 1(FBLIM1),is mainly expressed in fibroblasts and upregulated in CAFs compared to fibroblasts from normal tissue.FBLIM1-positive CAF subtype was correlated with increased TGFβ expression,higher mesenchymal marker level,and immunosuppressive tumor microenvironment.Finally,we demonstrated that FBLIM1 might serve as a poor prognostic marker for immunotherapy in clinical samples.In conclusion,we identified a novel CAF-based classifier with prognostic value in NSCLC patients and those treated with ICBs.Single-cell transcriptome profiling uncovered FBLIM1-positive CAFs as an aggressive subtype with a high abundance of TGFβ,EMT,and an immunosuppressive phenotype in NSCLC.