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. 2020 Oct;22(10):1264-1275.
doi: 10.1038/s41556-020-0575-z. Epub 2020 Sep 14.

PD-L1-mediated gasdermin C expression switches apoptosis to pyroptosis in cancer cells and facilitates tumour necrosis

Junwei Hou  1 , Rongce Zhao  1   2   3 , Weiya Xia  1 , Chiung-Wen Chang  1 , Yun You  1 , Jung-Mao Hsu  1   4 , Lei Nie  1 , Yeh Chen  4   5 , Yu-Chuan Wang  4 , Chunxiao Liu  1 , Wei-Jan Wang  1   6 , Yun Wu  7 , Baozhen Ke  1 , Jennifer L Hsu  1 , Kebin Huang  1   8 , Zu Ye  1 , Yi Yang  1 , Xianghou Xia  1 , Yintao Li  1 , Chia-Wei Li  1 , Bin Shao  1   9 , John A Tainer  1 , Mien-Chie Hung  10   11   12
Affiliations

PD-L1-mediated gasdermin C expression switches apoptosis to pyroptosis in cancer cells and facilitates tumour necrosis

Junwei Hou et al. Nat Cell Biol. 2020 Oct.

Erratum in

Abstract

Although pyroptosis is critical for macrophages against pathogen infection, its role and mechanism in cancer cells remains unclear. PD-L1 has been detected in the nucleus, with unknown function. Here we show that PD-L1 switches TNFα-induced apoptosis to pyroptosis in cancer cells, resulting in tumour necrosis. Under hypoxia, p-Stat3 physically interacts with PD-L1 and facilitates its nuclear translocation, enhancing the transcription of the gasdermin C (GSDMC) gene. GSDMC is specifically cleaved by caspase-8 with TNFα treatment, generating a GSDMC N-terminal domain that forms pores on the cell membrane and induces pyroptosis VSports手机版. Nuclear PD-L1, caspase-8 and GSDMC are required for macrophage-derived TNFα-induced tumour necrosis in vivo. Moreover, high expression of GSDMC correlates with poor survival. Antibiotic chemotherapy drugs induce pyroptosis in breast cancer. These findings identify a non-immune checkpoint function of PD-L1 and provide an unexpected concept that GSDMC/caspase-8 mediates a non-canonical pyroptosis pathway in cancer cells, causing tumour necrosis. .

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Conflict of interest statement

Declaration of Interests

The authors have no conflicts of interest to declare.

Figures

Extended Data Fig. 1
Extended Data Fig. 1. Establishment of stable cells in MDA-MB-231 cell line
(a) Deletion of endogenous Stat3 by CRISPR/Cas9. KO, knockout. Blots are representative of three independent experiments. (b) Stable re-expression of Stat3-WT or Stat3-Y705F in Stat3-knockout MDA-MB-231 cells. Validation of p-Y705-Stat3 expression level by immunoblotting. Blots are representative of three independent experiments. (c) Analysis of the nuclear localization signal (NLS) and nuclear export signal (NES) in the human PD-L1 amino acid sequence compared with that of other species. Sequence alignment of PD-L1 from human, mouse, cow, rat, and dog. The alignment was generated and presented by the ClustalW2 algorithm and ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Identical residues are indicated by the dark red background and conserved residues are in red. The NLS and NES sequences of PD-L1 are conserved across species. (d-f) Stable re-expression of PD-L1-WT (wild-type PD-L1), PD-L1-NLS (NLS-mutated PD-L1) or PD-L1-NES (NES-mutated PD-L1) in the PD-L1–knockout MDA-MB-231 cell line. Immunoblotting of total PD-L1 expression level in stable transfectants (d). Blots are representative of three independent experiments. Representative images of PD-L1 distribution of three independent experiments was determined by confocal assay under hypoxia (e-f). Scale bar, 20 μm. Unprocessed blots are provided in Source Data Extended Data Fig. 1.
Extended Data Fig. 2
Extended Data Fig. 2. nPD-L1-mediated pyroptotic cell death in cell lines of multiple cancer types.
(a) Immunoblotting analysis of PD-L1 expression levels in liver cancer and lung cancer cell lines. Blots are representative of three independent experiments. (b) Hypoxia-induced pyroptotic cell death in PD-L1-positive cells of multiple cancer types treated with p-Stat3 inhibitor, HO-3867. Red arrows indicate the pyroptotic cells with big bubbles. The experiment was repeated three times with similar results. Scale bar, 20 μm. Unprocessed blots are provided in Source Data Extended Data Fig. 2.
Extended Data Fig. 3
Extended Data Fig. 3. nPD-L1 and p-Y705-Stat3 are required for hypoxia-induced GSDMC expression.
(a) Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of mRNA levels of gasdermin family members in the indicated MDA-MB-231 stable transfectants. Data are shown as mean ± SD of n = 3 independent experiments. (b and c) GSDMC mRNA (b) and protein (c) levels in MDA-MB-231 cells under hypoxia. Data of (b) are shown as mean ± SD of n = 3 independent experiments. Blots are representative of three independent experiments. N, normoxia; H, hypoxia. (d) RT-qPCR analysis of mRNA levels of GSDMC in the indicated MDA-MB-231 stable transfectants. Data are shown as mean ± SD of n = 3 independent experiments. (e-g) MDA-MB-231 cells were cultured under hypoxia and treated with HO-3867 (20 μM) or ivermectin (25 μM). GSDMC mRNA (e) and protein (f) levels were analyzed by RT-qPCR and immunoblotting, respectively. GSDMC promoter luciferase reporter plasmids were transfected to cells, and luciferase activity was measured in (g). Data of (e) and (g) are shown as mean ± SD of n = 3 independent experiments. Blots are representative of three independent experiments. P values of all statistical analysis were determined by two-sided Student’s t-test. Statistical source data and unprocessed blots are provided in Source Data Extended Data Fig. 3.
Extended Data Fig. 4
Extended Data Fig. 4. The p-Y705-Stat3 binding site (in red) in the nucleic acid sequence of GSDMC promoter and the expression level of GSDME in MDA-MB-231 cells.
(a) GSDMC promoter sequence was obtained from GeneCopoeia and analyzed using the GPMiner program (http://gpminer.mbc.nctu.edu.tw/). Two putative p-Y705-Stat3–binding sites in the GSDMC promoter were predicted. p-Y705-Stat3-binding site was validated by screening with luciferase reporter assay. (b-d) Immunoblotting of GSDME in MDA-MB-231 cells. H226 cells as a positive control. Immunoblotting of the endogenous GSDME (b). Immunoblotting of GSDME in MDA-MB-231 cells under hypoxia (c). Immunoblotting of GSDME in MDA-MB-231 cells treated with daunorubicin, doxorubicin, epirubicin, and actinomycin-D (d). The experiments of (b-d) were repeated three times with similar results respectively. Unprocessed blots are provided in Source Data Extended Data Fig. 4.
Extended Data Fig. 5
Extended Data Fig. 5. Caspase-8, but not caspase-6, is required for TNFα-induced pyroptosis under hypoxia.
(a and b) MDA-MB-231 cells were cultured under normoxia (N) or hypoxia (H) and treated as indicated. LDH-released cell death was measured as shown in (a). Data are shown as mean ± SD of n = 3 independent experiments. P values were determined by two-sided Student’s t-test. Representative images of dying cell morphology of three independent experiments (b). Red arrows indicate cell swelling with big bubbles. Scale bar, 20 μm. Caspase-3i, caspase-3 inhibitor Z-DEVD-FMK; Caspase-6i, caspase-6 inhibitor Z-VEID-FMK; Caspase-8i, caspase-8 inhibitor Z-IETD-FMK (each at 10 μM). Statistical source data are provided in Source Data Extended Data Fig. 5.
Extended Data Fig. 6
Extended Data Fig. 6. The pyroptotic cell death in cell lines with different GSDMC and PD-L1 expression status.
(a) Immunoblotting of PD-L1 and GSDMC in Hs578T, BT549, MDA-MB-231 and MCF-7 cells. Blots are representative of three independent experiments. (b) LDH-released cell death in Hs578T, BT549, MDA-MB-231 and MCF-7 cells treated with TNFα under normoxia or hypoxia. Data are shown as mean ± SD of n = 3 independent experiments. P values were determined by two-sided Student’s t-test. Statistical source data and unprocessed blots are provided in Source Data Extended Data Fig. 6.
Extended Data Fig. 7
Extended Data Fig. 7. The N-terminal domain (1-365) of GSDMC is sufficient to induce pyroptosis
(a) 3-D structural modeling of GSDMC. The modeling was performed using SWISS-MODEL. The structural fold of GSDMC N- and C-terminal domains is in blue cartoon presentation. The linker region and the solvent-exposed loop region of the C-terminal domain of GSDMC are colored in green with numbered Asp/Glu residues colored in magenta. The structural figure was prepared using PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. (b) Confocal microscopy analysis of the distribution of GSDMC-FL, GSDMC (1¬–365), and GSDMD-N expressions in HeLa cells. GSDMD-N, N-terminal domain of GSDMD. Images are representative of three independent experiments. Scale bar, 20 μm. (c–d) HeLa cells were transfected with GSDMC-FL or GSDMC (1–365). Representative images of dying cell morphology of three independent experiments (c). Scale bar, 20 μm. Dying cells were stained with nucleic acid dye SYTOX green. Red arrows indicate cell swelling with large bubbles. LDH-released cell death is shown as mean ± SD of n = 3 independent experiments (d). P values were determined by two-sided Student’s t-test. (e) HEK293T cells were transfected with Flag-GSDMC-FL or Flag-GSDMC (1–365) for 16 h. Supernatants and cell pellets were collected and subjected to immunoblotting. The blot is representative of three independent experiments. (f) Cartoon diagram of GSDMC structure and the cleavage by caspase-8 across species. Statistical source data and unprocessed blots are provided in Source Data Extended Data Fig. 7.
Extended Data Fig. 8
Extended Data Fig. 8. The nuclear PD-L1 translocation in vivo causes poor survival in both immunocompetent and nude mice
(a) IHC staining of HIF1α, GSDMC, PD-L1, and p-Y705-Stat3 in MDA-MB-231 xenografts in nude mice (n = 8). MDA-MB-231 cells were injected subcutaneously into nude mice. Four weeks after tumor cell injection, all mice were sacrificed and tumors excised for IHC staining. The expression levels and patterns of HIF1α, GSDMC, PD-L1, and p-Y705-Stat3 in normoxic and hypoxic area in tumors were analyzed and indicated by green arrows. Scale bar, 100 μm. The experiment was repeated three times with similar results. (b) Survival analysis of 4T1 cells expressing PD-L1-WT or PD-L1-NLS in BALB/c nude mice. Data are shown of n = 10 mice. The experiment was repeated three times with similar results. Statistical significance for survival analysis was determined using a log-rank (Mantel–Cox) test. (c) Survival analysis of 4T1 cells expressing PD-L1-WT or PD-L1-NLS in immunocompetent BALB/c mice. Data are shown of n = 10 mice. The experiment was repeated three times with similar results. Statistical significance for survival analysis was determined using a log-rank (Mantel–Cox) test. Statistical source data are provided in Source Data Extended Data Fig. 8.
Extended Data Fig. 9
Extended Data Fig. 9. Chemotherapeutic drugs induce pyroptotic cell death by nPD-L1/GSDMC-mediated non-canonical pathway in breast tumor.
(a) Representative images of dying cell morphology by phase-contrast microscopy in MDA-MB-231 stable transfectants as indicated treated with daunorubicin, doxorubicin, epirubicin, and actinomycin-D. The experiment was repeated three times with similar results. Scale bar, 20 μm. (b) LDH-released cell death in MDA-MB-231 stable transfectants as indicated treated with daunorubicin, doxorubicin, epirubicin, and actinomycin-D. Data are shown as mean ± SD of n = 3 independent experiments. P values were determined by two-sided Student’s t-test. Statistical source data are provided in Source Data Extended Data Fig. 9.
Extended Data Fig. 10
Extended Data Fig. 10. A proposed model of nuclear PD-L1-mediated apoptosis-to-pyroptosis switch under hypoxia.
Under normoxia, caspase-3 can be activated by TNFα-induced caspase-8 to cause apoptotic cell death. However, hypoxia-activated p-Stat3 facilitates nPD-L1 translocation. nPD-L1 cooperates with p-Stat3 to transcriptionally activate GSDMC expression. GSDMC is specifically cleaved by macrophage-derived TNFα-activated caspase-8, which generates an N-terminal pore-forming fragment that causes pyroptotic cell death to induce tumor necrosis in breast cancer.
Fig. 1:
Fig. 1:. PD-L1 translocates into the nucleus in response to hypoxia.
(a) The relative ratio of nPD-L1 to total PD-L1 in MDA-MB-231 cells treated with various stimuli was evaluated on the basis of immunoblot quantification using the Image J software (version 1.38x). Data are shown of n = 3 independent experiments. TKI, tyrosine kinase inhibitor; PARPi, poly (ADP-ribose) polymerase inhibitor; PD-L1 Ab, PD-L1 antibody; MMC, mitomycin C; MMS, methyl methanesulfonate; 28-8, PD-L1 antibody from clone 28-8. (b) Immunoblotting of PD-L1 after cellular fractionation in MDA-MB-231 cells. N, normoxia; H, hypoxia. Blots are representative of three independent experiments. (c) Confocal microscopy analysis of PD-L1 expression in MDA-MB-231 cells. Scale bar, 20 μm. Images are representative of three independent experiments. (d) Percentage of MDA-MB-231 cells with nPD-L1 as determined in (c). Fifty nuclei were counted in each treatment group. Data are shown of n = 3 independent experiments. (e) 3D visualization of nPD-L1 (scale bar, 20 μm). Images are representative of three independent experiments. (f) Immunoblotting of PD-L1 in cellular fractions at the indicated time points under hypoxia. The experiment was repeated three times with similar results. (g) The percentage of PD-L1 nuclear translocation under hypoxia. Diagram showing the relative ratio of nPD-L1 to total PD-L1 based on the immunoblot quantification in (f) using the Image J software. Data are shown of n = 3 independent experiments. (h) Knockdown of HIF1α using shRNA and immunoblotting validation of its protein level. The experiment was repeated three times with similar results. (i) Comparison of nPD-L1 translocation in shControl stable transfectants to that in shHIF1α under hypoxia. The experiment was repeated three times with similar results. (j) Kinetics of PD-L1 nuclear translocation under hypoxia. Diagram showing the relative ratio of nPD-L1 to total PD-L1 based on immunoblot quantification in (i) using Image J software. Data are shown of n = 3 independent experiments. All error bars represent SD. P values were determined by two-sided Student’s t-test. Statistical source data and unprocessed blots are provided in Source Data Fig. 1.
Fig. 2:
Fig. 2:. p-Y705-Stat3 binds to PD-L1 and facilitates its nuclear translocation under hypoxia.
(a) Immunoprecipitation (IP) and Western blot analysis of PD-L1–p-Y705-Stat3 interaction under hypoxia in MDA-MB-231 cells. The experiment was repeated three times with similar results. (b) IP and Western blot analysis of the interaction of truncated PD-L1 with p-Y705-Stat3. PD-L1-FL, full-length PD-L1; PD-L1-ECD, PD-L1 extracellular domain; PD-L1-ICD, PD-L1 intracellular domain. The experiment was repeated three times with similar results. (c) Duolink assay (red dot: interaction between p-Y705-Stat3 and PD-L1) with antibodies specific for p-Y705-Stat3 and PD-L1. Scale bar, 20 μm. N, normoxia; H, hypoxia. The experiment was repeated three times with similar results. (d) 3D visualization of nPD-L1–p-Y705-Stat3 interaction. Scale bar, 20 μm. The experiment was repeated three times with similar results. (e) Confocal microscopy analysis of PD-L1 expression. Representative images shown. Wild-type Stat3 (Stat3-WT) or Stat3-Y705F mutant (Stat3-Y705F) were stably expressed in Stat3-knockout MDA-MB-231 cells. Scale bar, 20 μm. The experiment was repeated three times with similar results. (f) Immunoblotting of PD-L1 and p-Y705-Stat3 in the cellular fraction of the indicated MDA-MB-231 stable transfectants. The experiment was repeated three times with similar results. (g–i) MDA-MB-231 cells were treated with p-Y705-Stat3 inhibitor HO-3867 (20 μM) or importin α/β inhibitor ivermectin (25 μM). Localization of PD-L1 or PD-L1–p-Y705-Stat3 interaction (red dots) was analyzed by Duolink assay (g), confocal microscopy (h), and cellular fractionation (i). Scale bar, 20 μm. The experiments were repeated three times with similar results. Unprocessed blots are provided in Source Data Fig. 2.
Fig. 3:
Fig. 3:. nPD-L1 switches TNFα-induced apoptosis to pyroptosis under hypoxia.
(a) Stable re-expression of WT or NES-mutated PD-L1 in the PD-L1–knockout MDA-MB-231 cells. Images of PD-L1 distribution are representative of three independent experiments. Scale bar, 20 μm. (b) Fluorescent imaging of nPD-L1-induced apoptosis-to-pyroptosis switch under TNFα plus CHX treatment. Dying cells were stained with nucleic acid dye SYTOX green. Red arrows indicate cell swelling with large bubbles. Images are representative of three independent experiments. Scale bar, 20 μm. (c) LDH-released cell death is shown as mean ± SD of n = 3 independent experiments. (d) Time-lapse microscopy quantification of dying cells treated as described in (b). (e) LDH-released cell death in PD-L1-NES stable cells treated with TNFα plus CHX and/or HO-3867. Data are shown as mean ± SD of n = 3 independent experiments. (f) MDA-MB-231-PD-L1–KO cells with stable expression of WT or NLS-mutated PD-L1 were treated with TNFα plus CHX under hypoxia. LDH-released cell death is shown as mean ± SD of n = 3 independent experiments. (g and h) MDA-MB-231 cells were treated with TNFα plus CHX under hypoxia. Representative images of dying cell morphology of three independent experiments; red arrows indicate cell swelling with large bubbles (g). Scale bar, 20 μm. LDH-released cell death is shown as mean ± SD of n = 3 independent experiments (h). N, normoxia; H, hypoxia. (I and j) MDA-MB-231 stable transfectants were treated with TNFα plus CHX under hypoxia. Representative images of dying cell morphology of three independent experiments (i). Scale bar, 20 μm. LDH-released cell death is shown as mean ± SD of n = 3 independent experiments (j). (k) MDA-MB-231 cells were co-treated with TNFα plus CHX and the p-Y705-Stat3 inhibitor HO-3867 or the importin α/β inhibitor ivermectin under hypoxia. Representative images of dying cell morphology of three independent experiments. Scale bar, 20 μm. P values of all statistical analysis were determined by two-sided Student’s t-test. Statistical source data are provided in Source Data Fig. 3.
Fig. 4:
Fig. 4:. nPD-L1 as co-activator together with p-Y705-Stat3 transcriptionally activates GSDMC expression in response to hypoxia.
(a–d) GSDMC mRNA (a and c) and protein (b and d) levels in the indicated MDA-MB-231 stable transfectants were determined by RT-qPCR and immunoblotting, respectively. Data are shown as mean ± SD of n = 3 biologically independent experiments. Blots are representative of three independent experiments. N, normoxia; H, hypoxia. (e and f), GSDMC promoter luciferase reporter plasmids were transfected into the indicated MDA-MB-231 stable transfectants and assayed for luciferase reporter activities. Data are shown as mean ± SD of n = 3 biologically independent experiments. (g) GSDMC promoter luciferase reporter plasmids were co-transfected with PD-L1 and Stat3 in HEK293T cells and assayed for luciferase activities. Data are shown as mean ± SD of n = 3 biologically independent experiments. Pro-WT, wild-type GSDMC promoter; Pro-Mut, GSDMC promoter with predicted p-Y705-Stat3 binding site mutation (red). (h) Sequential ChIP-PCR analysis of interactions between PD-L1, p-Y705-Stat3, and GSDMC promoter. VEGF promoter which interacts with HIF1α and p-Y705-Stat3 under hypoxia as a positive control. The experiment was repeated three times with similar results. P, MDA-MB-231 parental cells; KO, MDA-MB-231-PD-L1-knockout cells. P values of all statistical analysis were determined by two-sided Student’s t-test. Statistical source data and unprocessed blots are provided in Source Data Fig. 4.
Fig. 5:
Fig. 5:. GSDMC cleavage by caspase-8 determines hypoxia-induced apoptosis-to-pyroptosis switch with TNFα treatment.
(a) % of LDH-released cell death in parental and GSDMC-knockout MDA-MB-157 cells treated with TNFα plus CHX under normoxia. Data are shown as mean ± SD of n = 3 independent experiments. (b) Representative images of dying cell morphology in (a) of three independent experiments. Red arrows indicate cell swelling with large bubbles. Scale bar, 20 μm. (c) GSDMC or vector control was transiently expressed in HeLa cells. Time-lapse microscopy quantification of dying cells treated with TNFα plus CHX under normoxia. The experiment was repeated three times with similar results. (d) LDH-released cell death in HeLa cells expressing GSDMC or vector control and treatment of TNFα plus CHX under normoxia. Data are shown as mean ± SD of n = 3 independent experiments. (e) Immunoblotting of GSDMC in MDA-MB-231 cells treated with TNFα plus CHX under hypoxia. N, normoxia; H, hypoxia. Blots are representative of three independent experiments. (f) Cleavage of human GSDMC by various active caspases. Red arrows indicate cleaved GSDMC. The experiment was repeated four times with similar results. (g) Immunoblotting of active caspase-6 and caspase-8 in MDA-MB-231 cells treated as indicated. Caspase-6i, caspase-6 inhibitor Z-VEID-FMK (10 μM); Caspase-8i, caspase-8 inhibitor Z-IETD-FMK (10 μM). Blots are representative of three independent experiments. (h) Deletion of caspase-8 by CRISPR/Cas9 in MDA-MB-231 cells. Blots are representative of three independent experiments. (i) Immunoblotting of GSDMC in parental and caspase-8–knockout MDA-MB-231 cells treated with TNFα plus CHX under hypoxia. Blots are representative of three independent experiments. (j) Representative images of dying cell morphology of three independent experiments. Parental and caspase-8-knockout MDA-MB-231 cells were treated with TNFα plus CHX under normoxia or hypoxia. Scale bar, 20 μm. (k) LDH-released cell death with and without caspase-8 knockout. Data are shown as mean ± SD of n = 3 independent experiments. P values of all statistical analysis were determined by two-sided Student’s t-test. Statistical source data and unprocessed blots are provided in Source Data Fig. 5.
Fig. 6:
Fig. 6:. GSDMC N-terminal domain (aa 1–365) binds to cell membrane and induces pyroptosis.
(a and b) Analysis of the pore-forming activity of caspase-8-cleaved GSDMC by high-resolution electron microscopy (a) and liposome leakage assay (b). Red arrows in (a) indicate pores. Triton X-100 was added at 20 min to achieve 100% leakage in (b). GSDMC-FL, full-length GSDMC. Scale bar, 100 nm. The experiments were repeated three times with similar results. (c) Membrane lipid-binding activity of GSDMC fragments, phosphatidylethanolamine (PE) as a negative control for liposomes. S, lipid-free supernatant; P, lipid-containing pellet; PI(4,5)P2, phosphatidylinositol-4,5-bisphosphate; GSDMC-N, N-terminal domain of GSDMC; GSDMC-C, C-terminal domain of GSDMC. The experiment was repeated three times with similar results. (d) Immunoblotting of wild-type (WT) and D365A-mutant (D365A) GSDMC proteins cleaved by caspase-8 with anti-His tag antibody. The experiment was repeated three times with similar results. (e) Fluorescent imaging of HeLa cells transfected with WT or D365A GSDMC genes and treated with TNFα plus CHX. Dying cells were stained with nucleic acid dye SYTOX green. Red arrows indicate cell swelling with large bubbles. Scale bar, 20 μm. The experiment was repeated four times with similar results. (f) LDH-released cell death is shown as mean ± SD of n = 3 biologically independent experiments. P values were determined by two-sided Student’s t-test. (g) Immunoblotting of GSDMC in HeLa cells expressing WT or D365A GSDMC and treated with TNFα plus CHX. The experiment was repeated three times with similar results. Statistical source data and unprocessed blots are provided in Source Data Fig. 6.
Fig. 7:
Fig. 7:. nPD-L1, caspase-8, and GSDMC are required for TNFα-induced tumor necrosis in hypoxic regions.
(a–e) The indicated MDA-MB-231 parental cells or stable transfectants were injected subcutaneously into nude mice (n = 8 mice/group). Mice in (c) were treated with CSF1R antibody to achieve macrophage depletion in vivo. Mice in (d) were treated with TNFα antibody to block TNFα in vivo. Representative images of tumor sections and H&E staining are shown. Green dashed line indicates areas of tumor necrosis. The percentage of necrotic area in tumor tissue is shown. Data are mean ± SD of n = 8 mice. P values were determined by two-sided Student’s t-test. Scale bar, 200 μm. (f) Representative images of tumor necrosis shown at 7 days and 21 days after inoculation of the indicated 4T1 stable transfectants in immunocompetent BALB/c mice (n = 8 mice/group). Data are mean ± SD of n = 8 mice. P values were determined by two-sided Student’s t-test. 4T1-WT, 4T1 cells stably expressing wild-type mouse PD-L1; 4T1-NLS, 4T1 cells stably expressing NLS-mutated mouse PD-L1. Yellow arrows indicate tumor necrosis. (g) IHC staining of HIF1α, GSDMC, PD-L1, and p-Y705-Stat3 in human breast cancer tissues. The expression levels and patterns of HIF1α, GSDMC, PD-L1, and p-Y705-Stat3 in normoxic and hypoxic area in human breast tumors were analyzed and indicated by green arrows. Scale bar, 100 μm. (h) Kaplan-Meier Plotter analysis of overall survival for human breast tumor tissue microarray in the indicated groups with different GSDMC expression levels. The patient number (n) for each group as indicated. Statistical significance for survival analysis was determined using a log-rank (Mantel–Cox) test. Statistical source data are provided in Source Data Fig. 7.
Fig. 8:
Fig. 8:. Chemotherapeutic drugs induce pyroptotic cell death via the nPD-L1/GSDMC-mediated non-canonical pathway in breast tumors.
(a) Immunoblotting of PD-L1, GSDMC, and cleaved caspase-6 and caspase-8 in MDA-MB-231 cells treated with various chemotherapy drugs as indicated. Blots are representative of three independent experiments. (b and c) Cell death induced by the indicated drugs was determined by LDH release and phase-contrast cell images in MDA-MB-231 cells. Data of LDH-released cell death are shown as mean ± SD of n = 3 biologically independent experiments. Phase-contrast cell images are representative of three independent experiments with similar results. Scale bar, 20 μm. (d and e) Phase-contrast cell images (d) and LDH release (e) in MDA-MB-231 cells treated with the indicated drugs in combination with caspase-6 or caspase-8 inhibition. Data of LDH-released cell death are shown as mean ± SD of n = 3 biologically independent experiments. Phase-contrast cell images are representative of three independent experiments with similar results. Scale bar, 20 μm. P values of all statistical analysis were determined by two-sided Student’s t-test. Statistical source data and unprocessed blots are provided in Source Data Fig. 8.
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"V体育ios版" Comment in

  • PD-L1 controls cancer pyroptosis.
    Blasco MT, Gomis RR. Blasco MT, et al. Nat Cell Biol. 2020 Oct;22(10):1157-1159. doi: 10.1038/s41556-020-00582-w. Nat Cell Biol. 2020. PMID: 32943765 No abstract available.

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