Inflammasome-Associated Gastric Tumorigenesis Is Independent of the NLRP3 Pattern Recognition Receptor

Inflammasomes are important multiprotein regulatory complexes of innate immunity and have recently emerged as playing divergent roles in numerous inflammation-associated cancers. Among these include gastric cancer (GC), the third leading cause of cancer-associated death worldwide, and we have previously discovered a pro-tumorigenic role for the key inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) in the spontaneous genetic gp130 F/F mouse model for GC. However, the identity of the specific pattern recognition receptors (PRRs) that activate tumor-promoting inflammasomes during GC is unknown. Here, we investigated the role of the best-characterized inflammasome-associated PRR, nucleotide-binding domain, and leucine-rich repeat containing receptor, pyrin domain-containing (NLRP) 3, in GC. In gastric tumors of gp130 F/F mice, although NLRP3 expression was elevated at the mRNA (qPCR) and protein (immunohistochemistry) levels, genetic ablation of NLRP3 in gp130 F/F:Nlrp3 -/- mice did not alleviate the development of gastric tumors. Similarly, cellular processes associated with tumorigenesis in the gastric mucosa, namely, proliferation, apoptosis, and inflammation, were comparable between gp130 F/F and gp130 F/F:Nlrp3 -/- mice. Furthermore, inflammasome activation levels, determined by immunoblotting and immunohistochemistry for cleaved Caspase-1, which along with ASC is another integral component of inflammasome complexes, were unchanged in gp130 F/F and gp130 F/F:Nlrp3 -/- gastric tumors. We also observed variable NLRP3 expression levels (mRNA and protein) among independent GC patient cohorts, and NLRP3 was not prognostic for survival outcomes. Taken together, these data suggest that NLRP3 does not play a major role in promoting inflammasome-driven gastric tumorigenesis, and thus pave the way for further investigations to uncover the key inflammasome-associated PRR implicated in GC.


INTRODUCTION
Gastric cancer (GC) has the fifth highest incidence of cancer and is the third leading cause of cancer-associated death worldwide (1). The major histopathological type of human GC is intestinaltype adenocarcinoma, accounting for up to 90% of all cases, which develops stepwise from gastric inflammation (gastritis) to intestinal metaplasia, dysplasia, and ultimately adenocarcinoma (2,3). Indeed, GC is among a growing number of cancers for which there is a link with chronic inflammation (at least 20%) triggered by dysregulated innate immune responses to microbial infection, in particular the Helicobacter pylori (H. pylori) bacterium that causes~75% of all GC cases (4)(5)(6)(7)(8)(9).
A role for innate immunity in GC was first suggested by the observation that polymorphisms in host genes that encode innate immune cytokines and/or their receptors, in particular IL1B encoding the pro-inflammatory cytokine interleukin-1 beta (IL-1b), conferred a marked increase in risk of H. pylori-infected individuals developing GC (10). Furthermore, transgenic overexpression of human IL-1b, or elevated endogenous expression of the IL-6 cytokine family member, IL-11, in mice can promote gastric tumorigenesis (11,12). More recently, we and others have reported that polymorphisms and/or elevated expression of key regulators of the innate immune system belonging to the pattern recognition receptor (PRR) superfamily are associated with the onset and/or progression of GC, as well as poor survival outcomes (13)(14)(15)(16)(17).
PRRs are classified into several large structurally and functionally conserved subfamilies, among which include the well-documented Toll-like receptors (TLRs), absent in melanoma 2 (AIM2)-like receptors (ALRs), and nucleotidebinding oligomerization domain-containing (NOD)-like receptors (NLRs) (18)(19)(20). Collectively, PRRs play a critical role in coordinating host innate immune responses elicited against pathogenic microorganisms (e.g., H. pylori), as well as host-derived stress or damage signals (e.g., extracellular matrix components, heat shock proteins, and DNA from damaged or necrotic cells) (21,22). In addition to their wide expression in immune cells of both innate (e.g., macrophages and neutrophils) and adaptive (e.g., B and T cells) arms of immunity, PRRs are also expressed in non-immune cells (e.g., epithelial, endothelial, and fibroblast). Indeed, with respect to this latter observation, it has emerged that PRRs can also directly elicit proliferation, survival, migration, and functional activation of these non-immune cells, which has relevance to numerous pathophysiological responses, including gut epithelial barrier integrity and oncogenic transformation (15,20,23). These diverse PRR-driven responses are transduced upon ligand engagement of PRRs via the activation of numerous signaling cascades, including nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB), phosphatidylinositol 3-kinase (PI3K)/ Akt, and mitogen activated protein kinases (MAPKs) (5,22).
A subset of NLR family members (i.e., NLRC4, NLRP1, NLRP3, NLRP6, and NLRP12), as well as the AIM2 cytosolic DNA sensor, are best known for their assembly of large multiprotein complexes called inflammasomes, containing apoptosis-associated speck-like protein containing a CARD (ASC) and pro-Caspase-1, which control production of mature biologically active IL-1b and IL-18 cytokines (20,(24)(25)(26). The role of inflammasomes in cancer is complex, as evidenced by numerous studies coupling mice deficient in either ASC or Caspase-1 with various experimentally induced or spontaneous cancer models (e.g., colorectal cancer), which have revealed contrasting anti-or pro-tumorigenic functions for inflammasomes (26). This is evident also for inflammasomeassociated PRRs, the best studied of which is NLRP3. For instance, Nlrp3 -/mice are more susceptible to chemicalinduced colitis-associated colon cancer (CAC), whereas conversely, Nlrp3 -/mice are protected against tumor initiation and progression in a chemical-induced skin carcinogenesis model (27,28). Although the mechanistic basis for the dual role of these ASC-containing inflammasome complexes remains to be fully elucidated, it is likely to be mediated by, at least in part, their differential expression and activity on distinct cell types (immune and non-immune), as well as usage of effector IL-1b and IL-18 cytokines.
In GC, we have recently reported a pro-tumorigenic role for ASC-containing inflammasomes in the gp130 F/F mouse model for intestinal-type GC (29). These mice spontaneously develop inflamed gastric antral tumors at 6 weeks of age with 100% disease penetrance, which is caused by a homozygous phenylalanine (F) knock-in substitution of the cytoplasmic Y 757 residue in glycoprotein (gp)130, the common signaling co-receptor for IL-6 family cytokines (30). Notably, genetic deficiency for ASC in gp130 F/F mice suppressed gastric tumorigenesis by selectively downregulating the production of mature IL-18, but not IL-1b, which in the gastric tumor epithelium signals via NF-kB to protect GC cells against Caspase-8-like apoptosis (29). However, the identity of the inflammasome-associated PRR that promotes tumorigenesis in this model remains unknown. Here, we investigate the role of NLRP3 in inflammasome-associated gastric tumorigenesis and reveal that genetic ablation of NLRP3 in gp130 F/F mice has no effect on early-or late-stage gastric tumorigenesis. In addition, NLRP3 was differentially expressed among various GC patient cohorts, yet did not correlate with patient prognosis. Taken together, these data suggest that inflammasome-driven GC is independent of NLRP3.

Human Biopsies
Human gastric tissue biopsies were collected from GC or GC-free patients enrolled at Monash Medical Centre (Melbourne, Australia) undergoing surgical resection or upper gastrointestinal endoscopy (16,17). Patients with a history of taking nonsteroidal antiinflammatory drugs, proton pump inhibitors, or antibiotics were excluded. Biopsies were either snap-frozen in liquid nitrogen, or fixed in formalin prior to tissue embedding (paraffin) and sectioning on slides. Histopathological assessment and H. pylori status was determined on fresh GC patient tissue biopsies used for NLRP3 immunohistochemical staining (n = 6) as before (31). Full and informed patient consent was obtained, and biopsy collections were approved by the Monash Health Human Research Ethics Committee (13058A).

Mice
The gp130 F/F mice have been previously reported (30). Mice homozygous null for Nlrp3 (32) were kindly provided by K. Fitzgerald (University of Massachusetts Chan Medical School, USA), and were used to generate gp130 F/F :Nlrp3 -/mice on a mixed 129Sv × C57BL/6 background. Mice were housed under specific pathogen-free conditions on a 12-h light/dark cycle, and all animal experiments were approved by the Monash University Monash Medical Centre "B" Animal Ethics Committee.

Human GC Dataset Bioinformatic Interrogation
RNA sequencing (RNA-seq) gene counts for NLRP3 and clinical data from The Cancer Genome Atlas (TCGA)-STAD cohort were obtained using the TCGAbiolinks R package. Counts were processed using the edgeR package. A DGEList object was created from the counts and gene annotation information was obtained using the Homo.sapiens package. Samples were grouped according to whether they were normal tissue (n = 32) or primary tumor (n = 375). Normalization factors were calculated using the TMM method and robust dispersions were estimated. Expression levels are expressed as log2 counts per million. The "Gastric Cancer Project '08 -Singapore Patient Cohort" (GSE15459) dataset was sourced for NLRP3 gene expression profiling in tumor and non-tumor tissue (33). GC patient overall survival in both patient cohorts was assessed by Kaplan-Meier analysis on patients stratified into "low" versus "high" tumoral gene expression levels for NLRP3 as described previously (13).

RNA Isolation and Gene Expression Analyses
Snap-frozen mouse stomach tissues were subjected to mechanical homogenization on ice using a T 10 ULTRA-TURRAX ® instrument (IKA), following which total RNA was isolated using TRI Reagent ® Solution (Sigma), and subsequent on-column RNeasy ® Mini Kit RNA clean-up and DNase treatment (Qiagen). Total RNA was transcribed with the Transcriptor High Fidelity cDNA Synthesis Kit (Sigma-Aldrich), and quantitative real-time PCR (qPCR) was performed on cDNA with SYBR Green chemistry (Life Technologies) using the 7900HT Fast Real-Time System (Applied Biosystems), and the ViiA 7 Real-Time PCR System (ThermoFisher Scientific). Data acquisition and analyses were undertaken using the Applied Biosystems Sequence Detection System Version 2.4 software and ViiA 7 software. Sequences for mouse and human primers have been previously published (17,29).

Statistical Analyses
All statistical analyses were performed using GraphPad Prism V8.0.2 software. Statistical significance (p < 0.05) between the means of two groups was determined using Student's t-tests (normal distribution) or Mann-Whitney U tests (abnormal distribution), and matched datasets involved Wilcoxon signedrank tests. Statistical significance between the means of multiple groups was determined using ordinary one-way ANOVA (normal distribution) or Kruskal-Wallis (abnormal distribution) tests. All data are presented as the mean ± standard error of the mean (SEM) from at least 3 technical replicates. The log-rank test was used to calculate the statistical significance of the difference in survival between two groups. All data are presented as standard error of the mean (SEM).

Differential Expression Status of NLRP3 Among GC Patient Cohorts Does Not Correlate With Survival Outcomes
Analysis of intestinal-type GC patient data within The Cancer Genome Atlas (TCGA) revealed that NLRP3 mRNA levels were comparable between total gastric tumor tissues of patients (n = 375) compared to non-tumor gastric tissues (n = 32) ( Figure 1A). In addition, NLRP3 expression was similar in gastric tumor tissues compared to their paired adjacent non-tumor tissues (n = 27) ( Figure 1B). Conversely, in a second independent intestinal-type GC patient cohort, the "Gastric Cancer Project '08 -Singapore Patient Cohort" (GSE15459) (33), comprising gastric tumor tissues (n = 177) and non-tumor gastric tissues (n = 92), NLRP3 mRNA levels were significantly upregulated in total gastric tumor tissues ( Figure 1C). Similarly, analysis of NLRP3 expression in 83 patients for which there were paired tumor and adjacent nontumor tissues indicated that NLRP3 was significantly upregulated in tumors from 82% (68/83) of patients ( Figure 1D). The altered tumoral expression of NLRP3 did not align with a specific disease stage, since NLRP3 mRNA expression levels were comparable in early (stage I and II) and advanced (stage III and IV) GC patients ( Figure 1E). We also observed that in these independent GC patient cohorts, the segregation of GC patient primary tumors into either low or high NLRP3 gene expression indicated that NLRP3 mRNA levels are not prognostic for overall patient survival outcomes ( Figure 1F). Furthermore, immunohistochemical staining on a third cohort (Australian) of GC patients (16,17) indicated that NLRP3 protein expression levels were elevated in patient tumors compared to matched non-tumor gastric tissues, with increased numbers of diffusely stained NLRP3-positive cells observed throughout the transformed glandular epithelium and in immune cells in the stroma of patient tumor tissues ( Figures 1G-I). Collectively, these divergent data suggest that NLRP3 may not have clinical significance in human GC.

Genetic Targeting of NLRP3 in the gp130 F/F GC Mouse Model Does Not Affect the Initiation or Progression of Tumorigenesis
Since we have previously shown that elevated expression of the ASC inflammasome adaptor promotes gastric tumorigenesis in the spontaneous gp130 F/F GC mouse model (29), we next assessed the expression of Nlrp3 in these mice by qPCR. In this model, antral gastric intestinal-type tumors spontaneously develop from 1.5 months of age onwards with 100% penetrance, and progressively increase in size through to 6 months of age (13,35). At 1, 3, and 6 months of age, gastric Nlrp3 mRNA levels were significantly elevated (6-to 8-fold) in gp130 F/F tumor-free gastric antrum (1 month old) or gp130 F/F gastric antral tumors (3 and 6 months old) compared to either adjacent non-tumor antral tissue, or normal antral tissues of wild-type (WT) mice (Figure 2A). In support of these gene expression data, immunohistochemistry indicated that the total number of NLRP3 positively stained cells was significantly greater in gp130 F/F antral tumors compared to normal antral tissue from WT mice, with predominant staining observed in the gastric tumor epithelium of gp130 F/F mice ( Figures 2B, C).
To determine whether increased gastric tumoral expression of NLRP3 contributed to the pathogenesis of GC, we generated NLRP3-deficient gp130 F/F mice (gp130 F/F :Nlrp3 -/-). At 3 months of age (early-stage tumorigenesis), the stomachs and gastric tumors of gp130 F/F :Nlrp3 -/mice were comparable in size and weight compared to age-matched gp130 F/F littermate mice, as was the incidence of tumors (Figures 2D-I). Similarly, gastric tumorigenesis was also comparable in gp130 F/F :Nlrp3 -/and gp130 F/F mice at 6 months of age (late-stage tumorigenesis), as evidenced by comparable stomach size and gastric tumor burden and incidence among age-matched mice of both genotypes ( Figures 3A-F). Since tumorigenesis in gp130 F/F mice is driven by IL-11-mediated hyper-activation of the STAT3 signaling pathway (11,30), we also investigated whether the loss of NLRP3 affected this pro-tumorigenic signaling axis in the stomach. However, the expression of STAT3-target genes Il11 and Socs3 was similar among gp130 F/F :Nlrp3 -/and gp130 F/F tumor and matched non-tumor gastric tissues at 3 and 6 months of age ( Figures 3G, H). Therefore, these data indicate that NLRP3 does not contribute the onset and growth of gastric tumors in the gp130 F/F GC model.

NLRP3 Deficiency in gp130 F/F Mice Does Not Reduce Gastric Inflammasome Activation
Considering that NLRP3 activity is mediated via inflammasome complexes, and the gastric tumor phenotype in gp130 F/F mice is associated with inflammasome activation (29), we investigated whether the ablation of NLRP3 affected inflammasome activation during gastric tumorigenesis. For this purpose, we compared the expression and/or activation status of key inflammasome components, ASC and Caspase-1, along with other inflammasome-associated PRRs, in gastric tissues from 6month-old (mo) gp130 F/F :Nlrp3 -/and gp130 F/F mice. Immunoblotting demonstrated that Caspase-1 activation levels, measured by detection of the cleavage of pro-Caspase-1 (p45) to its mature form (p20 subunit), were comparable between either gastric tumor or non-tumor tissues from gp130 F/F :Nlrp3 -/and gp130 F/F mice ( Figures 4A, B). Also, immunoblotting indicated comparable protein levels of ASC among gp130 F/F versus gp130 F/F : Nlrp3 -/gastric tumor lysates ( Figures 4A, C). Gene expression analyses by qPCR also indicated that NLRP3 deficiency did not significantly alter the mRNA levels of other inflammasomeassociated PRRs in tumor samples from gp130 F/F mice (Supplementary Figure 1). Furthermore, phosphorylation levels of NF-kB, which is activated downstream of inflammasomes to promote gastric tumorigenesis in the gp130 F/F model (29), were also similar between gp130 F/F and gp130 F/F :Nlrp3 -/gastric tumor or non-tumor samples (Figures 4A, D). Immunohistochemistry for cleaved Caspase-1 also confirmed comparable numbers and staining intensity for cleaved Caspase-1-positive cells throughout the tumor epithelium of gp130 F/F :Nlrp3 -/and gp130 F/F mice ( Figures 4E, F). Taken together, these findings further suggest that NLRP3 is not the primary inflammasome-associated PRR that contributes to gastric inflammasome activity and associated tumorigenesis in the gp130 F/F model.

NLRP3 Does Not Contribute to Gastric Tumor Cell Proliferation, Survival, or Inflammation During Tumorigenesis in the gp130 F/F GC Model
Despite NLRP3 ablation having no impact on gastric tumorigenesis in gp130F/F mice, it remained possible that NLRP3 may contribute to a subset of tumor-associated cellular processes in the gastric compartment. We first investigated whether NLRP3 deficiency in gp130 F/F mice impacted on tumor-associated inflammation. However, immunohistochemistry analyses indicated comparable numbers of infiltrating CD45+ leukocytes, B220+ B cells, and CD3+ T cells in the gastric mucosa of 3mo and 6mo gp130 F/F versus gp130 F/F :Nlrp3 -/mice ( Figures 5A-F and Supplementary  Figures 2A-D). The comparable gastric inflammation in gp130 F/F and gp130 F/F :Nlrp3 -/mice was also confirmed at the molecular level, with qPCR analyses indicating similar mRNA levels of inflammatory genes (Il6, Ifng, Il10, Il17a, and Tnfa) among both genotypes at 3 and 6 months of age ( Figure 5G and Supplementary Figure 2E).
Since elevated gastric epithelial cell proliferation and survival are prominent features of inflammasome-associated gastric tumorigenesis in gp130 F/F mice (29), we also investigated whether NLRP3 promoted these oncogenic cellular processes in the stomach. However, immunohistochemistry indicated similar PCNA + proliferating cell numbers in the epithelium of gastric tumors of gp130 F/F and gp130 F/F :Nlrp3 -/-3mo and 6mo mice ( Figure 6A, B and Supplementary Figures 3A, B). Also, similar numbers of apoptotic cleaved Caspase-3 + and cleaved Caspase-8 + cells were observed in 3mo and 6mo gp130 F/F and gp130 F/F :Nlrp3 -/mouse gastric tumors ( Figures 6C-F and Supplementary Figures 3C, D). We also note that the expression of several angiogenesis-related genes (Cxcl1, Cxcl2, Mmp2, Mmp9, and Vegf) was comparable among gastric tumor and non-tumor tissues from 3mo and 6mo gp130 F/F and gp130 F/F :Nlrp3 -/mice ( Figure 6G and Supplementary Figure 3E). Collectively, these data suggest that NLRP3 does not contribute to key inflammasome-driven cellular processes that promote gastric tumorigenesis.

DISCUSSION
In cancer, it has recently emerged that inflammasome complexes exhibit contrasting pro-and anti-tumorigenic activities. This is evident for the best characterized NLRP3-containing inflammasome, whereby mice deficient in NLRP3 are more susceptible to azoxymethane/dextran sodium sulfate (AOM/DSS)- induced colitis-associated colon cancer (CAC) (27,36). By contrast, comparable levels of tumor burden between wild-type and Nlrp3 -/mice when treated with AOM and DSS have also been observed (37). This dichotomy of function for NLRP3 in the intestine is also apparent in the chronic inflammatory setting of DSS-induced experimental colitis, in which Nlrp3 -/mice have been reportedly either more sensitive or resistant to intestinal inflammation and tissue damage (38,39). While these findings highlight the complexity of NLRP3 functions in the intestine, interpreting these findings must be treated with caution since the DSS-induced colitis and AOM/DSS-induced CAC models are known for experimental discrepancies due to variable gut microbiota present in different animal housing facilities. Further highlighting the complex functions of NLRP3 in cancer is a study demonstrating that Nlrp3 -/mice are protected against experimentally induced inflammation-associated skin carcinogenesis (28).
To further explore the role of NLRP3 in cancer, our current study suggests that NLRP3 does not play a major role in promoting the pathogenesis of gastric inflammation-associated tumorigenesis. Using multiple independent GC patient cohorts, we observed variable levels of NLRP3 expression that did not align with survival outcomes, suggesting limited prognostic potential of NLRP3 in GC. Interestingly, polymorphisms in the NLRP3 gene have been associated with increased GC risk in a Chinese patient cohort, and significantly upregulated NLRP3 expression levels in GC patient tumor biopsies have been correlated with poor prognosis, with progressively higher expression levels observed from gastritis to dysplasia and ultimately adenocarcinoma (40,41). While our current study suggests otherwise, these contrasting disease associations for NLRP3 in GC, as is commonplace for many other PRRs in cancers, are most likely explained by the high genetic and molecular heterogenicity in human GC, together with differences in study design relating to patient characteristics (e.g., geographical location, ethnicity, age, tumor stage, histological grade, and anatomical location). We also acknowledge that in the context of the NLRP3 inflammasome, NLRP3 expression levels alone do not necessarily correlate with levels of NLRP3 inflammasome complex formation and activation. Therefore, a more thorough investigation of the expression and/or activation levels of NLRP3 inflammasomeassociated components, namely, ASC, Caspase-1, and effector cytokines IL-1b and IL-18, is warranted in human GC patient samples.
Notably, our clinical findings were supported by data from the gp130 F/F preclinical GC model, in which NLRP3 deficiency had no impact on tumor burden or well-documented tumorpromoting cellular processes, namely, cell proliferation and survival, inflammation, and angiogenesis. The gp130 F/F mouse model was chosen for these studies since elevated inflammasome activation is a feature of ASC-driven gastric tumorigenesis in these mice (29). In addition, an advantage of gp130 F/F mice compared to other GC mouse models (e.g., INS-GAS and Gan) is the short tumor latency of 6 weeks post-birth, coupled with 100% disease penetrance on multiple genetic backgrounds (129Sv, C57BL/6, and BALB/c) (11,30).
Although the role of NLRP3 in GC has been poorly investigated to date, it is worth comparing our findings with a recent study using human cell lines, in which it was suggested that the NLRP3 inflammasome (via IL-1b) promoted GC cell proliferation and tumorigenesis by upregulating transcription of the cell cycle regulatory gene, CCND1 (41). However, one must caution the interpretation of these experimental findings since this study involved the use of mis-identified or crosscontaminated human GC cell lines (e.g., BGC-823 and SGC-7901) (42,43). Since our current in vivo data are generated from the gp130 F/F model for intestinal-type GC, which is the predominant subtype of GC, our findings now warrant further investigations on NLRP3 in additional genetically defined models for GC, including those for the diffuse type of GC.
At present, the role of PRRs in driving the pathogenesis of GC is not well defined. Current evidence from preclinical models suggests that TLR2 and the AIM2 cytosolic DNA sensor drive gastric tumorigenesis, independent of inflammation, by cancer cell autonomous mechanisms that augment the proliferation and survival (TLR2), as well as migration (AIM2), of the gastric tumor epithelium (13,17). Of note, AIM2 was shown to promote gastric tumorigenesis independent of inflammasomes in vivo, including in the gp130 F/F mouse model (17). Since gastric tumorigenesis in the gp130 F/F GC model is driven in part by ASC-containing inflammasomes, this genetic model provides an invaluable tool to dissect the contribution of distinct inflammasome-associated PRRs to GC. Since the global knockout of NLRP3 in gp130 F / F mice did not alter inflammasome activation, NLRP3 does not appear to be the primary inflammasome-activating PRR that is associated with gastric tumorigenesis. Our study therefore paves the way to further investigate the identity of the inflammasome-associated PRRs, for instance, other members of the NLR family (e.g., NLRP1 and NLRC4), that contribute to the pathogenesis of GC.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Monash Health Human Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Monash University MMC "B" Animal Ethics Committee.

AUTHOR CONTRIBUTIONS
Study conception and design: BJJ. Acquisition of study data: AlicW, VD, AlisW, LG, PT, and BJJ. Analysis and interpretation of data: AW, VD, LG, and BJJ. Writing and/or revision of the manuscript: AlicW and BJJ. Study supervision: BJ. All authors contributed to the article and approved the submitted version.