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. 2023 Jul 15;14(1):4223.
doi: 10.1038/s41467-023-39938-9.

mRNA lipid nanoparticle-mediated pyroptosis sensitizes immunologically cold tumors to checkpoint immunotherapy

Fengqiao Li  1 , Xue-Qing Zhang  2   3 , William Ho  1 , Maoping Tang  4   5 , Zhongyu Li  1 , Lei Bu  6 , Xiaoyang Xu  7   8
Affiliations

mRNA lipid nanoparticle-mediated pyroptosis sensitizes immunologically cold tumors to checkpoint immunotherapy

Fengqiao Li et al. Nat Commun. .

Abstract

Synergistically improving T-cell responsiveness is promising for favorable therapeutic outcomes in immunologically cold tumors, yet current treatments often fail to induce a cascade of cancer-immunity cycle for effective antitumor immunity. Gasdermin-mediated pyroptosis is a newly discovered mechanism in cancer immunotherapy; however, cleavage in the N terminus is required to activate pyroptosis. Here, we report a single-agent mRNA nanomedicine-based strategy that utilizes mRNA lipid nanoparticles (LNPs) encoding only the N-terminus of gasdermin to trigger pyroptosis, eliciting robust antitumor immunity. In multiple female mouse models, we show that pyroptosis-triggering mRNA/LNPs turn cold tumors into hot ones and create a positive feedback loop to promote antitumor immunity. Additionally, mRNA/LNP-induced pyroptosis sensitizes tumors to anti-PD-1 immunotherapy, facilitating tumor growth inhibition. Antitumor activity extends beyond the treated lesions and suppresses the growth of distant tumors. We implement a strategy for inducing potent antitumor immunity, enhancing immunotherapy responses in immunologically cold tumors.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1. Schematic of antitumor immunity via GSDMBNT mRNA@LNP-mediated pyroptosis and characterization of GSDMBNT mRNA@LNPs.
a Intratumoral administration of mRNA lipid nanoparticles encoding only the N-terminal domain of GSDMB triggers pyroptosis, eliciting antitumor immunity and facilitating anti-PD-1-mediated immunotherapy in immunologically cold tumors. b TEM image of GSDMBNT mRNA@LNPs. Scale bar = 200 nm. c The particle size, PDI, and zeta potential of GSDMBNT mRNA@LNPs were analyzed by DLS. Data are representative of four independent experiments. d DLS measurement of GSDMBNT mRNA@LNPs under the conditions indicated, including pH 6.5 buffer, pH 7.4 buffer, 10% or 20% plasma. Data are presented as means ± SD (n = 3). e Cellular uptake of FITC-labeled LNPs (Luc mRNA@FITCLNPs) was monitored in HEK 293, HeLa, 4T1, and B16F10 cells at different time points. Scale bar = 20 μm. f LNP-mediated endosomal/lysosomal escape and cytoplasmic release of Luc Cy5mRNA in HEK 293, HeLa, 4T1, and B16F10 cells 4 h after incubation. DAPI (blue), Endo/lysosome (green), Luc Cy5mRNA@LNPs (red), scale bar = 20 μm. Data shown in e, f are representative of two independent experiments. Source data are provided as a Source Data file. Cartoon in panel a was created with BioRender.com.
Fig. 2
Fig. 2. Lipid nanoparticles deliver mRNA encoding the N-terminal domain of GSDMB into cells to induce pyroptosis.
a Cell morphologies of the treated HEK 293, HeLa, 4T1 and B16F10 cells were detected using a confocal microscope. Before imaging, cells were treated with annexin V-FITC and propidium iodide (PI) and incubated for 15 min. Scale bars = 20 μm. Data are representative of three independent experiments. b LDH release-based cell death assay in HEK 293, HeLa, 4T1, and B16F10 cells after treatment with naked GSDMBNT mRNA, LNPs, or GSDMBNT mRNA@LNPs, respectively. Data are presented as means ± SD (n = 3). Statistical significance was calculated via one-way ANOVA. c Flow-cytometry analysis of cells positive for propidium iodide and annexin V. Data are presented as means ± SD (n = 3). Statistical significance was calculated using a two-tailed Student’s t test. Untreated cells served as the control (Ctrl) in all experiments. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. GSDMBNT mRNA@LNPs induce immunogenic pyroptosis and stimulate the maturation of bone marrow-derived dendritic cells (BMDCs).
a A confocal microscope was used to detect CRT expression in treated HEK 293, HeLa, 4T1, and B16F10 cells. Scale bar = 20 μm. Data are representative of three independent experiments. b, c Extracellular HMGB1 and ATP expression were analyzed by ELISA in HEK 293, HeLa, 4T1, and B16F10 cells after different treatments. Data in b are presented as means ± SD (n = 3). Data in c are presented as means ± SD (n = 4). Statistical significance was calculated using a two-tailed Student’s t test. d, e Immune stimulation of BMDCs by GSDMBNT mRNA@LNPs. B16F10 cells were pretreated with PBS (Ctrl), naked GSDMBNT mRNA, blank LNPs or GSDMBNT mRNA@LNPs, followed by coculture with BMDCs for 48 h. d Quantitative determination of proinflammatory cytokines IFN-γ, IL-1β and TNF-α using ELISA assay. Data are presented as means ± SD (n = 3). Statistical significance was calculated via one-way ANOVA. e Analysis of DC maturation biomarkers (MHC-II and CD86) using flow cytometry (n = 3). Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Treatment with GSDMBNT mRNA@LNPs promotes tumor control in an anti-PD-1-resistant 4T1 breast cancer mouse model.
a 6, 24, and 72 h after a single dose of GSDMBNT mRNA@LNPs (40 μg mRNA), cytokine concentrations were measured in tumor tissue or serum by ELISA. Data are presented as means ± SD (n = 4 mice per group). Statistical significance was calculated using a two-tailed Student’s t test. b Experimental timeline for treatment of 4T1 orthotopic tumor-bearing mice. s.c., subcutaneous; it, intratumoral; ip, intraperitoneal. c Individual growth curves of tumor size for mice treated as indicated. d, e The average tumor growth curves and survival percentages for mice treated as indicated (n = 7 mice per group). Data shown in e are represented as means ± SD. P values were determined by two-tailed unpaired Student’s t test in c, d or by log-rank (Mantel-Cox) test in e. Source data are provided as a Source Data file. Panel b was created with BioRender.com.
Fig. 5
Fig. 5. Treatment with GSDMBNT mRNA@LNPs induces antitumor immunity in an anti-PD-1-resistant 4T1 breast cancer mouse model.
a Immunofluorescence staining of tumors for CD8+ T cell infiltration and CRT expression after indicated treatments. Scale bar = 50 μm. b Quantitative analysis of immunofluorescence staining in terms of CD8+ and CRT intensities. Data are presented as means ± SD (n = 5). Statistical significance was calculated using a two-tailed Student’s t test. c ELISA analysis of IL-1β, IL-18, and HMGB1 in tumor and serum samples from 4T1 tumor-bearing mice receiving the treatments indicated. Data are presented as means ± SD (n = 4 mice per group). Statistical significance was calculated using a two-tailed Student’s t test. d Blood samples were collected on the second day after the final injection for aminotransaminase analyses. Data are presented as means ± SD (n = 4 mice per group). Source data are provided as a Source Data file.
Fig. 6
Fig. 6. Treatment with GSDMBNT mRNA@LNPs enhances potent antitumor activity in an aggressive melanoma mouse model.
a Experimental timeline for treatment of B16F10 tumor-bearing mice. b, c In vivo bioluminescence images and quantification of luciferase signals in mice treated as indicated for monitoring tumor growth. Imaging was performed every 5 days from the initial treatment day (day 7 after tumor inoculation) until day 23. d The survival percentages for mice treated as indicated. n = 8 mice for PBS, aPD-1, or GSDMBNT mRNA@LNP treatment groups, n = 7 mice for LNP treatment group, and n = 10 mice for aPD-1 + GSDMBNT mRNA@LNP treatment group. Survival analysis was analyzed using the log-rank (Mantel-Cox) test. e ELISA analysis of HMGB1 in the supernatant of B16F10 tumors excised from mice treated as indicated. Data are presented as means ± SD (n = 4 mice per group). f–h Representative images and quantitative analysis of immunofluorescence staining for CD8+ T cell infiltration and CRT expression in tumors after indicated treatments. Scale bar = 50 μm. Results are presented as means ± SD (n = 5). Statistical significance was calculated using a two-tailed Student’s t test. Source data are provided as a Source Data file. Panel a is created with BioRender.com.
Fig. 7
Fig. 7. Treatment with GSDMBNT mRNA@LNPs remodels the tumor microenvironment in an aggressive melanoma mouse model.
a The experimental timeline for the treatment of B16F10 tumor-bearing mice and cytokines and immune cell tests were conducted on day 18. b Cytokine concentrations were measured in tumor tissue or serum by ELISA. c Flow cytometry analysis results of the percentage of CD11c+ MHC-II+ DCs, CD3+ CD4+ T cells, CD3+ CD8+ T cells, CD3- NK1.1+ T cells, CD3+ NK1.1+ T cells, and monocytes isolated from lymph nodes or tumors. d Tumor weights of B16F10 tumor-bearing mice with different treatments. All results are presented as means ± SD (n = 4 mice per group). Statistical significance was calculated using a two-tailed Student’s t test. Source data are provided as a Source Data file. Panel a was created with BioRender.com.
Fig. 8
Fig. 8. Local combinational treatment of GSDMBNT mRNA@LNPs and aPD-1 controls tumor burden at distant sites.
C57BL/6 mice (n = 4 mice per group) were inoculated subcutaneously (s.c.) with 5 × 105 and 2.5 × 105 B16F10 cells in the left and right flanks, respectively. a Experimental timeline for treatment of B16F10 dual-tumor-bearing mice. b In vivo bioluminescence images of luciferase signals in mice treated as indicated for monitoring tumor growth. Imaging was performed every 5 days from the initial treatment day (day 7 after tumor inoculation) until day 23. c Individual curves of luciferase signals for mice treated as indicated. d, e Representative images and quantitative analysis of immunofluorescence staining for CD8+ T cell infiltration and CRT expression in tumors after indicated treatments. Scale bar = 50 μm. Results are presented as means ± SD (n = 5). Statistical significance was calculated using a two-tailed Student’s t test. Source data are provided as a Source Data file. Panel a is created with BioRender.com.
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