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Cancer immunogenic cell death via pyroptosis with CXCR4-targeted nanotoxins in hepatocellular carcinoma

Huang, Yingbin, Li, Yihu, He, Rui, Dong, Shuyi, Zhao, Zheng, Jiao, Xingyuan·2024·Frontiers in Bioengineering and Biotechnology
DOI 10.3389/fbioe.2024.1433126PMC PMC11570815
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Abstract

Introduction: Cytotoxic agents have shown limited benefits in hepatocellular carcinoma (HCC), mediated in part by the lack of targeting. As cell-penetrating peptides (CPPs) are capable of delivering various biologically active molecules into cells, including protein, peptides, small chemo-drugs, and nucleic acid with or without targeting, we developed T22-PE24, a CXCR4-targeted self-assembling cytotoxic nanotoxin, to effectively induce HCC pyroptosis. Methods: T22 incorporating enhanced green fluorescent protein (EGFP) or PE24 was purified from DE3 bacterial cells and characterized using transmission electron microscopy, the Zetasizer Nano®, and SEC-HPLC. The internalization effect of T22-EGFP was detected by flow cytometry system (FCS) in CXCR4+/LM3(CXCR4−) HCC cells. The CCK8, lactate dehydrogenase (LDH) release, Western blot, and nude mice HCC models were used to estimate the cell viability of T22-PE24. The complete-immunity HCC tumor-bearing mice model was used to assess the immune response of T22-PE24. Results: The round shape under transmission electron microscopy, 49.4 nm hydrodynamic diameter, and −33.33 mV zeta potential indicated that T22-PE24 self-assembled into nanoparticles. T22 incorporating EGFP selectively internalized in CXCR4+ HCC cells and showed no accumulation in CXCR4-knockout HCC cells. The T22-PE24 nanotoxin induced HCC pyroptosis via the caspase-3/GSDME signaling pathway and suppressed tumor growth in the absence of histological alterations in normal organs. Using the complete-immunity HCC tumor-bearing mice model, we found that T22-PE24 nanotoxin effectively induces the global reprogramming of cell components of the immune tumor microenvironment, leading to enhanced antitumor effects compared to those observed in immunodeficient mice. Conclusion: Our findings demonstrate the activation of the innate immune response in HCC by inducing pyroptosis with T22-PE24 nanotoxin treatment and support an implementation of this strategy for HCC treatment.

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FIGURE 1 High expression of CXCR4 in HCC cells and T22 targeting HCC. (A) CXCR4 expression in HCC cells and the normal tissue analyzed from TCGA. (B) CXCR4 expression in HCC cell lines determined by RT-PCR. (C) Cellular uptake in LM3 and LM3CXCR4− cells after treatment with various concentrations of T22-PE24 for 4 h by flow cytometry. The results are representative of three independent experiments. (D) Quantification analysis of T22-PE24 internalization in (C). (E) Cellular uptake in LM3 and LM3CXCR4− cells after treatment with T22-PE24 alone or in combination with CXCR4 inhibitor AMD3100. (F) Quantification analysis of T22-PE24 internalization in (E). Mean ± SEM of n = 3 repeats. *p < 0.05, **p < 0.01, ***p < 0.001 in a one-way ANOVA, followed by Tukey’s multiple-comparison test.
FIGURE 1 High expression of CXCR4 in HCC cells and T22 targeting HCC. (A) CXCR4 expression in HCC cells and the normal tissue analyzed from TCGA. (B) CXCR4 expression in HCC cell lines determined by RT-PCR. (C) Cellular uptake in LM3 and LM3CXCR4− cells after treatment with various concentrations of T22-PE24 for 4 h by flow cytometry. The results are representative of three independent experiments. (D) Quantification analysis of T22-PE24 internalization in (C). (E) Cellular uptake in LM3 and LM3CXCR4− cells after treatment with T22-PE24 alone or in combination with CXCR4 inhibitor AMD3100. (F) Quantification analysis of T22-PE24 internalization in (E). Mean ± SEM of n = 3 repeats. *p < 0.05, **p < 0.01, ***p < 0.001 in a one-way ANOVA, followed by Tukey’s multiple-comparison test.CC BY · PubMed Central
FIGURE 2 T22-PE24 inhibited the proliferation of HCC cells by activating pyroptosis. (A) IC50 of LO2, LM3, LM3CXCR4−, Hep3B, and Hep3BCXCR4− cells after incubation with T22-PE24. (B) Images of cell morphology in LM3 and LM3CXCR4− after incubation with 1 μM T22-PE24 determined by IncuCyte. Scale bar = 50 μm. (C) Quantification analysis of the LDH release in LM3, LM3CXCR4−, Hep3B, and Hep3BCXCR4− cells after incubation with 1 μM T22-PE24. (Mean ± SD shown. ***p < 0.001 in a two-way ANOVA, followed by Sidak’s multiple-comparison test.) (D) Flow cytometric analysis of pyroptosis induced by 1 μM T22-PE24. The upper right quadrant shows the pyroptotic cells.
FIGURE 2 T22-PE24 inhibited the proliferation of HCC cells by activating pyroptosis. (A) IC50 of LO2, LM3, LM3CXCR4−, Hep3B, and Hep3BCXCR4− cells after incubation with T22-PE24. (B) Images of cell morphology in LM3 and LM3CXCR4− after incubation with 1 μM T22-PE24 determined by IncuCyte. Scale bar = 50 μm. (C) Quantification analysis of the LDH release in LM3, LM3CXCR4−, Hep3B, and Hep3BCXCR4− cells after incubation with 1 μM T22-PE24. (Mean ± SD shown. ***p < 0.001 in a two-way ANOVA, followed by Sidak’s multiple-comparison test.) (D) Flow cytometric analysis of pyroptosis induced by 1 μM T22-PE24. The upper right quadrant shows the pyroptotic cells.CC BY · PubMed Central
FIGURE 3 T22-PE24, targeting CXCR4, activated pyroptosis via the caspase3/GSDME pathway. (A) Western blot analysis of the silence expression effect in LM3 transfected with siGSDMA, siGSDMB, siGSDMC, siGSDMD, or siGSDME for 48 h. (B) Images of cell morphology in LM3 transfected with siGSDMA, siGSDMB, siGSDMC, siGSDMD, or siGSDME after incubation with 1 μM T22-PE24. (C) Western blot analysis of LM3 treated with 0 μM, 0.25 μM, 0.5 μM, or 1 μM T22-PE24 for 24 h. (D) Western blot analysis of LM3 and LM3CXCR4− cells treated with or without 0.5 μM T22-PE24 for 24 h. (E) Western blot analysis of LM3 treated with 0.5 μM T22-PE24 and pretreated with or without 40 μM Z-VAD (OME)-FMK for 24 h. (F) Western blot analysis of Hep3B treated with 0 μM, 0.25 μM, 0.5 μM, or 1 μM T22-PE24 for 24 h. (G) Western blot analysis of Hep3B and Hep3BCXCR4− cells treated with or without 0.5 μM T22-PE24 for 24 h. (H) Western blot analysis of Hep3B treated with 0.5 μM T22-PE24 and pretreated with or without 40 μM Z-VAD (OME)-FMK for 24 h.
FIGURE 3 T22-PE24, targeting CXCR4, activated pyroptosis via the caspase3/GSDME pathway. (A) Western blot analysis of the silence expression effect in LM3 transfected with siGSDMA, siGSDMB, siGSDMC, siGSDMD, or siGSDME for 48 h. (B) Images of cell morphology in LM3 transfected with siGSDMA, siGSDMB, siGSDMC, siGSDMD, or siGSDME after incubation with 1 μM T22-PE24. (C) Western blot analysis of LM3 treated with 0 μM, 0.25 μM, 0.5 μM, or 1 μM T22-PE24 for 24 h. (D) Western blot analysis of LM3 and LM3CXCR4− cells treated with or without 0.5 μM T22-PE24 for 24 h. (E) Western blot analysis of LM3 treated with 0.5 μM T22-PE24 and pretreated with or without 40 μM Z-VAD (OME)-FMK for 24 h. (F) Western blot analysis of Hep3B treated with 0 μM, 0.25 μM, 0.5 μM, or 1 μM T22-PE24 for 24 h. (G) Western blot analysis of Hep3B and Hep3BCXCR4− cells treated with or without 0.5 μM T22-PE24 for 24 h. (H) Western blot analysis of Hep3B treated with 0.5 μM T22-PE24 and pretreated with or without 40 μM Z-VAD (OME)-FMK for 24 h.CC BY · PubMed Central
FIGURE 4 In vivo antitumor efficacy of T22-PE24 in tumor-bearing mice. (A) Tumor growth was measured over time. T22-PE24 was injected every 2 days for a total of six times. (B) Photographs of solid tumors in each treatment group after 17 days. (C) Tumor weight of different treatment groups at the end of the experiment. (D) IHC images of the tumor sections stained with cleaved caspase-3 or Ki67 after various treatments. (E) Quantification analysis of cleaved caspase-3-positive cells. Each dot represented a single tumor. (F) Quantification analysis of Ki67-positive cells. Each dot represented a single tumor. Mean ± SEM is shown. ns means no significance. *p < 0.05, ***p < 0.001, ****p < 0.0001. Tumor growth curves used two-way ANOVA followed by Tukey’s multiple-comparison test. Tumor weights, cleaved caspase-3, and Ki67 expression in tumor tissues used a one-way ANOVA, followed by Tukey’s multiple-comparison test.
FIGURE 4 In vivo antitumor efficacy of T22-PE24 in tumor-bearing mice. (A) Tumor growth was measured over time. T22-PE24 was injected every 2 days for a total of six times. (B) Photographs of solid tumors in each treatment group after 17 days. (C) Tumor weight of different treatment groups at the end of the experiment. (D) IHC images of the tumor sections stained with cleaved caspase-3 or Ki67 after various treatments. (E) Quantification analysis of cleaved caspase-3-positive cells. Each dot represented a single tumor. (F) Quantification analysis of Ki67-positive cells. Each dot represented a single tumor. Mean ± SEM is shown. ns means no significance. *p < 0.05, ***p < 0.001, ****p < 0.0001. Tumor growth curves used two-way ANOVA followed by Tukey’s multiple-comparison test. Tumor weights, cleaved caspase-3, and Ki67 expression in tumor tissues used a one-way ANOVA, followed by Tukey’s multiple-comparison test.CC BY · PubMed Central
FIGURE 5 In vivo immune activation efficacy of T22-PE24 in tumor-bearing mice. (A) Tumor growth was measured over time. T22-PE24 was injected every 2 days for a total of six times. (B) Photographs of solid tumors in each treatment group after 17 days. (C) Tumor weight of different treatment groups at the end of the experiment. (D) H&E and IF images of the tumor sections stained with CD3 and CD8 after various treatments. (E) Representative flow cytometry analysis of the CD3+ CD8+ T cell subgroup in PBMCs. (F) Quantification of CD3+ CD8+ population in PBMCs. Mean ± SEM is shown. *p < 0.05, ***p < 0.001, and ****p < 0.0001. Tumor growth curves used two-way ANOVA, followed by Tukey’s multiple-comparison test. Tumor weights used a one-way ANOVA followed by Tukey’s multiple-comparison test. Quantification of CD3+ CD8+ population analysis was performed by Student’s t-test.
FIGURE 5 In vivo immune activation efficacy of T22-PE24 in tumor-bearing mice. (A) Tumor growth was measured over time. T22-PE24 was injected every 2 days for a total of six times. (B) Photographs of solid tumors in each treatment group after 17 days. (C) Tumor weight of different treatment groups at the end of the experiment. (D) H&E and IF images of the tumor sections stained with CD3 and CD8 after various treatments. (E) Representative flow cytometry analysis of the CD3+ CD8+ T cell subgroup in PBMCs. (F) Quantification of CD3+ CD8+ population in PBMCs. Mean ± SEM is shown. *p < 0.05, ***p < 0.001, and ****p < 0.0001. Tumor growth curves used two-way ANOVA, followed by Tukey’s multiple-comparison test. Tumor weights used a one-way ANOVA followed by Tukey’s multiple-comparison test. Quantification of CD3+ CD8+ population analysis was performed by Student’s t-test.CC BY · PubMed Central
FIGURE 6 The self-assembling nanotoxin T22-PE24, specifically targeting CXCR4, was designed to selectively deliver the cytotoxic agent PE24 to HCC cells, leading to immunogenic pyroptotic cell death.
FIGURE 6 The self-assembling nanotoxin T22-PE24, specifically targeting CXCR4, was designed to selectively deliver the cytotoxic agent PE24 to HCC cells, leading to immunogenic pyroptotic cell death.CC BY · PubMed Central

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Metadata

Authors
Huang, Y., Li, Y., He, R., Dong, S., Zhao, Z., & Jiao, X.
Venue
Frontiers in Bioengineering and Biotechnology
Publisher
Frontiers Media SA
Published
2024-11-04
Volume
12
ISSN
2296-4185
DOI
10.3389/fbioe.2024.1433126
PMC
PMC11570815
Cited by
7
References
1
Licence
creativecommons.org/licenses/by/4.0/ (vor)

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