Construction And Efficacy Of Fibroblast Activation Protein-Targeted CAR-Macrophages In Lung Cancers

Background:Globally,lung cancer is the leading cause of cancer-related deaths,with 2.2 million new cases and 1.8 million deaths reported in 2020,and both incidence and mortality rates continue to rise,resulting in serious social burdens and economic losses.Non-small cell lung cancer(NSCLC)accounts for 80-85% of all lung cancer cases,and the majority of NSCLC patients are diagnosed at an advanced stage,even with metastases,severely affecting prognosis.The emerging of immunotherapy,especially immune checkpoint inhibitors(ICIs),has initiated a new era of anti-tumor treatment.Single or combination of ICIs have become the first-and second-line treatment strategies for NSCLC,with a 5-year overall survival rate as high as 40% for patients with high PD-L1 expression.Despite the significant improvement in overall survival rates for NSCLC patients with ICIs,oncologists still face treatment challenges due to primary-or secondary-resistance,and exploring strategies to improve tumor response to immunotherapy could represent a new leap forward for NSCLCs.Since the concept of "seed and soil" was proposed,the role of the tumor microenvironment(TME)in cancer resistance has received increasing attention.The TME may dynamically converse between the immunosuppressive TME and the immune-active TME,playing an important role in the development of immune resistance.Cancer-associated fibroblasts(CAFs)are an important component of the TME and have been proved to be involved in the development of resistance in various malignancies.Fibroblasts are in a resting state in healthy condition.However,in tumors,the constant accumulation of tumor cells causes continuous damage and induces chronic and irreversible activation of fibroblasts,ultimately resulting in the accumulation and proliferation of cancer-associated fibroblasts(CAFs)with strong proliferative characteristics and sustained self-activation ability.CAFs participate in the remodeling of blood vessels and the extracellular matrix(ECM)and secrete various cytokines to regulate immune responses,promoting tumor progression.The role of CAFs in resistance to immunotherapy has attracted widespread attention and may become a potential target for novel treatment strategies.Fibroblast activation protein(FAP)is a transmembrane serine protease and a member of the dipeptidyl peptidase 4(DPP4)protein family,approximately 97 k Da.Under physiological conditions,FAP expression is extremely low in normal adult tissues.However,its expression will be significantly increased in some chronic inflammatory diseases,fibrotic diseases,and tumors.In non-small cell lung cancer(NSCLC),FAP is highly expressed on CAFs and is associated with the formation of an immunosuppressive tumor microenvironment(TME),which in turn affects the efficacy of immunotherapy and promotes the development of tumor resistance.Therefore,targeting FAP for the treatment of activated CAFs may enhance the tumor’s response to immunotherapy and improve resistance to lung cancer immunotherapy.Chimeric antigen receptor(CAR)therapy is an emerging cell therapy,with CAR-T and CAR-NK treatments that have achieved remarkable efficacy in hematological malignancies.However,due to limited infiltration in solid tumors and significant side effects,the application of CAR-T and CAR-NK therapies has been restricted.Macrophages,being diverse in origin and heavily infiltrating tumor tissues,might overcome the infiltration issues faced by CAR-T and CAR-NK,while chimeric antigen receptor macrophages(CAR-M)have shown good safety during the process.Targeting FAP with CAR-M could further improve the TME by degrading ECM and phagocytosing cells expressing FAP,potentially effectively enhancing anti-tumor immune responses and overcoming TME-related resistance to tumor immunotherapy.This project aims to construct FAP-targeted CAR-M based on clarifying the expression of FAP in lung cancer and its relationship with lung cancer immune resistance,and to explore the therapeutic effects of FAP-CAR-M in a mouse lung tumor model and a lung fibrosis model related to lung cancer development,in order to provide guidance for lung cancer treatment strategies.Methods:1.Effects of FAP in immunotherapy resistance in lung cancer(1)Objects: Lung cancer patients data from the TCGA database and normal control data from the GTEx database.(2)Molding and Grouping: Subcutaneous tumor model was established using the LLCluc cell line in mice(3)Testing Methods: RNAseq data and corresponding clinical information of 1017 lung cancer patients and 578 normal controls were collected from public databases.With bioinformatics methods,including survival analysis,GO enrichment analysis,KEGG pathway analysis,co-expression analysis,EPIC algorithm,and TIDE scoring,the role of FAP in NSCLC,especially its relationship with immunotherapy resistance,was systematically investigated.IHC staining of subcutaneous tumors and clinical patient pathological tissues was performed to verify the impact of FAP on CD8+ T cell infiltration in lung cancer.2.Construction and in vitro functional testing of FAP-CAR-M(1)Objects: Mouse Ana-1 macrophage cell line.(2)Molding and Grouping: FAP was selected as the target protein,and a CAR sequence containing Flag,FAP-Sc Fv,CD8 hinge,CD8 TM,Fc Rγ,and CD19 fragments was designed and transduced into the Ana-1 cell line with a lentiviral vector.(3)Testing Methods: The successful construction of the sequence was verified by gel electrophoresis.The successful transduction of CAR was confirmed by Western blot and flow cytometry(FCM).Cell proliferation was assessed using the CCK-8 method.Cell phagocytic activity was assessed using a cell fluorescence phagocytosis assay.Cell oxidative stress capability was assessed by measuring reactive oxygen species(ROS)release.Cell imaging was used to detect the macrophages’ phagocytic ability on FAP-expressing K562 cells.3.Efficacy and safety of FAP-CAR-Ms in mouse lung cancer models(1)Objects: Female wild-type 6-week-old C57BL/6 mice.(2)Molding and Grouping: Subcutaneous tumor model and Orthotopic tumor model were established using the LLC-luc cell line in mice,with a self-designed in situ tumor tissue implantation kit.The mice were randomly divided into a tumor group and FAP-CAR-M treatment group.Tumor tissue specimens were collected on day 14.(3)Testing Methods: The differences in the immune microenvironment between subcutaneous and orthotopic tumors were compared using single-cell sequencing technology.Tumor growth was compared using in vivo imaging and measurement of tumor tissues.The infiltration of immune cells in tumor tissue was detected using pathological sections,immunohistochemistry,and FCM.Therapeutic effects and safety of FAP-CAR-Ms were assessed by measuring biochemical indicators and pathological sections.4.Effects of FAP-CAR-Ms in early intervention of the precancerous fibrotic process in lung cancer(1)Objects: Female wild-type 6-week-old C57BL/6 mice.(2)Molding and Grouping: A mouse pulmonary fibrosis model was established by bleomycin(BLM)intratracheal injection,and the mice were randomly divided into a normal control group,FAP-CAR-M treatment group,Pirfenidone treatment group,and combined treatment group.Specimens were collected on day 21.(3)Testing Methods: Co-culture with activated primary mouse fibroblast cells was performed to assess the phagocytic ability of FAP-CAR-Ms.Polarization state was determined via FCM,while the phosphorylation levels of signaling pathways were assessed through WB and cytokine release was measured using ELISA.The degree of fibrosis and collagen deposition in mice models was assessed using pathologoical sections and Masson staining.The infiltration of FAP-CAR-Ms in lung tissue was determined using FCM.Changes in the expression of α-smooth muscle actin(α-SMA)and FAP were detected using immunohistochemistry and Western Blot(WB).Blood biochemistry indicators were tested to clarify the safety of FAP-CAR-Ms.Results:1.Effects of FAP in immunotherapy resistance in lung cancerFAP expression was elevated in a variety of tumors.Within NSCLC,both adenocarcinoma and squamous cell carcinoma exhibited a significant increase in FAP expression.In adenocarcinoma,patients with high FAP expression had a shorter overall survival,while in squamous carcinoma,there was no significant difference.When focusing on the impact of FAP on immune function,it showed that FAP was positively correlated with the expression of multiple immune checkpoints,and that individuals with high FAP expression had higher TIDE scores,indicating a poorer response to immune checkpoint inhibitor treatments and suppressed CD8+ T cell infiltration.Co-expression analysis showed that there were 146 genes that were strongly correlated with FAP expression in NSCLC(Cor>0.6,p<0.05),with functions enriched in matrisome proteins,extracellular matrix formation and organization,cell adhesion and migration,angiogenesis,etc.Based on FAP expression levels,NSCLC could be divided into high and low FAP expression groups,and 690 differential genes had been identified(p<0.05).Functional enrichment analysis revealed that their functions were mainly in immune cell migration,extracellular matrix modification,and metabolic process.Mouse lung tumor model and patients’ pathological samples also demonstrated that high FAP expression correlates with reduced CD8+ T cell infiltration and increased PD-1 expression.2.Construction and in vitro functional testing of FAP-CAR-MThe FAP-targeted CAR sequence was successfully transduced into the Ana-1macrophage cell line using a lentiviral vector,and monoclonal strains were selected for further culture.Under normal conditions,FAP-CAR-Ms showed no significant differences in proliferation activity and phagocytic ability compared to Ana-1 cells,but released less ROS.In co-culture with FAP-positive cells,FAP-CAR-Ms showed stronger phagocytic ability than Ana-1 cells.3.Efficacy and safety of FAP-CAR-Ms in mouse lung cancer modelsIn subcutaneous and orthotopic tumor mouse models of lung cancer,there were differences in the infiltration of immune cells,with fewer T cells infiltrating in subcutaneous tumor models.However,in both tumor models,intravenous injection of FAP-CAR-M slowed tumor growth,inhibited FAP expression in the TME,increased CD8+ T cell infiltration.And FAP-CAR-M treatments did not affect the structure or function of other organs.4.Effects of FAP-CAR-Ms in intervention of precancerous fibrotic process in lung cancerWhen co-culturing with activated primary mouse fibroblasts,FAP-CAR-Ms showed stronger phagocytic capabilities,more significant phosphorylation of molecules in the PI3K/Akt pathway,and no significant difference in the release of TNF-α,IL-6,TGFβ1,while IL-10 release capacity was reduced compared to the Ana-1 cells.And this process did not change the polarization state of FAP-CAR-Ms.In BLM-induced mouse pulmonary fibrosis models,In BLM-induced mouse pulmonary fibrosis models,the control group showed significant collagen infiltration on day 21,with significantly increased expression of α-SMA and FAP,while FAP-CAR-M treatments significantly reduced the degree of fibrosis and α-SMA and FAP expression,comparable to the effects of pirfenidone.The combined treatment group showed even milder fibrosis,without affecting mouse liver and kidney function.Conclusions:1.High FAP expression in NSCLC may inhibit immune cell infiltration through regulating ECM components and metabolism,thereby weakening the effects of immune therapy,and increase tumor resistance to immune treatments.2.The introduction of the FAP-CAR sequence into macrophages to construct targeted FAP CAR-M does not affect the proliferation activity of macrophages and their phagocytosis of large particles,but enhances the phagocytic capacity for cells expressing FAP.3.There is organ-heterogeneity between the subcutaneous tumor model and the orthotopic tumor model in mice.In both models,intravenous injection of FAP-CAR-M can inhibit the accumulation of FAP-expressing CAFs,enhance CD8+ T cell infiltration,restrict tumor growth,and improve the effect of immune therapy.4.In BLM-induced mouse pulmonary fibrosis models,FAP-CAR-M treatments can alleviate the degree of pulmonary fibrosis,with efficacy comparable to pirfenidone,and may play an inhibitory role in reducing the likelihood of lung fibrosis patients developing lung cancer.