Checkpoint blockade and nanosonosensitizer-augmented noninvasive sonodynamic therapy combination reduces tumour growth and metastases in mice

Abstract

Combined checkpoint blockade (e.g., PD1/PD-L1) with traditional clinical therapies can be hampered by side effects and low tumour-therapeutic outcome, hindering broad clinical translation. Here we report a combined tumour-therapeutic modality based on integrating nanosonosensitizers-augmented noninvasive sonodynamic therapy (SDT) with checkpoint-blockade immunotherapy. All components of the nanosonosensitizers (HMME/R837@Lip) are clinically approved, wherein liposomes act as carriers to co-encapsulate sonosensitizers (hematoporphyrin monomethyl ether (HMME)) and immune adjuvant (imiquimod (R837)). Using multiple tumour models, we demonstrate that combining nanosonosensitizers-augmented SDT with anti-PD-L1 induces an anti-tumour response, which not only arrests primary tumour progression, but also prevents lung metastasis. Furthermore, the combined treatment strategy offers a long-term immunological memory function, which can protect against tumour rechallenge after elimination of the initial tumours. Therefore, this work represents a proof-of-concept combinatorial tumour therapeutics based on noninvasive tumours-therapeutic modality with immunotherapy.

Fig. 1

The design principle of nanosonosensitiser-augmented synergistic SDT and immunotherapy. Schematic illustration of antitumour immune responses induced by combined noninvasive SDT with immune-adjuvant-contained nanosonosensitisers and checkpoint blockade for effective cancer immunotherapy

Fig. 2

Synthesis and characterisations of the composite nanosonosensitisers. a Schematic illustration of the construction of HMME/R837@Lip nanosonosensitisers and their microstructures; b TEM image showing the quasi-spherical morphology of HMME/R837@Lip with high dispersity (scale bar = 100 nm); c hydrodynamic diameters of HMME/R837@Lip nanosonosensitisers in PBS as measured by DLS; d UV-vis absorbance spectra of Lip, HMME, HMME@Lip and HMME/R837@Lip, indicating the successful encapsulation of HMME into the nano-liposome; e zeta potential of Lip, HMME@Lip and HMME/R837@Lip, error bars are based on SD (n = 3); f scheme of US-triggered ¹O₂ production as assisted by HMME/R837@Lip; g ESR spectra of HMME/R837@Lip with or without US treatment; h time-dependent DPBF absorption spectra in the presence of HMME/R837@Lip under US irradiation for varied durations

Fig. 3

In vitro toxicity of HMME/R837@Lip upon US irradiation against 4T1 cancer cells and stimulation of in vitro immune response. a CLSM images of 4T1 cells stained with DCFH-DA after different treatments: control (without any treatment), HMME@Lip only, HMME/R837@Lip only, US only, HMME@Lip combined with US irradiation and HMME/R837@Lip combined with US irradiation (scale bar = 20 μm); b CLSM images of 4T1 cells stained with Calcein-AM and PI after various treatments: control (without any treatment), HMME@Lip only, HMME/R837@Lip only, US only, HMME@Lip combined with US irradiation and HMME/R837@Lip combined with US irradiation (scale bar = 20 μm); c the design scheme of the transwell system experiment. 4T1 cells and their residues were placed in the upper compartment and DCs were cultured in the lower compartment; d the percentage of mature DCs (CD11c⁺CD80⁺CD86⁺) was analysed by flow cytometry after different treatments for 20 h in the transwell system; e–g quantification of the level of DC maturation (e) and the secretion of IL-6 (f) and TNF-α (g) in DC suspensions. Data are expressed as means ± SD (n = 3). Statistical significances were calculated via Student’s t test, *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 4

In vivo HMME/R837@Lip-augmented SDT for inducing cell apoptosis and/or necrosis, promoting DC maturation and stimulating the expression of proinflammatory cytokines. a Schematic illustration of the experiment design to assess the in vivo sonotoxicity and the immune responses as triggered by HMME/R837@Lip-augmented SDT; b, c in vivo apoptosis and/or necrosis of the tumour induced by HMME/R837@Lip-augmented SDT, as shown by H&E staining (scale bar = 100 μm) and TUNEL assay (scale bar = 50 μm); d, e DC maturation in the tumour-draining lymph nodes induced by HMME/R837@Lip-augmented SDT on mice bearing 4T1 tumours, as assessed by flow cytometry after staining with CD11c, CD80, CD86 and live dead; f–h cytokine levels of TNF-α, IL-6 and IL-12p70 in sera from mice isolated from 24 to 72 h after HMME/R837@Lip-agumented SDT treatment. Data are expressed as means ± SD (n = 3). Statistical significances were calculated via Student’s t test, *p < 0.05, **p < 0.01

Fig. 5

Synergistic nanosonosensitiser-augmented SDT and immunotherapy for in vivo suppression of mimic distant tumours in a subcutaneous tumour model. a Schematic showing the experiment design for in vivo evaluations. Mice bearing 4T1 subcutaneous tumours on both sides were used in this experiment. Tumours on the right side of the check were designed as “primary tumour” for SDT, and those on the left side were set as “mimic distant tumours” without SDT; b in vivo fluorescence images to reveal the biodistribution of HMME/R837@Lip post i.v. injection into 4T1-tumour-bearing mice at the indicated time points. Yellow arrows indicate tumours; c the accumulation curve of HMME/R837@Lip in the tumour tissue by measuring the fluorescence intensity of tumours at different time points post i.v. injection, error bars are based on SD (n = 3); d the ex vivo fluorescence image of major organs and tumour dissected from the mouse 24 and 48 h post injection and (e) quantification analysis of the tissue content of HMME/R837@Lip by testing the corresponding fluorescence intensity; data are expressed as means ± SD (n = 3); f, h primary (f) and distant (h) tumours growth curves of different groups of tumour-bearing mice after various treatments as indicated in the figure. Error bars are based on SD (n = 5); average weights of g primary and i distant tumours at the end of treatment; time-dependent body temperature (j) and weight (k) surveillance of mice (n = 5) after different treatments. Statistical significances were calculated via Student’s t test, **p < 0.01 and ***p < 0.001

Fig. 6

Antitumour effect of HMME/R837@Lip-augmented SDT plus anti-PD-L1 immunotherapy in orthotopic tumour models. a Schematic illustration of HMME/R837@Lip-augmented SDT and anti-PD-L1 combination therapy to inhibit tumour growth at the distant sites on orthotopic tumour models; b in vivo fluorescence images to study the biodistribution of HMME/R837@Lip post i.v. injection in 4T1 orthotopic tumour-bearing mice at the indicated time points. Yellow arrows indicate tumours; c the accumulation curve of HMME/R837@Lip in the tumour tissue by measuring the fluorescence of tumours at different time points post i.v. injection, error bars are based on SD (n = 3); d–i primary (d) and distant (g) tumour-growth curves of different groups of orthotopic tumour-bearing mice after various treatments as indicated in the figure. Error bars are based on SD (n = 5); average weights of primary (e) and distant (h) tumours at the end of treatments; photographs of excised primary (f) and distant (i) tumours at the end of treatments; j in vivo bioluminescence images tracking the spreading and growth of i.v. injected fLuc-4T1 tumour cells in the mice after different treatments; k representative photographs to show the gross appearance of tumour nodules in the lungs; l the numbers of lung nodules were counted under anatomy microscope. Values are means ± standard error (SE) (n = 5); m representative haematoxylin and eosin staining analysis of the lung metastasis. Scale bar = 2 mm. Statistical significances were calculated via Student’s t test, *p < 0.05, ** p < 0.01 and ***p < 0.001

Fig. 7

HMME/R837@Lip-augmented SDT plus anti-PD-L1 therapy activating systematic antitumour immunity. a–c Representative flow-cytometry plots showing the tumour-infiltrating leucocyte cells, including CD45⁺ cells (CD45⁺), CD8⁺ T cells (CD45⁺CD3e⁺CD8⁺) and CD4⁺ FoxP3⁺ T cells (CD45⁺CD3e⁺CD4⁺ FoxP3⁺) in mimic distant tumours; d absolute quantification of the CD45⁺ cells, CD8⁺ T cells and CD4⁺ FoxP3⁺ regulatory T cells (Treg) in mimic distant tumours (gating on CD3⁺ cells), error bars are based on SD (n = 3); e CD45⁺ cells: Treg ratios and CD8⁺cells: Treg ratios in the distant tumours after various treatments to remove the first tumour. Values are mean ± SD (n = 3); f representative CLSM images of the mimic distant tumours after immunofluorescence staining (scale bar = 50 μm). Statistical significances were calculated via Student’s t test or Mann–Whitney U test. NS not significant. *p < 0.05, **p < 0.01 and ***p < 0.001

Fig. 8

Anticancer activity of HMME/R837@Lip-augmented SDT plus anti-PD-L1 therapy in murine colorectal cancer models. a Schematic illustration of the experiment design to assess the antitumour immune responses against mimic distant tumours and the immunological memory response triggered by HMME/R837@Lip-augmented SDT and anti-PD-L1 combination therapy; b, c primary (b) and mimic distant (c) tumour growth curves of different groups of tumour-bearing mice after various treatments as indicated in the figure. Error bars are based on SD (n = 6); d time-dependent body-weight surveillance of mice (n = 6) after different treatments; e morbidity-free survival of mice after different treatments (n = 6); f tumour-growth curves of the rechallenged tumours in the corresponding groups were stopped when the first mouse died. Error bars are based on SE (n = 8); g morbidity-free survival of mice after the indicated treatment (n = 8), statistical significance was calculated via the log-rank (Mantel–Cox) test; h TNF-α and IFN-γ levels in serum isolated from mice of the treatment group and native group 7 days after the second tumour was introduced. Error bars are based on SE (n = 5 for the native group, n = 4 for the combined treatment group); i representative flow-cytometry plots of splenic lymphocytes of CT26 tumour-bearing mice treated with the combined immunotherapy at day 29 right before mice rechallenged with the secondary tumours (gating on CD3⁺ cells); j, k absolute quantification of naive T cells, TCM and TEM in the spleen. TCM central memory T cells, TEM effector memory T cells. Error bars are based on SD (n = 4). Statistical significances were calculated via Student’s t test. *p < 0.05, **p < 0.01 and ***p < 0.001