Ultrsound Enhanced Tumor Perfusion Promotes Infiltration Of CD8~+T Cells And Potenciates Antitumor Efficacy Of PD-L1 Antibody

Poor infiltration of CD8⁺T cell is the critical problem for the insufficiency of immunotherapy in solid tumors,and hypovascular and hypoxic microenvironment formed by the aberrant tumor vessels is a major factor that blocks T cell infiltration.Hence,strategies to enhance tumor blood perfusion is of great clinical significance for promoting CD8⁺T cell infiltration and thus potentiating tumor immunotherapy.Previously,we have found that low intensity ultrasound stimulated microbubble cavitation（USMC）could enhance blood perfusion of solid tumors and reperfuse ischemic area,which was referred to as“ultrasound perfusion enhancement effect”.Whether the effect could induce CD8⁺T cell infiltration and further enhance tumor immunotherapy has not been reported before.In addition,the perfusion effect was not stable enough.Herein,we firstly needed to optimize the acoustic parameters for the effect,and then further explored the response of CD8⁺T cell in tumors caused by the perfusion effect.Finally,we investigated the synergistic anti-tumor efficacy and mechanism of tumor perfusion enhancement effect by USMC combined with PD-L1 antibody,which would provide a new method for enhancing tumor immunotherapy.Objective1.To select optimal acoustic parameters of ultrasound perfusion enhancement effect for further researches or applications.2.To explore the response of CD8⁺T cell in tumors aroused by the perfusion effect induced by USMC for providing theoretical feasibility of synergistic enhancement of tumor immunotherapy.3.To investigate the synergistic anti-tumor efficacy and mechanism of perfusion enhancement effect by USMC combined with PD-L1 antibody,which would provide a new method for enhancing immune checkpoint blockade therapy.Methods1.Mouse tumor modelMC38 cell suspension was diluted with phosphate-buffered saline into5×10⁶cells/ml and a 0.1 ml suspension was injected subcutaneously into the right flank of C57BL/6 female mice.2.Optimization and measurement of the acoustic parameters that may impact tumor perfusion enhancement effectMC38 colon cancer mice were randomly divided into seven groups according to the different combinations of mechanical index（MI）and pulse repetition frequency（PRF）.They were the Control,MI0.2-PRF200,MI0.2-PRF500,MI0.2-PRF1000,MI0.4-PRF200,MI0.4-PRF500 and MI0.4-PRF1000,each group had six mice.USMC treatment was performed with a VINNO 70 ultrasound system equipped with a Vflash cavitation regulation module and electronic focusing function.And the treatment worked with an X4-12L linear array transducer at the frequency of 4 MHz,MI of 0.2 or 0.4,pulse length of 18 cycles,pulse repetition frequency of 200 Hz or 500 Hz or 1000 Hz,pulse/interval time of 1s/1s,and the treatment duration was 10 min.Sonazoid^?microbubbles were slowly injected intravenously during treatment.Tumor blood perfusion was analyzed by contrast-enhanced ultrasound（CEUS）before and four different time points after treatment.In addition,the acoustic parameters like MI were measured by ONDA acoustic detection system.3.Exploration of the CD8⁺T cell response in tumors caused by perfusion enhancement effectMC38 Colon cancer bearing mice were randomly divided into four groups:Control（n=13）,USMCMI0.4（MI=0.4,n=13）,USMCMI1.2（MI=1.2,n=13）,USMI1.2（MI=1.2,n=13,treated with US only）.The treatment was operated with an X4-12L linear array transducer at the MI of 0.4 or 1.2,the PRF of 1000 Hz and the other parameters are the same as method 2.Sonazoid^?microbubbles were slowly injected intravenously during USMC.After treatment,flow cytometry（FCM）was used to detect the number of CD8⁺T cells and Tregs（Regulatory T cells）in tumors of each group.Secondly,tumor perfusion change was analyzed by CEUS.The corresponding histology was observed under light microscope and transmission electron microscopy（TEM）,as well as the expression of vascular endothelial marker CD31 was evaluated by immunofluorescence（IF）.4.Verification of the infiltration of CD8⁺T cells by the ultrasound perfusion enhancement effectThe tumor-bearing mice were randomly divided into two groups:Control（n=7）,USMC（n=7）.The USMC group was transfused with activated CFSE-labeled CD8⁺T cells via tail vein immediately after USMC treatment,while the control was only transfused with the same CD8⁺T cells.Thirty minutes later,IF and FCM were used to compare the amount of CFSE⁺CD8⁺T cells in tumors between USMC group and the control.5.Investigation of the synergistic anti-tumor efficacy and the mechanism of USMC combined with PD-L1 antibodyAgain,the tumor-bearing mice were divided into four groups:Control（n=35）,USMC（n=36）,anti-PD-L1（n=36）,USMC+anti-PD-L1（n=36）.The anti-tumor efficacy was assessed by the tumor growth curve and the survival time of mice.The synergistic mechanisms of USMC combined with anti-PD-L1 were explored from the following four aspects.（1）The increased delivery of PD-L1 antibody in tumors was determined by the concentration of anti-PD-L1 in tumors using ELISA.（2）The normalization of tumor vessels was indicated by CEUS of tumor perfusion,and the IF expression of CD31 and ICAM-1（intercellular adhesion molecule-1）.⑶The improvement of immune microenvironment:FCM was used to analyze the number of CD8⁺T cells as well as its expression of Ki67,IFN-γ（Interferon-gamma）and Grzm-B（Granzyme B）,and also the proportion of Th1,Th2,Tregs,MDSC（Myeloid-derived suppressor cells）,M1-like TAM（Tumour-associated macrophage）and M2-like TAM in tumors;ELISA was used to detect the secretion of CXCL9（Chemokine（C-X-C motif）ligand 9）and CXCL10.⑷The amelioration of tumor hypoxia:The content of hypoxia inducible factor-1α（HIF-1α）in tumor was detected by ELISA.The expression of vascular endothelial growth factor（VEGF）in tumor tissues was analyzed by IF.Results1.The effects of MI and PRF on tumor perfusion enhancement.CEUS showed that the acoustic parameters MI of 0.4 combined with PRF of 1000 Hz presented the best tumor perfusion enhancement effect among all the groups.Quantitative analysis showed that the peak intensity（PI）and area under curve（AUC）values of MI0.4-PRF1000 were higher than those of the control,MI0.2-PRF200,MI0.2-PRF500 and MI0.2-PRF1000 immediately after treatment（P<0.0001 or P<0.05）,and even higher than those of the other six groups 30 min,2h and 4h after treatment respectively（P<0.0001 or P<0.001）.Additionally,we observed that this perfusion enhancement effect induced by USMC appeared immediately after treatment,peaked at 30 min,began to decrease 2h later,and lasted about 4h.2.The measurements of acoustic parameters for USMC.The results showed that the on screen values of central frequency（4MHz）,pulse length（18 cycles）and PRF（200 Hz,500 Hz,1000 Hz）used in USMC treatment were consistent with the actually measured values.But,the measured value was inconsistent with the on screen MI value.The optimized MI 0.4 corresponded to a peak negative pressure of 0.46 MPa,and so the actual measured MI value calculated by the formula was 0.23.3.Comparison of the tumor perfusion effect and corresponding histological changes between high MI and low MI USMC.Our results showed that the USMC with low MI（0.4 on screen and0.23 actual）significantly enhanced tumor blood perfusion visually and also increased the values of PI and AUC consistently（P<0.01,P<0.05）,while the USMC with high MI（1.2 on screen and 0.68 actual）reduced blood perfusion with consistent PI and AUC decline（P<0.001,P<0.05）when compared with the control.Vasodilation of tumor was found under light microscopy and TEM after treated by the USMC of MI 0.4,while microvascular damage in tumor tissues was observed in the USMC of MI1.2.Interestingly,USMC of MI 0.4 did not change CD31 expression of tumors.However,USMC of MI1.2 reduced CD31 expression by IOD（Integrated optical density）analysis compared with the control（P<0.01）.The results indicated that the USMC perfusion effect with low MI was due to the vasodilation of tumor microvessels rather than the formation of neovasculature.4.Response of CD8⁺T cells in tumor after USMC treatment.FCM showed that the percentage and absolute number of infiltrating CD8⁺T cells in the USMCMI0.4 group were higher and USMCMI1.2 group were lower than those of the control respectively（all P<0.001）.No significant differences of Tregs numbers occurred among all groups.However,the ratio of CD8⁺T cells to Tregs in USMCMI0.4 group was higher than USMCMI1.2 group（P<0.01）.The results indicated that low MI USMC effect could promote the infiltration of CD8⁺T cells,in the meantime the effect did not increase the number of immunosupressive cell Tregs.However,the perfusion reduction effect induced by high MI USMC resulted in a decrease in the number of CD8⁺T cells,which was the reverse verification of promoting CD8⁺T cell infiltration by the perfusion effect.5.Promotion of exogenous activated CD8⁺T cells by perfusion enhancement effect.IF showed that the CFSE-labeled exogenous activated CD8⁺T cells were more extensively distributed in the USMC treated tumors than that of the control.FCM also showed that the number of CFSE-labeled CD8⁺T cells in the USMC group was 1.96 times than that of the control（P<0.01）.This result further verified that the perfusion enhancement effect could promote CD8⁺T cell infiltration.6.Anti-tumor effect of USMC combined with anti-PD-L1.We observed that the tumor volume and weight after treatment with USMC combined with anti-PD-L1 were significantly lower than those of anti-PD-L1,USMC and the control（P<0.05,P<0.0001,P<0.0001）.Kaplan-Meir survival curve analysis showed that the 70-day survival rate of mice in the combined group was 66.7%,significantly higher than that of the other three groups（P<0.05,P<0.001,P<0.001）.The results indicated that the combination therapy did has a synergistic effect.7.The synergistic mechanisms of combination therapy.The synergistic mechanisms of combination therapy were analyzed as the following four aspects.⑴USMC increased the concentration of anti-PD-L1 in tumors,which was 1.31-fold higher in the combined group than in the monotherapy group.⑵The combination therapy might promote tumor vascular normalization.CEUS proved tumor perfusion enhancement was found in the combined group,consistent with the PI and AUC values which were higher than those of the other groups（all P<0.0001）.IF showed that the CD31 IOD value in the combined group was lower than that of the other three groups（P<0.01,P<0.0001,P<0.0001）,while the ICAM-1 IOD value higher than that of the other three groups（P<0.05,P<0.0001,P<0.0001）.Therefore,the combination therapy exhibited not only the best tumor perfusion enhancement,but the lowest vessel density in this group,indicating that the combination therapy might induce tumor vascular normalization.⑶The combination therapy improved the tumor immune microenvironment.A:FCM showed not only the percentage and absolute number of CD8⁺T cells higher in the combined group than those of the other three groups,but the expression of CD8⁺T cell proliferation antigen Ki67 was also higher than that of the other three groups,so did the IFN-γand Grzm-B secreted by CD8⁺T cells.These results suggested that the combination therapy could not only increase the number of CD8⁺T cells,but also enhance their proliferation and killing function.B:The proportion of Th1 and Th17 were higher while Tregs were lower in combined group than those of the control respectively（P<0.01）,which suggested that the combination therapy might benefit the differentiation of CD4⁺T cells into antitumor immunophenotypes.C:The proportion of immunosupressive cell MDSC were lower than those of the control group（P<0.01）.D:The proportion of M1-like TAM while M2-like TAM in combined group was respectively higher and lower than that of the control（P<0.05）.The result suggested that the combination therapy might promote the polarization of TAM to M1 type,which could be helpful for antitumor immunotherapy.E:ELISA analysis showed that the concentration of CXCL9 and CXCL10in tumors in the combined group were higher than those of the other three groups.They were the most important chemokines for promoting CD8⁺T cells to migrate toward tumor.⑷The combination therapy ameliorated tumor hypoxia.ELISA analysis showed that the HIF-1αcontent in combined group was significantly lower than that in control group（P<0.05）.Meanwhile,the expression of VEGF was significantly lower than that of the control group by IF analysis（P<0.01）.These results suggested that the combination therapy might inhibit the HIF-α/VEGF signaling pathway by alleviating hypoxia in tumor microenvironment,thereby promoting tumor vascular normalization and improving tumor immune microenvironment.Conclusions1.Low intensity USMC with low MI（0.4 on screen and 0.23 actual）and high PRF（1000 Hz）could produce effective and stable tumor blood perfusion enhancement effect,which could last for 4h.2.Tumor perfusion enhancement effect induced by USMC of low MI could increase the number of tumor infiltrating CD8⁺T cells,which might be conducive to enhancing tumor immunotherapy.While,the perfusion reduction effect induced by USMC of high MI could decrease the number of tumor infiltrating CD8⁺T cells,which might be the reverse verification of promoting CD8⁺T cell infiltration by perfusion enhancement effect.3.The perfusion enhancement effect by USMC combined with anti-PD-L1 therapy presented improved anti-tumor efficacy.The synergistic mechanisms of combination therapy included:USMC promoted delivery of anti-PD-L1,normalized tumor vessel,improved immune microenvironment and ameliorated tumor hypoxia.Together,the perfusion enhancement effect produced by USMC could be an effective method for potentiating PD-L1 antibody tumor therapy.