The Role And Potential Mechanism Of Anti-fibrotic Agent Pirfenidone In Tumor And Tumor Immune Microenvironment

Part ? The effect of single-agent pirfenidone on tumor growth and tumor immune microenvironment in miceObjective Pirfenidone is an orally active pyridone compound with a broad-spectrum anti-fibrotic effect.It has been approved for the treatment of idiopathic pulmonary fibrosis.In our previous study,we proved that pirfenidone can effectively inhibit the TGF-? signaling pathway,thereby inhibiting radiation-induced pulmonary fibrosis.It is known that TGF-?signaling pathway plays an important role in tumor growth and immune escape.Does pirfenidone affect the tumor and tumor immune microenvironment?Therefore,in this part of the study,we will explore the effect of single-agent pirfenidone on tumor and tumor immune microenvironment.Methods In this study,immunodeficient tumor-bearing mice were used to evaluate the effect of pirfenidone on tumor growth;immunocompetent tumor-bearing mice were used to evaluate the effect of pirfenidone on the tumor immune microenvironment.Flow cytometry was used to detect the changes of CD8+T cells and MDSCs in the tumors between the treatment group and the control group.Transcriptome sequencing was used to detect the differential expressed genes between the treatment group and the control group,and GO and KEGG enrichment analysis were used to perform functional analysis of the differential genes.Tgfb1 knockdown tumor cells were used to test whether the anti-tumor effect of pirfenidone depends on the TGF-? signaling pathway.Results In immunodeficient tumor-bearing mouse models,pirfenidone had no significant effect on tumor growth;in tumor-bearing mouse models with normal immunity,pirfenidone effectively inhibited tumor growth,enhanced CD8+T cells infiltration,and reduced the aggregation of MDSCs.RNA-seq analysis showed that the differential expressed genes between the treatment group and the control group were mainly enriched in immune-related pathways such as "immune system process","immune response",and"positive regulation of interferon-gama pathway".Knockdown of Tgfb1 in tumor cells counteracted the anti-tumor effect of pirfenidone.Conclusion Pirfenidone can not only reduce pulmonary fibrosis,but also enhance the anti-tumor effect of PD-L1 inhibitors.For cancer patients with comorbid pulmonary fibrosis,the combined application of PFD and immune checkpoint inhibitors may be an optional treatment strategy.Part ? The effect of pirfenidone combined with PD-L1 inhibitor in immunocompetent tumor-bearing mouse modelObjective In the first part of this study,we proved that in the immunocompetent tumor-bearing mouse model,pirfenidone can inhibit tumor growth by activating the anti-tumor immune microenvironment.In this part,we evaluate the anti-tumor effect of pirfenidone combined with PD-L1 inhibitors.Methods Immunocompetent tumor-bearing mice were randomly divided into four groups,and given PBS,single-agent pirfenidone,single-agent PD-L1 inhibitor and combined treatment,respectively.Tumor growth status was observed.Transcriptome sequencing was used to detect the changes in the differential expressed genes between the four groups.Flow cytometry was used to detect the changes of immune cells infiltrating in the tumor.Immunohistochemistry was used to detect the infiltration of CD3+T cells in the tumor.The body weight,blood biochemistry,and histopathological section of each organ in each group after treatment were observed to test whether the combination treatment is safe.Results Combination of pirfenidone with PD-L1 inhibitors can effectively inhibit tumor growth and prolong the survival time of mice.Transcriptome sequencing showed that the combination treatment significantly activated immune-related pathways and induced an inflammation-related immune phenotype.Flow cytometry and immunohistochemistry showed that combination treatment increased CD3+T cell infiltration and reduced MDSCs infiltration.No obvious toxic and side effects of the combination treatment were observed.Conclusion Combining pirfenidone with PD-L1 blockade induced synergic antitumor effects in mouse models.Combination treatment of the two drugs was safe in vivo.Part ? Pirfenidone combined with PD-L1 inhibitors in tumor mouse model with comorbid pulmonary fibrosisObjective Lung cancer and pulmonary fibrosis are common diseases of the respiratory system,and they often appear together.Pirfenidone is an anti-fibrotic drug used clinically.In the first two parts of the study,we proved that pirfenidone could enhance the anti-tumor immunity and synergy with PD-L1 inhibitors to inhibit tumor growth.In this part of the study,we plan to construct a tumor-bearing mouse model with comorbid pulmonary fibrosis,evaluate the effects of pirfenidone combined with PD-L1 inhibitors,and perform a side-to-side comparison with the TGF?RI inhibitor galunisertib.Methods C57BL/6 mice were given bleomycin intratracheally,and tumors were implanted subcutaneously 7 days later.After the tumor volume grew to 100 mm3,the mice were randomly divided into three groups:PBS control,pirfenidone+PD-L1 inhibitor and galunisertib+PD-L1 inhibitor.Changes in tumor volume and lung fibrosis were observed in mice.Results Compared with galunisertib at 37.5 mg/kg BID,pirfenidone at 500 mg/kg/day more effectively inhibited tumor growth and reduced lung fibrosis.Conclusion Pirfenidone not only reduced pulmonary fibrosis,but also enhanced the anti-tumor effect of PD-L1 inhibitors.For cancer patients with comorbid pulmonary fibrosis,the combination treatment of PFD with immune checkpoint inhibitors may be an optional treatment strategy.