Cisplatin-induced Pyroptosis Enhances the Efficacy of PD-L1 Inhibitor in Small-Cell Lung Cancer via GSDME/IL12/CD4Tem Axis

Abstract

The combination therapy of platinum-based chemotherapy and PD-L1 inhibitors but not the single anti-PD-L1 therapy has significantly improved the prognosis of patients with small-cell lung cancer (SCLC). However, the synergistic mechanism of combination therapy has not been fully elucidated. In this work, we identified a positive correlation between the expression of pyroptosis-related proteins Gasdermin E (GSDME) and the survival rates of patients with SCLC. Importantly, it was shown that human SCLC cell lines with high expression of GSDME showed more sensitivity to cisplatin, as well as cisplatin plus anti-PD-L1 treatment both in vitro and in vivo. Mechanically, cisplatin induced the activation of GSDME and the release of cytokines including IL-12, which enhance the expression of IFN-γ in T cells in the tumor immune microenvironment (TME) and subsequently improve anti-PD-L1 response. Altogether, our work demonstrates that cisplatin could induce GSDME-dependent cell pyroptosis to improve the response of anti-PD-L1 therapy though switching the TME from "cold" to "hot" in SCLC, indicating GSDME as a response biomarker for combination therapy of anti-PD-L1 and chemotherapy, as well as a potential target to sensitize the response to PD-L1 inhibitor therapy in future.

Keywords: GSDME; IL-12; Pyroptosis; SCLC; Tumor immune microenvironment.

Figure 1

SCLC patients with high expression of GSDME in tumor cells have better prognosis. (A) PFS of GSDME^hi and GSDME^lo SCLC patients receiving chemotherapy (Data from cBioportal database). (B) OS of patients with GSDME^hi and GSDME^lo in SCLC patients receiving chemotherapy. (C) Representative images of immunohistochemical staining tumors from GSDME^hi and GSDME^lo SCLC patients. (D) PFS of GSDME^hi and GSDME^lo SCLC patients receiving chemotherapy (Data from Shanghai Chest Hospital). (E) PFS of patients with GSDME^hi and GSDME^lo SCLC patients receiving chemotherapy plus immunotherapy (Data from Shanghai Chest Hospital).

Figure 2

The immune microenvironment of SCLC patients with high expression of GSDME is more similar to "hot tumor". (A) ImmuneScore of patients with GSDME^hi and GSDME^lo SCLC patients (Data from cBioportal database). (B) CIBERSORT analysis compared 22 immune cell classifications between the GSDME^hi and GSDME^lo SCLC patients (Data from cBioportal database). (C) Immunofluorescence, representative staining images of CD4⁺ cells in the GSDME^hi and GSDME^lo groups, and pearson correlation was performed (Data from Shanghai Chest Hospital). (D) Immunofluorescence, representative staining images of CD4⁺Ki67⁺ cells in the GSDME^hi and GSDME^lo groups, and pearson correlation was performed (Data from Shanghai Chest Hospital). (E) Immunofluorescence, representative staining images of FOXP3⁺ cells in the GSDME^hi and GSDME^lo groups, and pearson correlation was performed (Data from Shanghai Chest Hospital). (F) Enrichment differential bubble plot, significantly up-regulated top 20 KEGG pathways in the GSDME^hi group than GSDME^lo.

Figure 3

The antitumor effects of GSDME depends on immunocompetent system. (A) Overexpression of GSDME in small cell lung cancer cell lines were measured via western blot. (B) Overexpression of GSDME in small cell lung cancer cell lines were measured via RT-qPCR. (C) Knockout of GSDME in small cell lung cancer cell lines were measured via western blot. (D) Knockout of GSDME in small cell lung cancer cell lines were measured via RT-qPCR. (E) The growth curve of GSDME-KO and GSDME-NC NSG mice bearing tumors (DMS114). (F) The growth curve of GSDME-KO and GSDME-NC humanized NSG mice bearing tumors (DMS114). (G) Inhibition rate of GSDME-OE human SCLC cell lines and their GSDME-NC group when co-cultured with human PBMC via RTCA. (H) Crystal violet staining image of live GSDME-OE human SCLC cell lines and their GSDME-NC group co-cultured with human PBMC. (I) Inhibition rate of GSDME-KO human SCLC cell lines and their GSDME-NC group when co-cultured with human PBMC via RTCA. (J) Crystal violet staining image of live GSDME-KO human SCLC cell lines and their GSDME-NC group co-cultured with human PBMC.

Figure 4

Chemotherapy induces GSDME mediated pyroptosis, and overexpression of GSDME significantly enhances cisplatin sensitivity. (A) Bright-field images of SCLC cell lines treated with 5 μM cisplatin for 24 and 48 hours via phase-contrast microscope. (B) LDH release activity was detected after 5 μM cisplatin after 24, 48 and 72 hours, respectively. (C) RT qPCR was used to detect the mRNA expression level of Gasdermin family proteins in SCLC cell lines treated with 5 μM cisplatin. (D) Western blot showed that 5 μM cisplatin was applied to small cell lung cancer cell lines, and the expression levels of GSDME full-length protein and GSDME-N-terminal protein were detected at 0, 8, 24 and 72 hours, respectively. (E) RTCA displays an inhibition rate of 5 μM cisplatin in GSDME-OE and GSDME-NC GLC16 cell lines. (F) RTCA displays an inhibition rate of 5 μM cisplatin in GSDME-KO and GSDME-NC GLC16 cell lines. (G) The degree of cell death in SHP77 GSDME-OE cell and GSDME-NC group after 5 μM cisplatin for 24 hours via Annexin V/PI. (H) The degree of cell death in DMS114 GSDME-KO cell and GSDME-NC group after 5 μM cisplatin for 24 hours via Annexin V/PI. (I) CCK8 showed a curve fitting the inhibition rate 72 hours after the gradient concentration of cisplatin acted on SHP77 GSDME -OE and GSDME-NC group. (J) CCK8 showed a curve fitting the inhibition rate 72 hours after the gradient concentration of cisplatin acted on GLC16 GSDME -KO and GSDME-NC group.

Figure 5

The expression of GSDME regulates the cisplatin activated IL12RB1-IL12 pathway. (A) Enriched differential bubble plot, showing the top 20 KEGG pathways significantly upregulated in the GSDME-OE group compared to GSDME-NC group after cisplatin induction. (B) GSEA plot reveals the regulatory effect of Cytokine-Cytokine receptor interaction (ko04060) in the GSDME-OE group and GSDME-NC after cisplatin induction. (C) Enriched differential bubble plot, showing the top 20 KEGG pathways significantly upregulated in the GSDME-KO group compared to GSDME-NC group after cisplatin induction. (D) GSEA plot reveals the regulatory effect of Cytokine-Cytokine receptor interaction (ko04060) in the GSDME-KO group and GSDME-NC after cisplatin induction. (E) Immunofluorescence, representative staining images of IL-12⁺ cells in the GSDME^hi and GSDME^lo groups, and pearson correlation was performed (Data from Shanghai Chest Hospital). (F) Immunofluorescence, representative staining images of IL12RB1⁺ cells in the GSDME^hi and GSDME^lo groups, and pearson correlation was performed (Data from Shanghai Chest Hospital). (G) The concentration of IL-12P70 in the cell culture supernatant of GSDME-OE/KO and GSDME-NC SCLC lines induced by cisplatin via ELISA. (H) Luminex detection of plasma IL-12P70 concentrations in patients with extensive stage small cell lung cancer in the GSDME high expression group and GSDME low expression group. (I) The concentration of plasma IL-12P70 in GSDME-high and GSDME-low patients with ES-SCLC via Luminex.

Figure 6

Overexpression of GSDME significantly increases the efficacy of chemo-immunotherapy. (A) The growth curve of GSDME-OE and GSDME-NC humanized NSG SCLC mice treated with cisplatin (GLC16). (B) The growth curve of GSDME-OE and GSDME-NC humanized NSG SCLC mice treated with cisplatin and/or Durvalumab (GLC16). (C) Tumor size of GSDME-OE/KO and GSDME-NC humanized SCLC NSG mice. The GSDME-KO group received human recombinant IL-12, while the GSDME-OE group received human recombinant neutralizing IL-12 protein intravenously. (D) The growth curve of GSDME-OE/KO and GSDME-NC humanized SCLC NSG mice. The GSDME-KO group received human recombinant IL-12, while the GSDME-OE group received human recombinant neutralizing IL-12 protein intravenously. (E) In vitro, PBMC was co-cultured with GSDME-OE/KO and GSDME-NC DMS114 cells, and treated with cisplatin. Recombinant IL-12 and neutralizing IL-12 protein was administered in GSDME-KO and GSDME-OE group respectively. PBMC was collected for proliferation test via CFSE. (F) In vitro, GSDME-OE/KO and GSDME-NC DMS114 cells were co-cultured with PBMC, and treated with cisplatin. Recombinant IL-12 and neutralizing IL-12 protein was added to GSDME-KO and GSDME-OE group respectively. Cell killing ability was quantified by LDH release from the co-culture supernatant. (G) CIBERSORT analyzed the 22 immune cell ratios of tumors from GSDME-OE and GSDME-NC humanized SCLC NSG mice ungergoing cisplatin induction.

Figure 7

Graphical Abstract. In SCLC, GSDME improves the efficacy by reshaping the tumor microenvironment induced by cisplatin. GSDME-OE SCLC tumors activate the IL12RB1-IL12 pathway, release more active components of IL12, subsequently promote differentiation towards CD4 effector memory T cell. Thus, T cells were stimulated to release IFN-γ, so that dendritic cells were able to release more IL-12, transforming a vicious anti-tumor cycle. Exogenous IL-12 restores the weakened chemotherapy efficacy caused by knocking out GSDME, while neutralizing IL-12 antibodies restores the enhanced chemotherapy efficacy caused by overexpression of GSDME. In conclusion, GSDME reshapes the cisplatin-induced SCLC tumor microenvironment through the IL12RB1-IL12-CD4 effector memory T cell pathway, thereby improving the efficacy of chemo-immunotherapy.