NLRP3 Promotes Immune Escape by Regulating Immune Checkpoints: A Pan-cancer Analysis

NLRP3 plays a pathogenic role in tumorigenesis by regulating innate and acquired immunity, apoptosis, differentiation, and intestinal microbes in tumors. In different tumors, NLRP3 plays different roles, and its mechanism is complex. Our research aimed to comprehensively investigated the role of NLRP3 in pan-cancers based on multi-omics data in the TCGA database. We found the expression of NLRP3 was changed in tumors compared with paired non-tumor specimens. Most types of tumors showed increased expression of NLRP3. Among them, the overexpressed NLRP3 in liver hepatocellular carcinoma (LIHC) and ovarian cancer (OV) indicated worse overall survival (OS). Further analysis also conrmed overexpressed NLRP3 in colon cancer (COAD) indicated a high probability of microsatellite instability (MSI) and low tumor mutational burden (TMB), which indicated a better response to immune checkpoint inhibitors (ICIs). We also analyzed the association between NLRP3, immune inltration, and immune checkpoints. Interestingly, overexpression of NLRP3 was closely related to high inltration of immune cells (T cells, B cells, etc.) and overexpressed immune checkpoints (PD-1, PD-L1, LAG3, etc.). These results demonstrated NLRP3 promoted immune escape in cancers. Finally, we investigated the expression of various immune checkpoints by treating NLRP3 inhibitor MCC950 during the co-culture of peripheral blood mononuclear cells (PBMC) and LIHC cell line Hep3B. We found MCC950 signicantly repressed the expression of PD-L1 and LAG3, and promoted the apoptosis rate of Hep3B. In conclusion, our research comprehensive demonstrated the role of NLRP3 in pan-cancer, especially in LIHC. We conrmed inhibition of NLRP3 promoting the immune killing effect to cancer cells by repressing the expression of immune checkpoints.


Introduction
In ammation and persistent infection may contribute to various types of cancers 1 . It has been con rmed that in ammation plays key role in cancer initiation, development, progression, angiogenesis, and invasion 2 . In ammation may be associated with a series of cancer regulatory pathways, including hypoxia, chemotherapy, radiotherapy, or immune attack. As an important component of innate immunity, NLRP3 in ammasome plays an important role in the body's immune response. A large amount of evidence shows that in ammasomes play an important role in pathogen infection and autoimmune diseases. However, their role in tumor progression remains unclear.
In ammasome induces pro-caspase-1 activation and in ammatory cytokine maturation in the innate immune system. In addition to being related to autoimmune diseases, the overexpression of IL-1β may also lead to the occurrence of tumors. Several in ammasomes, including NLRP3, NLRP6, NLRC4, NLRP1 and AIM2, may play a pathogenic role in tumorigenesis by regulating innate and acquired immunity, apoptosis, differentiation and intestinal microbes. The role of NLRP3 in tumor progression is very complex. Studies have shown that in various cancers, NLRP3 has both cancer-promoting and anti-tumor effects 3,4 . For example, NLRP3 in ammasome protects against colon cancer attributed to the effector function of caspase-1 to mediate secretion of IL18 and inhibit liver metastatic growth by enhancing NK cell tumoricidal action 5,6 . However, the NLRP3-dependent release of IL-1β induces immune cells, primarily CD4 + T cells, to express and release IL-22, which has been associated with the aggressive growth of multiple cancers including breast, gastric, lung, and skin cancers 7 .
Although NLRP3 has been reported in certain cancers, these studies focused on a single or a few types of cancer; there have been no studies comparing multiple types of cancer. We carried out a pan-cancer analysis of the NLRP3 in ammasome across various cancer types based on the TCGA datasets. We also investigated the association between NLRP3 and immune in ltration, immune escape, immune checkpoints, DNA methyltransferase (DNMT) levels, and mismatch repair (MMR). These results provide important insights into the roles of NLRP3 in cancer immunology.

Data Acquisition
We identi ed and downloaded the Processed level 3 RNA sequencing data and corresponding clinical annotations from the TCGA database which comprises over 20,000 samples from 33 types of cancer and corresponding non-carcinoma samples.

The expression and survival analysis of NLRP3 in pancancer
The data of NLRP3 transcript for 33 cancer types and adjacent non-carcinoma tissues were extracted from TCGA and used to compare the expression of NLRP3 in pan-cancer and normal tissues. Kaplan-Meier analysis was used to carry out the association of NLRP3 and OS in pan-cancer. Cox analysis was used to calculate correlations between gene expression and OS, progression-free interval (PFS), diseasespeci c survival (DSS), and disease-free survival (DFS). [EPCAM]) and NLRP3 expression levels. DNA methylation has been implicated in tumorigenesis and cancer progression. As DNMT1, DNMT2, DNMT3A, and DNMT3B are the major enzymes involved in DNA methylation 9 , we analyzed the correlation between their expression levels and those of NLRP3.

Analysis of immune in ltration
Tumor Immune Estimation Resource (TIMER; https://cistrome.shinyapps.io/timer/) allows systemic analysis of immune in ltrates in different cancer types using a deconvolution statistical approach to infer tumor-in ltrating lymphocyte counts based on gene expression data 10 . Using the TIMER algorithm, the associations between NLRP3 levels and the lymphocytes in the tumor microenvironment were examined. Estimation of Stromal and Immune Cells in Malignant Tumor Tissues Using Expression Data (ESTIMATE) uses gene expression pro les to predict the purity of a tumor based on the in ltration of stromal cells/immunocytes 11 . The ESTIMATE algorithm yields 3 scores based on GSEA of single samples, including (1) stromal score, which indicates the in ltration of stromal cells ; (2) immune score, which describes the degree of immune in ltration; and (3) estimate score, which re ects impurity degree of tumors. We used the algorithm to estimate both immune and stromal scores for a variety of tumor tissues and evaluated the associations between the scores and NLRP3 levels.

Cell Culture and cell proliferation assay
Human hepatocellular cell line Hep3B and Huh7 were both acquired from the University of Colorado Cancer Center Cell Bank and cultured in DMEM medium, supplemented with 10% FBS (Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO 2 atmosphere.
For cell proliferation assay, 2000 cells per well were seeded into 96 well plates (5 multiple wells were set up). 10 µl CCK-8 solution was added into each well at 1, 2 and 3 days, and incubated for 2 hours. The absorbance values of each well were measured at 450 nm (OD450). Biosciences). After 24h, the chambers were xed by 4% paraformaldehyde for 30 min and then stained by 0.1% crystal violet for additionally 30 min. Finally, at least ve elds of chambers were captured and cell number was counted.

Co-culture of PBMC and Hep3B
Peripheral blood mononuclear cells (PBMC) were extracted from healthy volunteers by density gradient centrifugation in a lymphocyte separation medium (MP Biomedicals, Irvine, CA, USA). T cells in PBMC were activated and expanded with CD3, CD28 antibody, and 10 ng/ml IL-2 (Thermo Fisher Scienti c, Waltham, MA, USA), and then co-cultured with Hep3B cells at a ratio of 10:1 in the presence of a uorescent caspase 3 substrate (BD Biosciences, San Jose, CA, USA).

Western Blotting Analysis
The cells of each group were digested and lysed by 100ul RIPA lysate. After completely lysed, the lysate were centrifuged at 4 ℃ for 15 minutes. The supernatants were collected as the total protein extract. Then, the BCA assay was performed to quantify the proteins (Thermo Fisher Scienti c, Waltham, MA, USA
3.2 The association between NLRP3 and TMB, MMR.
Tumor mutational burden (TMB) re ects the number of mutations contained in tumor cells and is a quanti able biomarker. Previous report had demonstrated patients with high TMB showed satis ed response to treatment of ICIs 12 . We analyzed the relationship between gene expression and TMB as follows, using Spearman's rank correlation coe cient. 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 con rmed MSI patients showed stronger resistance to chemotherapy 13 . 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 ve 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 coexpression of NLRP3 and methyltransferases in various cancers, including LIHC, LAML, KIRP, KIRC, KICH, BRCA, BLCA, UVM, TGCT, SARC, PRAD and PAAD (Fig. 2D).

The association between NLRP3 and immune in ltration.
As NLRP3 was closely related to in ammation, we investigated the association of NLRP3 and immune in ltration ( Fig. 2A). Interestingly, NLRP3 was strongly associated with the in ltration 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 (in ltration of stromal cells), immune score (immune in ltration), and Estimate score (impurity degree of tumors). These results suggested NLRP3 promoted the immune in ltration of tumors.

The association between NLRP3 and immune checkpoints.
We examined the association between immune checkpoints and NLRP3 expression in pan-cancer and found a signi cantly 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 signi cantly 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.
As described above, NLRP3 showed signi cantly 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 signi cantly repressed the expression of LAG-3 in T cells (Fig. 6A). Meanwhile, MCC950 signi cantly repressed the expression of PD-L1 in Hep3B, and this repression was concentrationdependent (Fig. 6B). Finally, we investigated to apoptosis rate of Hep3B induced by T cells killing effect. As expected, 0.02uM MCC950 signi cantly promoted the apoptosis rate of Hep3B during co-culture with PBMC (Fig. 6C).

Discussion
In ammasome is a multimeric protein platform that induces pro-caspase-1 activation and in ammatory cytokine maturation in the innate immune system. Several in ammasomes, including NLRP3, NLRP6, NLRC4, NLRP1 and AIM2, may play a pathogenic role in tumorigenesis by regulating innate and acquired immunity, apoptosis, differentiation and intestinal microbes. The role of NLRP3 in tumor progression is very complex 14 . Studies have shown that in various cancers, NLRP3 has both a cancer-promoting effect and an anti-tumor effect 15 . To comprehensively demonstrated the role of NLRP3 in cancers, our study analyzed multi-omics data derived from 33 types of cancers based on the TCGA database.
Firstly, there was a signi cant difference in the expression of various types of cancers compared with paired normal tissues. Combined with further survival analysis, we con rmed overexpression of NLRP3 in LIHC and OV showed strong association with OS. Other types of tumors (DLBC, KICH, HNSC and GBM) also showed close relationship with PFS, DFS and DSS. These results tallied with previous studies, and indicated NLRP3 play key roles in tumor progression 16 .
Furthermore, we investigated the correlation between NLPR3 and MSI. At present, MSI detection and research are mostly carried out in COAD. Previous research has con rmed COAD-MSI patients have a better prognosis, higher survival rate compared with microsatellite stability (MSS) patients, but they cannot bene t from 5-FU chemotherapy 17 . Our research con rmed overexpression of NLRP3 in COAD was closely related to MSI in COAD. TMB was another index for predicting prognosis and drug response. Tumors with highly non-synonymous TMB express a large number of abnormal proteins, which are recognized by the immune system as neoantigens. Therefore, ICIs are more likely to recognize TMB induced new antigens, achieving the effect of attacking and killing tumors 18 . Our research con rmed overexpression of NLRP3 was closely related to high TMB. In general, overexpression of NLRP3 indicated high probability of MSI (resistance to chemotherapy) and high TMB (sensitivity to ICIs). In another word, high expression of NLRP3 in COAD preferred treatment of ICIs to chemotherapy etc.). Furthermore, we also demonstrated high expression of NLRP3 was closely related to mutations of MMR genes and high expression of DNA methyltransferases. This result suggested NLPR3 was associated with the instability of cancer genome.
Due to the close relationship between NLPR3 and immune regulation, we further investigated the role of NLRP3 in tumor immunology. Interestingly, overexpression of NLRP3 was closely related to high in ltration of various types of immune cells, including T cells, B cells, and so on. This result indicated NLRP3 activated tumor immune response. In general, activated tumor immune response indicated killing effect on the tumor, but this contradicted the survival analysis (Overexpression of NLRP3 indicated worse OS in LIHC and OV). We suggested it may be due to immune escape caused by overexpression of immune checkpoints 19 . Therefore, we further analyzed the correlation between NLRP3 and different immune checkpoints. Interestingly, NLRP3 was signi cantly co-expressed of various immune checkpoints. Subsequently, we investigated the expression of several main immune checkpoints with treating NLRP3 inhibitor. As expect, PD-L1 and LAG3 were both signi cantly repressed by NLRP3 inhibitor, accompanied with obvious apoptosis of tumor cells. This result con rmed NLPR3 regulated the expression of immune checkpoints, especially PD-L1 and LGA3. Previous study had con rmed inhibition of NLRP3 repressed PD-L1 in lymphoma 20 . However, rare research reported NLRP3 regulated LAG3. LAG-3 negatively regulates the proliferation, activation and homeostasis of T cells. Blocking LAG-3 in human CD4 + cells promoted cell proliferation and increase the expression levels of IL-2, IL-4, IFN-γ and TNFα 21 .
In conclusion, our research comprehensively investigated the role of NLRP3 in pan-cancer. We determined overexpression of NLRP3 was closely related to immune escape by regulating PD-L1 and LAG3. Our research disclosed the therapy and diagnosis potential of NLRP3 in cancers. Availability of data and materials

Abbreviations
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions YD and ZY contributed to the experiments performing. YY and YD contributed to the statistical analysis of the data. JX and WS contributed to animal model. XY and QZ contributed to the design of the study. All authors read and approved the nal version of the manuscript.
Ethics approval and consent to participate All procedures involving human participants were performed in accordance with Shanghai Pudong Hospital ethical committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. All patients provided their written informed consent. The study protocol was approved by the Pudong Hospital Committee on human research.

Patient consent for publication
Not applicable.