The Effect And Potential Mechanism Of Nintedanib In Tumor Immunotherapy

Part Ⅰ The effect of nintedanib combined with anti-PD-L1 antibody in tumor-bearing mouse modelObjective Nowadays,immune-checkpoint inhibitors(ICIs),such as anti-programmed cell death protein 1 antibody(αPD-1)/anti-programmed death-ligand 1 antibody(αPD-L1),have been widely applied in clinical and scientific research.Despite their effective antitumor effects in clinical tumor therapy,most tumors are still resistant to ICIs,and long-term benefits are lacking.Therefore,for these cases,there is an urgent need to develop new methods to enhance the efficacy of ICIs therapy.Nintedanib,a potent tyrosine kinase inhibitor,is known to be an excellent anti-angiogenic agent.It has been reported that anti-angiogenic drugs combined with immunotherapy can effectively increase the efficacy of immunotherapy.However,there are few studies on nintedanib combined with immunotherapy in cancer treatment.This part aims to explore the anti-tumor and anti-metastatic effect and safety evaluation of nintedanib combined withαPD-L1 in tumor-bearing mouse models,and to provide a new combination strategy for tumor immunotherapy.Methods Two mouse tumor cell lines,MC38 and LLC,were used to construct colorectal and lung tumor subcutaneous xenograft models,respectively,while the triple-negative breast tumor cell line 4T1 was used to construct a mouse model of spontaneous lung metastasis of orthotopic breast cancer.Mice were randomly divided into four groups and received PBS,nintedanib,αPD-L1,and combination therapy.The therapeutic efficacy was evaluated by the tumor growth curve,mouse survival,the number of lung metastatic nodules,the TUNEL immunofluorescence staining,and ki67 histochemical staining of tumor tissue sections.The expression of epithelial-mesenchymal transition(EMT)-related proteins in 4T1 tumor tissues and different tumor cell lines after nintedanib treatment was detected by Western Blot.After different treatments,the safety evaluation was detected by recording the body weight changes of mice,collecting serum for blood biochemical assay and main organs for paraffin section.Results In the MC38 and LLC tumor-bearing models,nintedanib combined with αPD-L1 group had significant anti-tumor effects compared to other groups.Among,in the MC38 model,some tumors had complete regression.The overall survival of tumor-bearing mice was effectively prolonged in both animal models after combined therapy.In the 4T1tumor-lung metastasis model,the combination group also showed a good anti-tumor effect.What’s more,the results showed that combined therapy significantly reduced the number of pulmonary metastatic nodules,while nintedanib alone has only a partial effect.Nintedanib can inhibit the expression of EMT-related interstitial proteins in 4T1 tumor tissues and multiple tumor cell lines.The safety evaluation results revealed that there were no obvious toxic effects on major organs and blood biochemical indicators after nintedanib application.Conclusion The combination of nintedanib and αPD-L1 showed significant anti-tumor and anti-metastatic effects,which effectively prolonged the overall survival of tumor-bearing mice and was no obvious side effects.Due to its effectiveness and safety,this synergistic therapy provides a new combination drug regimen for tumor immunotherapy.Part Ⅱ The effect of nintedanib combined with anti-PD-L1 antibody on tumor microenvironment Objective In the first part,we found that nintedanib could significantly enhance the antitumor efficacy of αPD-L1 and improve the survival of tumor-bearing mice.As we all know,the efficacy of immunotherapy is inseparable from the tumor microenvironment state.However,the effect of nintedanib on the tumor microenvironment is pending discussion.This part aims to investigate the effect of nintedanib combined with αPD-L1 on the tumor microenvironment and the underlying mechanism of enhanced immunotherapy.Methods The LLC and MC38 tumors were embedded for further experiments.The immunofluorescence staining(CD31 and α-SMA)was used to explore the intratumoral angiogenesis status;The immunohistochemical staining of T cell surface markers(CD3 and CD8)was performed on tumor tissue sections to detect the T cells infiltration in tumor tissue;Flow cytometry was used to detect the activation state and infiltration of different immune cells in the tumor immune microenvironment between different treatment groups.Results The immunofluorescence staining results showed that nintedanib had a good anti-tumor angiogenesis effect,and the proportion of functional perfusion vessels(α-SMA/CD31)was significantly increased.In the MC38 model tumor,the combination group significantly increased the number of CD3+ and CD8+ T cells I tumor area compared to other groups.Flow cytometry results showed that the combination group could effectively increase the infiltration and activation ratio of CD8+ T cells and dendritic cells(DCs).The proportion of M2-type polarization of macrophages and the proportion of infiltrating myeloid-derived suppressor cells(MDSCs)were significantly reduced.Conclusion The nintedanib combined with αPD-L1 treatment group can promote tumor vessels normalization,reshape the tumor immune microenvironment,improve the tumor immunosuppressive microenvironment,and activate the anti-tumor immune response,thus exerting an excellent anti-tumor effect.Part Ⅲ Study on the mechanisms of nintedanib improve the anti-tumor efficacy of anti-PD-L1 antibody Objective Previous research has demonstrated nintedanib combined with αPD-L1 has significant anti-tumor and anti-metastatic effects and effectively activates the tumor immune microenvironment.However,the specific pathway to reshape tumor immune microenvironment and the mechanism to enhance the efficacy of immunotherapy are unclear.This part aims to further explore the mechanism by which nintedanib activates the anti-tumor immune response and enhances the therapeutic efficacy of αPD-L1,thus providing a more reliable basis for the clinical application of nintedanib combined with αPD-L1.Methods MC38 tumor tissues were used to perform RNA sequence to detect gene expression differences between different treatment groups.To further explore the pathway and function statues,GO,KEGG and GSEA were used for enrichment analysis.Moreover,validation of differential genes by RT-qPCR and cytotoxicity detected by CCK-8 were performed.Immune-related proteins after nintedanib treatment expression were detected by Western Blot and flow cytometry.Results RNA-seq analysis showed that combination group differential genes were mainly enriched in immune-related pathways such as "RESPONSE TO INTERFERON-GAMMA" and "ANTIGEN PROCESSING AND PRESENTATION",compared to other groups.Among,genes related to immune chemokines,such as: Ccl6,Ccl8,Ccl9,Cxcl2 and Cxcl3,were significantly upregulated after combined treatment and verified by RT-q PCR.In addition,KEGG enrichment analysis showed that there was activation of JAK-STAT signaling pathway in the combined group.At the protein level,nintedanib combined with αPD-L1 can significantly activate the phosphorylation of signal transducer and activator of transcription 3(STAT3)in vivo,and upregulate the expression of PD-L1,β2 microglobulin(β2M)and antigen processing-related transporter(TAP1);In vitro experiments,low concentrations(1 μM)nintedanib can also activate STAT3 phosphorylation,upregulate PD-L1,β2M rather than TAP1 in different tumor cell lines(A549,MC38 and LLC).In addition,nintedanib combined with IFN-γ treatment further activates the expression of these proteins.Conclusion Nintedanib combined with αPD-L1 can further activate immune-related pathways in vivo,improve IFN-γ responsiveness,and MHC-1-mediated antigen presentation process,thereby improving the αPD-L1 efficacy.These effects may be partly attributed to phosphorylation of STAT3,and upregulation of PD-L1 and MHC-I expression in tumor cells.Part Ⅳ The antitumor effect of nintedanib combined with anti-PD-L1 antibody in mice with pulmonary fibrosis Objective With the popularization of immunotherapy,the incidence of immune-related adverse events such as pneumonia has increased significantly.We found that clinically patients with interstitial lung disease(ILD)will increase the risk of lung cancer,and some tumor patients have suffered from underlying lung diseases of varying degrees,such as chronic obstructive pulmonary disease and ILD,including idiopathic pulmonary fibrosis(IPF),ICIs therapy-related ILD,and radiation-induced pulmonary fibrosis.After ICIs treatment,these patients are more likely to cause ICIs-related lung disease and aggravate pulmonary symptoms,thus limiting the application of ICIs and leading to treatment discontinuation and tumor progress.Therefore,there is an urgent need to develop new approaches that can both alleviate pulmonary complications and improve the efficacy of ICIs therapy.In this part,we intend to explore the anti-pulmonary fibrosis and anti-tumor effects of nintedanib combined with αPD-L1 in an IPF-tumor bearing model.Methods IPF model was induced by intratracheal nebulization of bleomycin sulfate(1.5 mg/kg),and luciferase-MC38 cells were implanted subcutaneously after 7 days.Mice were randomly divided into three groups(tumor volume reached around 100 mm3)and received PBS,αPD-L1,nintedanib combined with αPD-L1 treatment.The survival and tumor volume of mice were observed by in vivo imaging technology,and the pulmonary fibrosis was evaluated by Mirco-CT imaging,H&E,and Masson staining of tissue sections.Results To mimic the tumor patients with pulmonary complications,IPF-tumor bearing model was established and received different treatment.The combination therapy significantly inhibited tumor growth and prolonged survival compared with other groups.Although the αPD-L1 monotherapy group partially inhibited tumor growth,this group had the highest mortality rate.The results of lung CT scan and staining showed that the control and the αPD-L1 group had apparent significant pulmonary fibrosis,while the combination group significantly reduced pulmonary fibrosis.Conclusion Nintedanib combined with αPD-L1 can play an excellent anti-tumor and anti-pulmonary fibrosis effect,providing a safer and more effective combination strategy for immunotherapy in cancer patients with pulmonary fibrosis.