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. 2024 Jun;11(22):e2400485.
doi: 10.1002/advs.202400485. Epub 2024 Mar 29.

Regulation of CTLs/Tregs via Highly Stable and Ultrasound-Responsive Cerasomal Nano-Modulators for Enhanced Colorectal Cancer Immunotherapy

Jinxia Zhang  1   2 , Lihong Sun  2 , Ling Jiang  2 , Xinxin Xie  2 , Yuan Wang  2 , Ruiqi Wu  2 , Qingshuang Tang  2 , Suhui Sun  2 , Shiwei Zhu  2 , Xiaolong Liang  1   2 , Ligang Cui  1   2
Affiliations

Regulation of CTLs/Tregs via Highly Stable and Ultrasound-Responsive Cerasomal Nano-Modulators for Enhanced Colorectal Cancer Immunotherapy

Jinxia Zhang et al. Adv Sci (Weinh). 2024 Jun.

Abstract

Immunotherapy is showing good potential for colorectal cancer therapy, however, low responsive rates and severe immune-related drug side effects still hamper its therapeutic effectiveness. Herein, a highly stable cerasomal nano-modulator (DMC@P-Cs) with ultrasound (US)-controlled drug delivery capability for selective sonodynamic-immunotherapy is fabricated. DMC@P-Cs' lipid bilayer is self-assembled from cerasome-forming lipid (CFL), pyrophaeophorbid conjugated lipid (PL), and phospholipids containing unsaturated chemical bonds (DOPC), resulting in US-responsive lipid shell. Demethylcantharidin (DMC) as an immunotherapy adjuvant is loaded in the hydrophilic core of DMC@P-Cs. With US irradiation, reactive oxygen species (ROS) can be effectively generated from DMC@P-Cs, which can not only kill tumor cells for inducing immunogenic cell death (ICD), but also oxidize unsaturated phospholipids-DOPC to change the permeability of the lipid bilayers and facilitate controlled release of DMC, thus resulting in down-regulation of regulatory T cells (Tregs) and amplification of anti-tumor immune responses. After intravenous injection, DMC@P-Cs can efficiently accumulate at the tumor site, and local US treatment resulted in 94.73% tumor inhibition rate. In addition, there is no detectable systemic toxicity. Therefore, this study provides a highly stable and US-controllable smart delivery system to achieve synergistical sonodynamic-immunotherapy for enhanced colorectal cancer therapy.

Keywords: cerasomes; immunoregulation; immunotherapy; sonodynamic therapy; ultrasound‐responsive.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Fabrication illustration of DMC@P‐Cs and the delivery of sonosensitizers and immunosuppressants to the tumor to achieve the sonodynamic therapy of CT26 tumor combined with Immunotherapy.
Figure 2
Figure 2
Characterization of the DMC@P‐Cs. A) TEM image of DMC@P‐Cs. Scale bar: 50 nm. B) Dynamic light scattering (DLS) measurement of DMC@P‐Cs. C) Zeta potential of P‐Cs and DMC@P‐Cs. D) UV−vis absorption spectra of PL, P‐Cs, DMC, and DMC@P‐Cs in PBS solutions. E) Fluorescence spectra of P‐Cs and DMC@P‐Cs in aqueous solutions, while PL dissolved in organic solvents were used as a control (Ex = 405 nm). F) Comparison of ROS generation ability among DI water group, US group, PL group, P‐Cs group, DMC@P‐Cs group, P‐Cs + US group, and DMC@ P‐Cs + US group (equivalent concentration of PL: 20 µg/mL). G) Fluorescence emission spectra of the DMC@P‐Cs incubated with SOSG under US irradiation for different periods (equivalent concentration of PL: 20 µg/mL, Ex: 480 nm). H) DMC release behaviors of various samples in physiological status. I) DMC release behaviors of various samples with US irradiation.
Figure 3
Figure 3
In vitro cellular uptake and ROS production capacity of DMC@P‐Cs. A) The time‐dependence of CLSM images of CT26 cells after incubation with DMC@P‐Cs (equivalent concentration of PL: 20 µg mL−1). Scale bar: 40 µm. B) Flow cytometry analysis for cellular uptake of CT26 cells incubated with DMC@P‐Cs at the PL concentration of 20 µg mL−1 for different time. C) CLSM images of CT26 cells after incubation with various materials: PL, P‐Cs, and DMC@P‐Cs (equivalent concentration of PL: 20 µg mL−1). Blue signals indicated the location of cell nucleus, red signals indicated the internalization of DMC@P‐Cs, and green signals indicated the lysosomes stained with lysotracker green. Scale bar: 40 µm. D) Fluorescence images of CT26 cells treated in various ways: Control (without any treatment), US, P‐Cs, DMC@P‐Cs, P‐Cs + US, and DMC@P‐Cs + US (equivalent concentration of PL: 20 µg mL−1). Scale bar: 200 µm.
Figure 4
Figure 4
Experiments on the killing ability and mechanism of DMC@P‐Cs on CT26 cells. A) Fluorescence images of CT26 cells stained with the LIVE/DEAD activity/cytotoxicity kit. Scale Bar: 100 µm. B) Viability of CT26 cells incubated with different concentrations of DMC@P‐Cs, DMC@P‐Ls and Free DMC. C) Viability of CT26 cells treated in different ways: P‐Cs + US, DMC@P‐Cs + US. D) Fluorescence images of Rodamine 123‐labeled CT26 cells treated in the same way as the above‐mentioned evaluation of intracellular ROS production. Scale bar: 200 µm. E) The invasion ability of CT26 cells was analyzed by Transwell. Scale bar: 200 µm.
Figure 5
Figure 5
SDT‐Triggered ICD mediated by DMC@P‐Cs and inhibition of Tregs formation by DMC@P‐Cs combined with US. A) CRT exposed on CT26 cell surface after different treatments as observed by CLSM. Scale bar: 20 µm. B) Quantification of CRT signal intensity for different groups in (A). Data were presented as mean ± SD (n = 3). C) HMGB1 released from CT26 cells after different treatments as observed by CLSM. Scale bar: 20 µm. D) Quantification of HMGB1 signal intensity for different groups in (C). Data were presented as mean ± SD (n = 3). E) HSP70 exposed on CT26 cell surface after different treatments as observed by CLSM. Scale bar: 20 µm. F) Quantification of HSP70 signal intensity for different groups in (E). Data were presented as mean ± SD (n = 3). G) PP2A activity of CD4+ T cells after various treatments with control, free DMC, P‐Cs, P‐Cs + US, DMC@P‐Cs, and DMC@P‐Cs + US for 8 h. H) Flow cytometry analysis of intracellular FOXP3 expression of CD4+ T cells after incubation with different treatments for 3 days in the presence of anti‐CD3 and CD28. I) Quantification of CD4+FOXP3+ T cells after incubation of naive CD4+ T cells with different treatments for 3 days in the presence of anti‐CD3 and CD28.
Figure 6
Figure 6
Evaluation of the anti‐tumor effect in vivo. A) In vivo NIR fluorescence images of CT26 tumor‐bearing mice after i.v. administration of DMC@P‐Cs/DMC@P‐Ls (PL: 4mg/kg) (n=3). B) Quantification of NIR signals of tumor sites of CT26 tumor‐bearing mice at different time‐points showed in (A) C) Fluorescence images of major organs and tumors from mice at 48 h post injection of DMC@P‐Cs/DMC@P‐Ls. D) Quantitative analysis of fluorescence intensity of tumors and main organs showed in (C). E) Schematic diagram of the treatment route. F) Tumor volume change after different treatments. G) Body weight change of mice in the 19 days' treatment. H) H&E staining, TUNEL staining, Ki67 immunohistochemical staining and Caspase‐3  immunofluorescence staining of the tumor tissue slices after different treatments (PBS, US, P‐Cs, DMC@P‐Cs, P‐Cs+ US, and DMC@P‐Cs + US). Scale bar: 100 µm.
Figure 7
Figure 7
DMC@P‐Cs mediated antitumor immune response. A) Percentages of CD4+/CD8+ T cells within the tumors of mice receiving the indicated treatments. B) Representative flow cytometric plots and the percentages of mature DCs (CD11c+CD80+CD86+) in tumor‐draining lymph nodes. C) Percentages of FOXP3+ T cells within the tumors of mice receiving the indicated treatments. Data were presented as mean ± SD (n = 3). D) Quantification the percentage of CD8+ T cells shown in (A). Data were presented as mean ± SD (n = 3). E) Quantification the percentage CD80+CD86+cells shown in (B). Data were presented as mean ± SD (n = 3). F) Quantification the percentage CD25+FOXP3+cells shown in Figure 7C. Data were presented as mean ± SD (n = 3). Cytokine levels of G) IFN‐γ, H) IL‐6, and I) TNF‐α in the serum collected from CT26 tumor‐bearing mice after different treatments at day 13 (n = 3).
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