| Immune checkpoint blockade(ICB)represents a breakthrough in cancer therapy and acts by reinvigorating the cytotoxic potential of the imm une system.However,the clinical efficacy of ICB remains very limited,leaving most patients unable to benefit from such treatment.Increasing evidence has revealed primary resistance mechanisms,which include insufficient tumor immunogenicity,immunosuppression and inadequate T cell activation.It has been reported that a sufficient preexisting population of antitumor CD8+ T cells within the tumor microenvironment(TME)favors tumor susceptibility to PD1-based ICB therapy.Among patients with melanoma who receive anti-PD1 therapy,the presence of sufficient T cell numbers in biopsy specimens prior to treatment was associated with a better response to therapy,and the CD8+ T cell density at the t umor margin was predictive of a good response,suggesting a positive correlation between intratumoral T cell infiltration and sensitivity to ICB therapy.Previo us studies have demonstrated that the lack of immunogenicity of solid tumors is an obstacle to their sensitivity to ICB therapy,but how to improve immunogenicity remains largely unknown.The recent classification of “hot” and “cold” tumors has been increasingly advocated as an aid to the clinical application of ICB.“Cold” tumors,such as prostate cancer and colon cancer,do not respond to single-agent ICB therapies because of their low immunogenicity.Combination therapy has been utilized in the clinic to turn cold tumors into hot tumors in order to increase the efficacy of immunotherapy in recent years;such combination therapies include chemotherapy or radiation therapy combined with immunotherapy.Chemotherapy and radiation can mainly lead to t umor cell apoptosis by disrupting cell division or destroying mitochondria.However,apoptosis is a typ e of physiological cell death associated with poor immunogenicity.It is of vital importance to identify new combination therapies to improve the benefit of ICB.Lipid microbubbles(MB)encapsulating inert gas were originally used as contrast agents for low-power ultrasound imaging.It has been reported that ultrasound-stimulated MB cavitation can promote tumor perfusion,thereby increasing CD8+ T cell infiltration and enhancing the efficacy of ICB in an MC38 tumor model.Since MB can only minimally enter the TME because the gaps between the endothelial cells in tumor blood vessels are smaller than 2 um,their application to tumor immunogenicity remains limited.Compared with MB,nanobubbles(NB)can easily pass through the blood vessel endothelium and accumulate in the tumor mass.In addition,we have demonstrated that NB destroyed by sufficient ultrasonic power show minor thermal effects.Previous studies have proven that NB carrying Ce6(a sonosensitizer)suppress cancer cells through sonosensitizer-induced ROS generation,thereby achieving a synergistic effect with PD1 blockade;however,the impact of ultrasound-stimulated nanobubbles(USNB)on the tumor-immune microenvironment and their implication in tumor immunogenicity remain largely elusive.Here,we revealed for the first time that USNB can effectively transform part of the“cold” tumors into “hot” tumors,thereby exerting a synergistic effect with anti-PD1 treatment on “cold tumors”.Importantly,we demonstrated that USNB significantly increase the release of antigens and damage-associated molecular patterns(DAMPs)to activate the inflammatory response in the TME and draining lymph nodes(d LN),resulting in enhanced systemic antitumor immunity and immune memory and causing long-lasting inhibition of tumor growth and recurrence.These findings strongly suggest a promising role of USNB in improving the efficacy of anti-PD1 therapy in “cold” tumors in the clinic.1.USNB synergistically improved the efficacy of anti-PD1 therapy1.1 Methods1.1.1 Mouse tumor models were established by using mouse colon cancer cells MC38,prostate cancer cell RM1 and melanoma cell B16 and the mouse were treated with anti-PD1,USNB,USNB+anti-PD1 or left untreated(10 mice in each group).Compare the tumor growth rate and mouse survival rate between different groups to verify the synergistic effect of USNB with anti-PD1 treatment.1.1.2 The number and proportion of CD44+CD8+T cells,CD4+T cells,Th1 cells and Treg cells in TME,d LN and spleen in mouse of each treatment group were detected by flow cytometry and compared to verify the effects of USNB on tumor infiltrating lymphocytes and systemic immunity.1.1.3 We applied NK,CD4+ and CD8+ T cell-depleting antibodies in combination with USNB treatment and separated the mice into the USNB+αNK,USNB+αCD4,USNB+αCD8,USNB+αCD4+αCD8 and USNB groups(10 mice in each group).To further investigate whether CD4+ T cells or CD8+ T cells play the dominant role in USNB treatment effects.1.2 Results1.2.1 USNB treatment slowed the growth of tumors;however,anti-PD1 treatment exerted a weak therapeutic effect when used alone.The USNB and anti-PD1 combination treatment significantly inhibited tumor growth and prolonged the survival rate of tumorbearing mice.1.2.2 Compared with that in the untreated group,the number of CD44+CD8+ T cells in TME,d LN and spleen was increased in both the USNB and USNB+anti-PD1 groups but not in the anti-PD1 group.There was an increased number of CD44+CD8+ T cells in the d LN and spleens after anti-PD1 treatment,but no significant change was observed in the tumors.1.2.3 The proportions and numbers of total CD4+ T cells were comparable among the different treatment groups.Similar results were observed for Th1 and Treg cells.1.2.4 The mice in the USNB+αCD4+αCD8 and USNB+αCD8 groups exhibited the fastest tumor growth rates,and the mice in the USNB+αCD4 and USNB+αNK groups exhibited a moderate tumor growth rate compared with those in the other groups.1.3 Conclusion1.3.1 USNB treatment can delay tumor growth and enhance the therapeutic effect of PD1 blockade.1.3.2 USNB treatment can promote the infiltration of CD8+ T cells and strengthen systemic T cell immunity.1.3.3 The effect of USNB relies mainly on CD8+ T cells.2 USNB endows CD8+ T cells with competence for immunotherapy2.1 Methods2.1.1 Mouse tumor model was established with RM1 and MC38 cells,and the grouping was the same as before.2.1.2 The number of GZMB+CD8+ T cells,CD107+CD8+T cells,IFNγ+CD8+ T cells in TME of each group and the MFI(average fluorescence intensity)of GZMB,CD107,IFNγ and KLRG1 and Tbet expression levels of CD44+CD8+ T cells in each group were detected by flow cytology and compared to investigate the effect function of infiltrating CD8+ T cells in the different treatment groups.2.1.3 The number of PD1+Tim3+CD8+T cells and the MFI of PD1 and Tim3 of that in each treatment group were detected by flow cytology to verify whether USNB+antiPD1 can reduce the exhaustion of CD8+ T cells within the tumor.2.2 Results2.2.1 USNB combined with PD1 blockade significantly increased the numbers of GZMB+CD8+ T cells,CD107+CD8+ T cells and IFNγ+CD8+T cells compared with USNB or PD1 blockade treatment alone.2.2.2 The MFIs of GZMB,CD107,IFNγ and KLRG1 in CD44+CD8+T cells w ere also increased after combination treatment.2.2.3 Both the percentage and number of PD1+Tim3+CD8+ T cells were decreased after USNB and combination treatment and the mean fluorescence intensity(MFI)of PD1 and Tim3 also decreased in the combination group.2.3 Conclusion2.3.1 USNB treatment can enhance the effector function of tumor-infiltrating CD8+T cells and further enhance the effect of PD1 blockade.2.3.2 USNB+anti-PD1 treatment can significantly alleviate the exhaustion of CD8+T cells.3 USNB promotes the immune response by facilitating the release of tumor antigens and DAMPs3.1 Methods3.1.1 Mouse tumor model was established with RM1 and MC38 cells,and the grouping was the same as before.3.1.2 The changes of antigen-presenting cells(APCs)within TME and d LN at different time points after USNB treatment were detected by flow cytology,including CD11c+(dendritic cell),CD68+(macrophages),CD11b+(monocytes),CD45+(leukocytes),and CD49b+(NK cells),to investigate whether the augmentation of the infiltration and effect function of CD8+ T cells is caused by APC-induced CD8+ T cell activation.3.1.3 We cultured RM1-OVA cells in vitro and treated them with NB,US,or USNB or left them untreated.Then,we examined OVA and DAMPs(HMGB1,ATP,Calreticulin and HSPA2)levels in the supernatant by Western blot.The morphology of cells after treatment was viewed by transmission electron microscopy.Mito Tracker/7AAD staining of USNB-treated MC38 cells was then performed and evaluated under confocal microscopy to verify whether USNB treatment can promote the release of tumor antigens and DAMPs.3.1.4 We cocultured splenocytes with the supernatants of NB,US and USNB treated RM1-OVA cells.Then,the activation degree and killing efficiency of CD8+ T cells in each group were detected by flow cytometry to verify whether USNB therapy enhances the immunity of CD8+ T cells by promoting the release of tumor antigens as well as DAMPs.3.1.5 RNAseq verifies whether USNB therapy can promote antigen presentation and CD8+ T cell activation at the transcriptional level,thereby enhancing the efficacy of anti-PD1 therapy.3.2 Results3.2.1 In the TME,the numbers of leukocytes(CD45+),DCs,monocytes,NK cells and macrophages increased after USNB treatment alone or in combination with anti-PD1 treatment on both day 1 and day 4.3.2.2 In d LN,the numbers of DCs,monocytes and macrophages were increased in the USNB and USNB+anti-PD1 groups compared to those in the untreated and anti-PD1 groups at day 1,but this difference disappeared at day 4.3.2.3 The results showed that at both 0 h and 4 h after USNB treatment,OVA,ATP,HMGB1,Calreticulin and HSPA2 levels were released obviously higher than that released by other cells.3.2.4 USNB-treated cells were severely damaged,as evidenced by the lo ss of cell membrane integrity,swollen mitochondria,fragmented nuclei and leakage of cell contents into the extracellular space.However,neither US nor NB treatment affected the morphology of tumor cells.3.2.5 USNB-treated cells exhibited substantial changes in cell morphology and high levels of 7AAD+ nuclei fragments combined with some mitochondrial leakage from the cells.3.2.6 Flow cytometric staining analysis showed that CD8+ T cells expressed higher levels of activation(PD1,CD69,CD25)and killing(TNFα,IFNγ)markers.3.2.7 After USNB treatment,the TME exhibited a high level of “acute inflammation response” signaling pathway and other pathways related to “cell death” and “response to stimulation and wound”,which confirmed that USNB can cause tumor cell injury and promote inflammation and the immune response.Moreover,the USNB+anti-PD1 group also exhibited a high level of signaling pathways related to “immune response”,“T cell activation” and “antigen processing and presentation” compared to the USNB,anti-PD1 or untreated groups.3.3 Conclusion3.3.1 The augmentation of the infiltration and effect function of CD8+ T cells is caused by APC-induced CD8+ T cell activation.3.3.2 USNB induces antigen and DAMP release to enhance the therapeutic effect of PD1 blockade by improving antigen presentation in immunogenic “cold” tumors and further increasing the effector function of CD8+ T cells.4 USNB causes necrosis of tumor cells to facilitate the release of tumor antigens and DAMPs4.1 Methods4.1.1 The nanobubbles were stained and co-cultured with tumor cells,and the cell uptake was observed by confocal microscopy.4.1.2 RM1 cells were cultured in vitro and were treated with NB,US,or USNB or left them untreated.0h and 24 h after treatment,the cell morphology and growth status were observed under the microscope.Cells were stained with 7AAD,annexin V and Caspase3 at 0h and 4h after treatment,and the apoptosis signal was detected by flow cytometry.Intracellular ROS levels were detected by flow cytometry 4 h after treatment.RM1 cells were labeled with CFSE and divided into 4 groups(grouped as before)for different treatments,and the cell proliferation status was detected by flow cytometry 24 h after treatment.4.1.3 Mouse tumor model was established with RM1 and MC38 cells,and the grouping was the same as before.After treatment,tumor tissue is taken for fixation and embedding and HE staining,and tissue necrosis is observed under the microscope.4.2 Results4.2.1 NB could be taken up by RM1 cells and MC38 cells as soon as 30 min after being added to the cell medium,and NB remained stable after internalization into the cytoplasm.4.2.2 The majority of cells broke into fragments after USNB treatment.After 24 h,the cells in the US,NB and untreated groups had reattached to the flask and had grown to confluence,while the USNB-treated cells remained suspended and dying.4.2.3 At 0 h,approximately 65% of the USNB-treated cells were annexin V-and7AAD+,while in the other treatment groups,the percentage of these cells was less than1%.Four hours after treatment,the annexin V-7AAD+ population of USNB-treated cells was reduced to 7.84%,and the annexin V+ 7AAD+ population was increased to approximately 21.8%.4.2.4 At 0 h,Caspase3 expression was comparable among these groups,but 4 h after treatment,Caspase3 expression was increased by 43.3% in the USNB group compared to that in other groups.ROS production in MC38 cells was obviously increased 4 h after USNB treatment.4.2.5 The USNB-treated cells did not grow,whereas other treatments had no apparent impact on cell growth.4.2.6 A larger necrotic region was produced under USNB treatment conditions,while the US and untreated groups exhibited little necrosis in both the RM 1 and MC38 tumor models.4.3 ConclusionUSNB can lead to tumor necrosis both in vivo and in vitro,and the effect of USNB on cells relies on the mechanical force of NB stimulated by US.5 USNB treatment in combination with PD1 blockade enhances systemic antitumor immunity and immune memory5.1 Methods5.1.1 We inoculated RM1 tumors on both legs of mice,and treated the mice with USNB,anti-PD1 or USNB+anti-PD1,with USNB applied to the tumor in the right leg.Then we injected 0.3×10 6 MC38 tumor cells intravenously 3 days after the subcutaneous implantation of 0.7×10 6 MC38 cells into the right leg,and USNB was applied to the subcutaneous tumor.5.1.2 Mice with subcutaneous MC38 tumors were treated with USNB+anti-PD1 to complete remission(CR),and subcutaneously implanted MC38 tumors again(Rechallenge)60 days after CR.Observe the trend of tumor growth.5.2 Results5.2.1 The growth pattern of the USNB-treated tumors on the right leg was consistent with previous data.The growth curve of the untreated tumors exhibited the same trend.The number of metastatic nodes was significantly lower in the USNB+anti-PD1 group than in the untreated and anti-PD1 antibody treated groups.5.2.2 Mice with CR acquired immune memory protection and completely eliminated the MC38 tumor cells.5.3 Conclusion5.3.1 USNB treatment exerts an abscopal effect and can effectively promote systemic antitumor immunity,leading to elimination of distal untreated tumors,w hich is crucial for advanced cancer patients in the clinic.5.3.2 USNB treatment in combination with PD1 blockade enhanced systemic antitumor immunity and promoted the acquisition of immune memory after complete remission. |
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