FUS sti. spleen suppressed orthotopic H22/Hepa1-6-HCC proliferation.

A, the diagram of ultrasonic platform applied for FUS sti. spleen. B, the scheme of whole spleen irradiated by FUS transducer carried by the XYZ motorized positioning stage. C, ultrasound focal field scanning after calibration by a hydrophone. D, experimental protocol for the use of FUS sti. spleen after H22 cancer cell implantation into the liver. E, images of spleen and liver bearing H22-HCC tumor. F-G, statistics of H22-HCC tumor weight and volume, respectively. H, H22 malignant ascites volume. I-K, liver weight (total weight of liver parenchyma and H22-HCC tumor), spleen weight, and mice weight, respectively. L, Kaplan-Meier survival curves of G1-G3 groups. M, schematic diagram of the experimental protocol for FUS sti. spleen following orthotopic implantation of Hepa1-6 cancer cells in the liver. N, representative images showing the spleen and liver with Hepa1-6-HCC tumor. O-P, statistics of Hepa1-6-HCC tumor weight and volume, respectively. Q-S, evaluation of total liver weight (including both parenchyma and Hepa1-6-HCC tumor), spleen weight, and body weight, respectively. T, Kaplan-Meier survival curves of T1-T3 groups. U-V, immunohistochemical staining of cyclin-D1, Ki67, NK1.1, and CD8a positive cells in the spleen and tumor from G2-G3 groups, and the corresponding quantification of positive area ratio, respectively. W-X, immunohistochemical staining and corresponding quantitative analysis of cyclin-D1, Ki67, NK1.1, and CD8a expression in both splenic and tumor tissues from T2-T3 groups. G1 and T1 group with normal mice classified as negative control (named normal), G2 group with orthotopic H22-HCC mice and T2 group with orthotopic Hepa1-6-HCC mice used as the positive control (named control), and G3 group with orthotopic H22-HCC mice and T3 group with orthotopic Hepa1-6-HCC mice respectively subjected to FUS stimulation (named FUS sti. spleen).

Statistical significance of FCM results of various immunocytes across the spleen, blood, tumor and para-carcinoma tissues from orthotopic H22/Hepa1-6-HCC mice subjected to FUS sti. spleen, non-treated murine tumor models, and normal mice.

G1, normal mice; G2, control group of xenograft H22 HCC in situ; G3, FUS sti. spleen-treated orthotopic H22 HCC. T1, normal mice; T2, control group of xenograft Hepa1-6 HCC in situ; T3, FUS sti. spleen-treated orthotopic Hepa1-6 HCC. The red asterisk indicates a significant increase in the immunocyte proportions, while the blue asterisk indicates a significant decrease in the immunocyte proportions, and the ns represents no significant difference. The representative FCM plots and statistical diagram are shown in Supplementary Figure 2-9.

FUS sti. spleen significantly improved the proportion and physiological function of splenic T_NK cells.

A, dot plots of canonical marker genes applied for T_NK cell subclusters identification. B, UMAP projection of T_NK cells from both the control and FUS sti. spleen group. C, comparison of T_NK cell proportion between the control group and FUS sti. spleen group. D, H, and L, pseudotime-ordered analysis of CD4 T cells, CD8 T cells, and NK cells, respectively, from the control group and FUS sti. spleen group. E, I, and M, pseudotime heatmap showing the dynamic changes in gene expression of CD4 T cell, CD8 T cell, and NK cell subclusters, respectively, based on their common kinetics through pseudotime using Monocle 2. Genes (rows) were clustered into 4 modules, and cells (columns) were ordered according to pseudotime. F, J, and N, distinct GO terms of biological process and their p value associated with each module in CD4 T cells, CD8 T cells, and NK cells respectively. G, K, and O, KEGG signaling pathway enrichment of signal transduction in CD4 T cells, CD8 T cells, and NK cells respectively.

SUS significantly modified T_NK cells in TME from a negative to a positive tumor suppressive state.

A, dot plots of signature markers applied for intratumoral T_NK cell subclusters identification. B, UMAP projection of intratumoral T_NK cells from the control group and FUS sti. spleen group. C, cell proportion of intratumoral T_NK cell subclusters between the control group and FUS sti. spleen group. D, H, and L, pseudotime-ordered analysis of CD4 T cells, CD8 T cells, and NK cells, respectively, from the control group and FUS sti. spleen group. E, I, and M, pseudotime heatmap showing the dynamic changes in gene expression of CD4 T cell, CD8 T cell, and NK cell subclusters, respectively, based on their common kinetics through pseudotime using Monocle 2. Genes (rows) were clustered into 4 modules, and cells (columns) were ordered according to pseudotime. F, J, and N, distinct GO terms of biological process and their p values associated with each module in CD4 T cells, CD8 T cells, and NK cells, respectively. G, K, and O, KEGG signaling pathway enrichment of signal transduction in CD4 T cells, CD8 T cells, and NK cells, respectively.

Characterization of epithelial cell landscape to evaluate tumor progression, pseudotime trajectory analysis of CD8 T cell and NK cell trafficking between spleen and tumor, and cytokine profiling in plasma.

A, violin plot showing CNV level of distinct cell compositions in TME between the G2 and G3 groups. B, dot plot of various expression profiles of signature genes across epithelial cell subtypes. C, UMAP plot of epithelial cells stratified into seven distinct subclusters. D, the proportion of epithelial cell subclusters in the G2 group and G3 group. E, violin plot of CNV level across epithelial cell subclusters between the G2 group and G3 group. F, pseudotime identifying 4 states of epithelial cell subclusters along the trajectory, and their cell proportion analysis. G, pseudotime-ordered analysis of epithelial cells from the G2 group and G3 group. H, pseudotime heatmap showing the dynamic gene expression in the epithelial cell subclusters based on their common kinetics through pseudotime using Monocle 2. Genes (rows) were clustered into 4 modules, and cells (columns) were ordered according to pseudotime. I, distinct GO terms of biological process and their p values associated with each module in epithelial cells. J, KEGG signaling pathway enrichment of signal transduction in epithelial cells. K, UMAP projection of CD8 T cells from both spleen and tumor of both G2 and G3 groups. L-M, scVelo plot of CD8 T cell subclusters from both spleen and tumor of both G2 and G3 groups. N, scVelo plot of CD8 T cell subtypes in the G3 group compared to G2 group, which demonstrating CD8 T cell transition from spleen to tumor in response to FUS sti. spleen. O, UMAP projection of NK cells in from both spleen and tumor of both G2 and G3 groups. P-Q, scVelo plot of NK cell subclusters from both spleen and tumor from both G2 and G3 groups. R, scVelo plot of NK cell subclusters in the G2 group or G3 group, which demonstrating NK cell transition from spleen to tumor in response to SUS. S, heatmap of cytokine levels in plasma from orthotopic H22-HCC mice with or without FUS sti. spleen treatment. T, protein-protein network analysis of cytokines and target genes based on STRING database. U, KEGG pathway enrichment analysis of the target genes of cytokines.

FCM analysis of activated CD8 T cells and NK cells in orthotopic H22-HCC mice subjected to FUS sti. spleen, and differential analysis of KEGG signaling pathways, as well as the results of Ca2+ enhanced cancer cell suppression in vitro.

A, heatmap showing FCM results of “naive”, “inhibitory” or “cytotoxic” CD8 T cell subtypes in G1-G3 groups based on the signature genes of CCL5 and CCR7 displayed in Figure 2 A and 3 A, and “immature” or “mature” NK cell subtypes according to the signature genes of CCL5 and CD159a (Klrc1). Additionally, the classical markers CD137 (4-1BB) and IFN-γ were used to distinguish the “cytotoxic” CD8 T cells or not, and perforin was used to distinguish “cytotoxic” NK cells or not. The corresponding flow charts and statistical analysis are shown in Supplementary Figure 12. B-C, GSVA analysis of partial KEGG pathways enrichment analysis in splenic CD8 T cell subtypes and NK cell subtypes (as clustered in Figure 2 A-B), respectively. D-E, GSEA analysis of partial KEGG pathways enrichment analysis in splenic CD8 T cells. F-G, GSEA analysis of partial KEGG pathways enrichment analysis in splenic NK cells. H, schematic diagram of FUS enhancing calcium influx and regulating TNF, NFκB, MAPK, and other signaling pathways. I, flow charts of Fluo-4 labeled splenic cells from orthotopic H22-HCC mice with or without FUS sti. spleen treatment, and the corresponding quantification. J, assay of calcium content in the whole spleen. K-L, heatmaps showing qRT-PCR results in the spleen, splenic CD8 T cells, and splenic NK cells of orthotopic H22-HCC mice with or without FUS sti. spleen treatment. M-N, relative cell viability of CD8 T cells, NK cells, or H22 cells processed with ultrasound irradiation and mixed cultivation in transwells. O-P, relative cell viability of CD8 T cells, NK cells, or Hepa1-6 cells processed with ultrasound irradiation and mixed cultivation in transwells. Q, FCM results of Fluo-4 fluorescent CD8 T cells. C1, H22/Hepa1-6 cancer cells; C2, CD8 T/NK cells; C3, CD8 T/NK cells cocultured with H22/Hepa1-6 cancer cells in the transwell; C4, FUS stimulated CD8 T/NK cells; C5, CD8 T/NK cells subjected to FUS irradiation, and cocultured with H22/Hepa1-6 cancer cells in the transwell; C6, CD8 T/NK cells mixed with H22/Hepa1-6 lysate and cocultured with H22/Hepa1-6 cancer cells in the transwell; C7, CD8 T/NK cells in 300 nM calcium culture medium and cocultured with H22/Hepa1-6 cancer cells in the transwell; C8, CD8 T/NK cells mixed with H22/Hepa1-6 lysate, subjected to FUS irradiation, and cocultured with H22/Hepa1-6 cancer cells in the transwell; C9, CD8 T/NK cells mixed with H22/Hepa1-6 lysate in 300 nM calcium culture medium and cocultured with H22/Hepa1-6 cancer cells in the transwell; C10, CD8 T/NK cells in 300 nM calcium culture medium subjected to FUS irradiation, and cocultured with H22/Hepa1-6 cancer cells in the transwell; C11, CD8 T/NK cells mixed with H22/Hepa1-6 lysate in 300 nM calcium culture medium, subjected to FUS irradiation, and cocultured with H22/Hepa1-6 cancer cells in the transwell.

Characterization of STNDs@Ca2+ and therapeutic efficacy evaluation of FUS plus STNDs@Ca2+.

A, structural diagram of the STNDs@Ca2+. B, the process and mechanism of FUS plus STNDs@Ca2+ facilitated Ca2+ controlled-release in the spleen, leading to enhanced immune cell activation and antitumor effects. C, zeta potential changes during the preparation of STNDs@Ca2+, showing a transition from negative to neutral. D, size distribution of STNDs@Ca2+. E, LCFM and cryo-TEM images demonstrating spherical morphology and uniform size (∼247 ± 23 nm). F, calcium-loading capacity of STNDs@Ca2+, measured at ∼2 mmol per 1 mL STNDs@Ca2+. G, dynamic changes in STNDs@Ca2+ size over time at 37°C. H, B-mode ultrasound imaging of FUS-triggered phase transition and cavitation of STNDs@Ca2+ under varying peak negative pressures. I, ILLIS monitoring splenic accumulation of the STNDs@Ca2+ within the 0-3 hour time window. J, ILLIS imaging of in vivo biodistribution of STNDs@Ca2+ after intravenous injection. K, microscope images showing fluorescent STNDs@Ca2+ distribution in spleen and the corresponding positive area ratio analysis. L-S, therapeutic outcomes in orthotopic H22-HCC mice, including anatomical pictures, tumor weight, tumor volume, ascites volume, liver weight, spleen weight, mice weight and survival curves. T, total calcium content in the spleen. U, microscope images of Von Koussa staining and the corresponding positive area ratio analysis. V-W, FCM analysis of the proportions of NK cells and CD8+ T cells, particularly calcium-positive activated subsets.

The results of serum biochemical analysis to evaluate the toxicity of STNDs@Ca2+.

O1, the saline injected group; O2, the STNDs@Ca2+ injected group.