TGF-β Inhibitor Enhances Pancreatic Cancer Response To Combined Irreversible Electroporation And Anti-PD1 Antibody

Background and Aim: Pancreatic cancer is one of the most lethal human malignancies with little response to radiation and chemotherapy.Irreversible electroporation(IRE)has attracted increasing attention as an effective intervention in locally advanced pancreatic cancer,IRE combined with checkpoint blockade has shown promising efficacy in clinical trials.However,the long-term survival rate remains a concern.Our previous study demonstrated that neutrophils were recruited into pancreatic cancer following the IRE treatment.Tumor-associated neutrophils(TAN)can be polarized into a pro-tumor N2 phenotype by the transforming growth factor β(TGF-β)in the tumor microenvironment.We aimed to investigate the effect of TAN modulated by the TGF-βinhibitor SB525334 on the efficacy of IRE combined with anti-PD1 antibody(αPD1)in treating pancreatic cancer.Methods: Data were extracted from TCGA databases to identify the association between TAN and prognosis in pancreatic cancer patients.Neutrophil depletion was conducted to investigate its impact on the therapeutic outcome of IRE+αPD1 treatment.The effect of phenotype changes of TAN on pancreatic cancer progression in IRE-treated mice was examined by immunohistochemistry and q PCR.The toxicity of SB525334 was tested in mice.Efficacy of IRE+αPD1+SB525334 on pancreatic cancer was evaluated in the mouse.Glutathione-responsive mesoporous silica nanoparticles that could encapsulate SB525334(d MSN-SB)were prepared to reduce the toxic and side effects of SB and increase the ability to modulate the TAN phenotype in the tumor microenvironment,and its properties were further tested.CCk8 was used to test the viability of cells treated by SB or d MSN-SB.The efficacy of IRE+αPD1+d MSN-SB was tested using subcutaneous models and orthotopic Panc02 tumor models.To further explore the mechanism of enhanced anti-tumor efficacy,immunohistochemistry,western blot,q PCR,and flow cytometry were used to examine the changes in TAN,immune cells,chemokines,and stromal components in the tumors following indicated treatment,and the interaction between TAN and T cells was identified in vitro.Results:(1)Tumor infiltration by neutrophils was unfavorable for the outcome of patients.Increased median survival was observed in Panc02-bearing mice with depleted neutrophils.(2)A substantial infiltration of neutrophils was observed in the tumor,and tumor progression accelerated as TAN shifted toward the N2 phenotype over time.(3)SB showed a high incidence of toxic and side effects in mouse models.The d MSN-SB exerted the same biological performance as SB in blocking the TGF-β pathway and modulating TAN,and it can effectively maintain the local drug concentration.Further research found that the polarization of TAN into N2 phenotype was effectively prevented,the number of CD8+ tumor-infiltrating T cells was increased,while a reduction of Treg cells and fiber content was observed in the tumor microenvironment following the treatment of IRE+d MSN-SB+αPD1.IRE+d MSN-SB+αPD1 is a safe treatment.(4)d MSN-SB can further enhance the efficacy of IRE+αPD1 in the treatment of pancreatic cancer.Long-lasting anti-tumor immunity was inducted in mice treated by IRE+SB+αPD1.IRE+d MSN-SB+αPD1 also significantly improved the survival of 4T1 breast cancer-bearing mice.Conclusion: N2 phenotype TAN correlates with poor prognosis in pancreatic cancer.Sustained inhibition of TGF-β signaling using d MSN-SB within the tumor promotes neutrophil polarization into an anti-tumor phenotype.d MSN-SB enhances pancreatic cancer response to the combined IRE and αPD1 therapy by modulating immunity in the tumor microenvironment.IRE+d MSN-SB+αPD1 is a safe treatment,and it may also be a promising treatment strategy for other “cold” tumors.