Ultrasound-Stimulated Microbubbles Cavitation Enhances Anti-Tumor Efficacy of PD-L1 Antibody Therapy

This study aimed to investigate the synergistic anti-tumor efficacy and mechanism of perfusion enhancement effect by ultrasound stimulated microbubbles cavitation (USMC) combined with PD-L1 antibody, which would provide a new method for enhancing immune checkpoint blockade therapy.

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). USMC treatment was performed with a VINNO 70 ultrasound system equipped with a Vflash cavitation regulation module and electronic focusing function. The treatment used an X4-12L linear array transducer at a frequency of 4 MHz, MI of 0.4, pulse length of 18 cycles, pulse repetition frequency of 1 kHz, pulse/interval time of 1s/1s, and the treatment duration was 10 min. Sonazoid® microbubbles were slowly injected intravenously during treatment.

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. (3) The improvement of immune microenvironment: Flow cytometry (FCM) was used to analyze the number of CD8+ T cells as well as its expression of Ki67, IFN-γ and 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 and CXCL10. (4) The amelioration of tumor hypoxia: The content of HIF-1α in tumor was detected by ELISA. The expression of VEGF in tumor tissues was analyzed by immunofluorescence (IF).

Results:

1. The combination therapy achieved greater tumor growth inhibition and longer survival than those of other groups.
2. The synergistic mechanisms of combination therapy were analyzed as the following four aspects.

(1) USMC increased the concentration of anti-PD-L1 in tumors, which was 1.31-fold higher in the combined group than in the monotherapy group. (2) The 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<0.05). IF showed that the CD31 IOD value in the combined group was lower, while the ICAM-1 IOD value higher than that of the other three groups (all P<0.05). 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. (3) The 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-γ and Granzyme 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 anti-tumor 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 anti-tumor immunotherapy. E: ELISA analysis showed that the concentration of CXCL9 and CXCL10 in 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. (4) The combination therapy ameliorated tumor hypoxia. ELISA analysis showed that the HIF-1α content 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.

Conclusion: 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.