3.1 The expression and survival analysis of NLRP3 in pan-cancer
First of all, we extracted the expression of NLRP3 in pan-cancer based on TCGA-database. We found NLRP3 was overexpressed in CHOL, COAD, ESCA, GBM, HNSC, KIRC, KIRP, LAML, LGG, LIHC, OV, PAAD, SKCM, STAD, TGCT, THCA, UCS, but decreased in ACC, BLCA, LUAD, LUSC, READ, UCEC compared with paired normal specimens (Fig. 1A). Subsequently, we investigated the association of NLRP3 and survival in pan-cancer via Kaplan-Meier analysis. Higher expression of NLRP3 indicated worser overall survival (OS) in LIHC, OV, and TGCT patients (Fig. 1B). Meanwhile, we also investigated the association of NRLP3 and OS, progression-free interval (PFS), disease-specific survival (DSS), and disease-free survival (DFS) via Cox analysis respectively (Fig. 1C). Higher expression of NLRP3 indicated worser PFS in DLBC and GBM patients, worser DSS in DLBC, KICH, and TGCT patients, worser DFS in HNSC patients. In general, the overexpression of NLRP3 indicated worser survival in various cancers.
3.2 The association between NLRP3 and TMB, MMR.
Tumor mutational burden (TMB) reflects the number of mutations contained in tumor cells and is a quantifiable biomarker. Previous report had demonstrated patients with high TMB showed satisfied response to treatment of ICIs12. We analyzed the relationship between gene expression and TMB as follows, using Spearman's rank correlation coefficient. The expression of NLRP3 showed positive correlation with TMB in THYM, SARC, and COAD; whereas showed negative correlation with TMB in UVM, THCA, STAD, PRAD, LUSC, LUAD, LIHC, HNSC, and DLBC (Fig. 2A). It indicated high expression of NLRP3 was associated with high TMB in most cancers, thus showed better response to treatment of ICIs. Microsatellite instability (MSI) refers to any change in the length of a microsatellite caused by the insertion or deletion of a repeat unit in a tumor compared with normal tissues, and the appearance of new microsatellite alleles genetic phenomenon. Previous studies also confirmed MSI patients showed stronger resistance to chemotherapy13. We determined higher expression of NLRP3 showed negative correlation with MSI in UCEC, TGCT, SKCM, LUSC, HNSC and DLBC; whereas showed positive correlation with MSI only in COAD (Fig. 2B). Mutation of mismatch repair system (MMR) caused DNA replication errors to be unable to be repaired, which will lead to higher somatic mutations. We evaluated the correlation of NLRP3 and the mutation of five MMR genes: MLH1, MSH2, MSH6, PMS2, EPCAM. We found high expression of NRLP3 was closely related to the mutation of MMR in various cancers, including KICH, KIRC, LIHC, PAAD, PRAD, SKCM and UVM (Fig. 2C). DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability and the way that DNA interacts with proteins, thereby controlling gene expression. we analyzed the correlation between NLPR3 and the four methyltransferases (DNMT1: red, DNMT2: blue, DNMT3A: green, DNMT3B: purple). We found a strong co-expression of NLRP3 and methyltransferases in various cancers, including LIHC, LAML, KIRP, KIRC, KICH, BRCA, BLCA, UVM, TGCT, SARC, PRAD and PAAD (Fig. 2D).
3.3 The association between NLRP3 and immune infiltration.
As NLRP3 was closely related to inflammation, we investigated the association of NLRP3 and immune infiltration (Fig. 2A). Interestingly, NLRP3 was strongly associated with the infiltration of the main lymphocytes in the tumor microenvironment (TME), including B cell, CD4 + T cells, CD8 + T cells, neutrophils, and dendritic cells (DCs). Subsequently, we further assessed the association of NLRP3 and TME via the ESTIMATE algorithm (Fig. 2B). Higher expression of NLRP3 showed a strong correlation with stromal score (infiltration of stromal cells), immune score (immune infiltration), and Estimate score (impurity degree of tumors). These results suggested NLRP3 promoted the immune infiltration of tumors.
3.4 The association between NLRP3 and immune checkpoints.
We examined the association between immune checkpoints and NLRP3 expression in pan-cancer and found a significantly positive correlation between NLRP3 and various immune checkpoints (LAG3, ICOS, CTLA4, TIM3, PD-1, PD-L2, PD-L1, and TIGIT, Fig. 4A) in almost all types of cancers. Here we further showed the correlation scatter diagram of NRLP3 and multiple immune checkpoints in LIHC, which has a strong correlation (Fig. 4B).
3.5 Inhibition of NRLP3 showed no effect to the cell proliferation and invasion of LIHC.
Based on all our described above, NLRP3 played key roles in various cancers, especially in LIHC. Firstly, we investigated whether NLRP3 directly affects the proliferation and invasion of LIHC. NLRP3 inhibitor MCC950 was adopted to repress the expression of NLRP3 in two LIHC cell lines Hep3B and Huh7. MCC950 significantly repressed the expression of NLRP3 from 0.02uM to 0.5uM (Fig. 5A). However, the proliferation ability of neither LIHC cells was not repressed by MCC850 (from 0.02uM to 0.5uM). Similarly, a high concentration of MCC950 (0.5uM) also showed no effect on the invasion ability of LIHC cell lines. These results suggested NLRP3 did not directly affect tumor proliferation and invasion ability.
3.6 Inhibition of NRLP3 repressed immune escape by inhibiting LAG-3 and PD-L1.
As described above, NLRP3 showed significantly positive co-expression with various immune checkpoints. We further suggested NLRP3 plays key roles in immune escape by regulating the expression of immune checkpoints. MCC950 was added in the medium during co-culture of PBMC and Hep3B (10:1 ratio). We found MCC950 significantly repressed the expression of LAG-3 in T cells (Fig. 6A). Meanwhile, MCC950 significantly repressed the expression of PD-L1 in Hep3B, and this repression was concentration-dependent (Fig. 6B). Finally, we investigated to apoptosis rate of Hep3B induced by T cells killing effect. As expected, 0.02uM MCC950 significantly promoted the apoptosis rate of Hep3B during co-culture with PBMC (Fig. 6C).