Marie-Claude JAURAND ^1* Fiona Murphy ² Emanuela Felley-Bosco ^3*

• ¹ Université de Paris, Paris, Ile-de-France, France
• ² University of Strathclyde, Glasgow, Scotland, United Kingdom
• ³ Université de Lausanne, Lausanne, Switzerland

engineered materials as a result of advances in (nano)technological developments of manufactured fibers (Nel, 2023). • Discovery of asbestos-related diseases Asbestos mining on an industrial scale started from the end of the 1800's with the development of mesothelioma in exposed miners first reported 50 years later (Wagner et al., 1960). The use of asbestos had been regulated in many countries since 1995, however several developed and developing countries continue to mine, export and use asbestos in high volumes (Frank, 2020) with approximately 1,200,000 metric tons of asbestos used worldwide in 2021 (USGS 2022 https://pubs.usgs.gov/publication/mcs2022). Over time, the risk of exposure identified at the workplace has been extended to the use of asbestos-containing material, and during efforts to remove asbestos from existing structures (Gottesfeld, 2024). Secondary exposure scenarios have become an increasing cause for concern as many asbestos-containing buildings constructed in the mid-twentieth century are reaching end-oflife and will require significant repair, reconstruction or demolition in the near future. These existing and emerging threats have the potential to increase asbestos exposure in more diverse populations and continue the risk for development of asbestos-related disease (Alpert et al., 2020) (Singh and Frank, 2023) (Metintas et al., 2024).Our historical experience with asbestos has left us with a heavy burden of disease. Lessons, however, have been learnt relevant to both the scientific context, including an increased understanding of the mechanism underpinning fiber toxicity, and from an ethical perspective. Recognition of the long latency period from initial exposure to disease development and the lack of compensatory mechanisms within the body to neutralize pathogenic fibers, highlights the need to address the humanitarian concern of on-going exposure and anticipate health risks before developing new industrial procedures utilizing EMP and during the design of novel fiber-like materials.• The remarkable characteristics of asbestos fibers related to toxicity Experimental studies have revealed that fiber morphology and physico-chemical properties modulate the biological effects of asbestos fibers, emphasizing a role of fiber dimensions, especially length (Stanton et al., 1981). Additionally, in vitro acellular systems were used to quantify fiber dissolution rate and confirm the role of fiber durability in the biopersistence of pathogenic fibers. Epidemiological studies have substantiated these findings, especially on the relevance of fiber dimensions. From this research it can be summarized that size, chemistry and surface reactivity are basic parameters that govern toxicity. They are involved in biological responses such as fiber uptake and phagocytosis, interactions with biological molecules, genetic alterations, inflammation, immunity, translocation processes and biopersistence (Sayan and Mossman, 2015) (Nagai and Toyokuni, 2010) (Donaldson et al., 2010) (Kuroda, 2021) (Huang et al., 2011). Further research has added several fiber parameters as involved in the toxicological effects such as rigidity. This information has been used to develop models to predict a mesothelioma potency hazard based on fibers dimensions (Nel, 2023) (Wylie and Korchevskiy, 2023) or, with further refinement, the "fiber potential toxicity/pathogenicity index (FPTI)" which includes 18 parameters associated with an adverse effect in the pathological process (Gualtieri, 2018) (Wylie and Korchevskiy, 2023) .The FPP is now being applied to assess the carcinogenic potential of fibrous particles such as glass and refractory fibers, carbon nanotubes (CNTs), metallic fibers and new manufactured particles (high aspect ratio nanomaterials, HARNs) (Nel, 2023) (Murphy et al., 2021) (Kane et al., 2018).The FPP has left us with the legacy of continuing research into asbestos toxicity mechanisms and studying EMPs and HARNs to avoid health damage. Experimental research has demonstrated a translocation of inhaled asbestos fibers towards the pleural space although the mechanism is not fully understood (Miserocchi et al., 2008). The retention of asbestos fibers in the pleural cavity seems partly related to the size of stomata or pores through which pleural fluid drains to the lymphatic system (from 0.8µm in mice to 10µm in human) (Schinwald et al., 2012). Subsequent accumulation of long fibers in the pleural cavity leads to direct irritation of the mesothelial layer, frustrated phagocytosis of pleural macrophages and inflammation. Direct instillation of CNTs and other high aspect ratio nanomaterials (HARN) into the pleural space have demonstrated a similar pathogenicity and mechanism of action to asbestos fibers in terms of production of oxidative stress, inflammation and genotoxicity (Donaldson et al., 2013) (Yoshida, 2019) (Nagai and Toyokuni, 2010).The mesothelial cell response to asbestos fibers was studied in cell culture models, including a SV40-immortalized, non-tumorigenic human mesothelial cell line. Normal mesothelial cells internalize the fibers, and chrysotile fibers were found in phagolysosomes, with a lysosome degranulation (Nagai and Toyokuni, 2012). Inflammatory factors shown to be released in vitro by mesothelial cells may propagate a chronic inflammatory environment in vivo subjecting mesothelial cells to on-going oxidative stress which may eventually result in cell transformation (Sayan and Mossman, 2015). In this Research Topic, Leinardi et al provide a comprehensive review of the role inflammatory components released from cells after cell death can contribute to chronic disease development in the context of silica exposure. While silica induce disease by the release of pro-inflammatory damage associated molecular patterns including HMGB1 from macrophages (Leinardi et al., 2022), asbestos carcinogenesis can be promoted by the release of HMGB1 directly from mesothelial cells (Suarez et al., 2023).To examine early changes along the mesothelium in response to fibers a transcriptomic kinetic analysis of mesothelial cells exposed by injection of fibers into the pleural cavity of C57BL/6 mice was carried out in at timepoints between one week up to 20 months after exposure (Chernova et al., 2017). Samples consisted of long and straight CNTs, short CNTs, and a long (carcinogenic) and short (lower pathogenicity) amosite asbestos fibers (Chernova et al., 2017). Inflammatory lesions studied from 1 week to 6 months after injection were similar in mice exposed to both long samples in terms of cell components in the pleural cavity and expression of inflammatory response genes, whereas gene expression from mice exposed to short fibers and controls clustered together. Inflammatory response pathways and activation of kinases involved in pro-oncogenic pathways that were identified in early lesions with dysregulation maintained through to tumor development. The status of Cdkn2a gene (encoding two proteins p16 Ink4a and p19 ARF ) ortholog of the key tumor suppressor genes (TSGs) in human mesothelioma CDKN2A (encoding P16 INK4a and P14 ARF ) was examined in inflammatory lesions at 1-year post injection before tumors developed and in tumors induced by exposure to long CNT and amosite asbestos (Chernova et al., 2017). The authors reported silencing of Cdkn2a (Ink4a/Arf) by hypermethylation and co-deletion of the proteins in the fiber-induced inflammatory lesions that increased in tumors, which also acquired p19/Arf deletion. This shows that epigenetic changes are present early in an inflammatory, pre-tumoral stage and emphasizes the similarities with human pleural mesothelioma (PM) (Chernova et al., 2017).Transcriptome analyses of asbestos-induced inflamed tissue was investigated in heterozygous Nƒ2 +/-C57Bl6 mice intraperitoneally exposed to crocidolite fibers for 12 weeks and assessed up to 33 weeks after the last exposure (Rehrauer et al., 2018). They revealed a decreased level of Nƒ2 expression and an activation of Yap/Taz localized in cell nucleus in inflamed mesothelium, that increased in tumors indicating a deregulation of Hippo pathway in these mice (Rehrauer et al., 2018). Although conducted in a genetically modified mouse model to increase mesothelioma susceptibility, this study highlights the potential role dysregulation of the Hippo pathway due to Nf2 mutation plays in progression of mesothelioma. Although Nf2 loss is regularly identified in mesothelioma tumors, reviewed in the article by Sekido and Sato as part of this Research Topic (Sekido and Sato, 2023), in human disease NF2 mutation appears to be a late event, indicating that genomic damage of NF2 may not be a direct asbestos effect but result from the chronic inflammatory and oxidative environment.• Histopathology and molecular genetic characterization of PM The characteristics of human PM are continuously evolving and concern several fields of cell and tumor biology, from histological classification, genetic, epigenetic and chromosomal status, state of regulatory pathways, cell to cell interactions, to the immunological microenvironment.The recent histological classification of PM retains the three main histologic subtypes: epithelioid, biphasic and sarcomatoid, with biphasic including epithelioid and sarcomatoid elements. Mesothelioma subtypes show a variety of architectures, cellular aspects and stroma, and their prognosis is different, with a worse survival for the sarcomatoid subtype compared to epithelioid (Sauter et al., 2022) (Husain et al., 2024). There was no epidemiological evidence of an association between the histological classification of mesothelioma and exposure to a given type of asbestos fibers (Vorster et al., 2022) (Franklin et al., 2016). However, in a genetically engineered conditional mouse model, where mostly sarcomatoid mesothelioma develop spontaneously after co-deletion of Nf2, p53 and Cdkn2a in mesothelial cells, asbestos exposure accelerates the onset of tumors which are mostly epithelioid (Farahmand et al., 2023). One notable feature of the response to asbestos exposure in this genetically modified mouse model is an increased recruitment of macrophages observed as a precursor to mesothelioma development. This raises the possibility the tumor microenvironment and epigenetic events downstream of asbestos exposure provides cues favoring proliferation when compared to the tumor developing in the absence of asbestos. This is also supported by the observation that different methylation levels of CpG sites were detected within tumors and reflective of intra-tumor heterogeneity of histological subtype. DNA methylation was preferentially located in CpG islands for sarcomatoid sub-type, while mainly located in non-CPG islands for the areas with high epithelioid histology suggesting that histo-molecular gradients are linked to epigenetic regulation (Blum et al., 2019).While there are limited studies showing the effects of asbestos fibers on the regulation of gene expression after short-term exposure of mesothelial cells to asbestos fibers, there is currently a large body of data on the pathological and biomolecular characteristics of PM. They are usually investigated long after the onset of the tumors, which are biopsied a long time since the beginning of exposure, possibly several decades.Investigations of the molecular landscape of mesothelioma go back to the 2,000s with the development of methodologies for large-scale analytical methods that provide highthroughput analysis of biological data. The genetic and epigenetic modifications of the tumors were studied using multi-omic approaches such as next-generation sequencing and microarrays. As observed in histology, PM is a heterogeneous tumor, with a rather low number of somatic gene mutations compared to other cancers, but with a high number and types of chromosomal rearrangements, copy number alteration, genome duplication and mutations in a number of key genes, most of them being TSGs (CDKN2A, BAP1, NF2, SETD2, LATS2, TP53) and a mutation in the TERT promoter (Bueno et al., 2016) (Meiller et al., 2021)(Febres-Aldana et al., 2024) (Creaney et al., 2022) (Nair et al., 2023) (Mangiante et al., 2023). At a lower rate, mutations were detected in genes of the SWI/SNF family (ARID1A, ARID2, and SMARCA4), and genes related to histone methylation (KMT2D, SETD2) were mutually exclusive (Quetel et al., 2020) (Febres-Aldana et al., 2024).Transcriptomic analyses revealed heterogenous molecular profiles of PM could be identified allowing a molecular classification of pleural PM. This refined classification identified several subgroups characterized by different molecular profiles and gene alterations that distinguish the epithelioid from the sarcomatoid phenotype and were linked to the patients' survival with a better outcome for epithelioid molecular profile than sarcomatoid (Bueno et al., 2016) (Blum et al., 2019). Investigation of the intra-tumor heterogeneity showed that in reality tumors from an individual patients are composed of a combination of epithelioid-like and sarcomatoid-like components (defined by E/S score) (Blum et al., 2019) (Alcala et al., 2019). There was a significant enrichment of BAP1 and SETD2 mutations in tumors of the highest E.score, of TERT_prom, NF2 and TP53 alterations with the tumors of the highest S-score, and LATS2 were more frequently altered in nonepithelioid PM and positively associated with the S-score (Quetel et al., 2020) (Blum et al., 2019).A genetic predisposition was suggested in families of mesothelioma cases and a high incidence of mesothelioma related to BAP1 tumor predisposition syndrome multifunctional gene (BRCA1-associated protein, BAP-1 gene) was discovered (Testa et al., 2011). Other studies reported a significant proportion of frequency of germline mutations and most pathogenic variants in DNA repair and TSGs (Panou et al., 2018) (Belcaid et al., 2023).Additional evidence for the influence of genetic predisposition in mesothelioma pathogenesis is suggested by experimental model such as the Cross Collaborative MexTAg mouse model, where 72 different genetic background where tested resulting in the identification of genetic variants predictive of different disease latency (Fisher et al., 2024).Knowing that asbestos induces inflammation and chromosome damage, including chromosome missgegregation in mesothelial and other mammalian cells, research was performed to detect mutation signature in PM (Huang et al., 2011). Recently, an analysis of clinical genomic profiling of patients with PM identified near-haploidization in an aggressive biphasic subtype that occurred in younger patients without asbestos (Yang SR, et al 2024). Losses in chromosomes 14q have been reported in a few studies, for example recurrent loss in 14q11.2-q21 was found in asbestos-exposed compared to patients not exposed, losses in chromosomes 14q was similarly lost in fiber-induced murine mesothelioma (Björkqvist et al., 1999) (Jean et al., 2011). While DNA oxidation was reported in asbestos-exposed animals, no ROS-induced mutation signature was reported in human PM. Recently data on PM were reinvestigated with a new statistical analysis, signature variability analysis (SVA), which considers the heterogeneity of the tumor mutations and the variability of mutations within and across tumors (Morrison et al., 2023). While there was no difference in copy number alterations and single base substitutions signature between exposed and unexposed patients, tumors from patients exposed to asbestos have more within-sample signature diversity and less across-sample heterogeneity than those from unexposed patients suggesting that SVA could be used to generate a footprint of asbestos exposure. Interestingly, analysis of biopsies taken at distinct anatomical sites revealed intra-tumor heterogeneity (Meiller et al., 2021) (Zhang et al., 2021). In this study, NGS performed on key mesothelioma genes showed heterogenous variants, especially NF2, which appears to be a late event.A recent meta-analysis of DNA methylation in PM investigated 53 studies for DNA methylation of genes in mesothelioma in a total of 97 genes including microRNAs (miRNA) analyzed at CpG methylation sites (Vandenhoeck et al., 2021). In this study, methylation was more frequent in mesotheliomas of the epithelioid subtype. The number of aberrantly methylated genes was also positively linked to asbestos bodies count in the lung, which is a signature of asbestos exposure. The majority of genes significantly hypermethylated in mesothelioma in comparison with normal tissue are CDH1, ESR1, miR-34b/c, PGR, RARb, SFRP1, and WIF1, and one, APC is hypomethylated. A promoter hypermethylation of cell cycle control-associated genes: CDKN2A, CDKN2B, RASSF1, CCND2, APC and HPPBP1 was found in patients with high asbestos body burden in asbestos-exposed patients after control of confounding factors (Christensen et al., 2008). Asbestos bodies counts were also positively linked to methylation at CpG sites (Christensen et al., 2009).Dysregulation of both miRNA and long non coding RNAs (LncRNAs) has been identified in mesothelioma tumors (Abd-Elmawla et al., 2023) (Xu et al., 2023). Many miRNAs targeted epithelial or mesenchymal markers and their expression is dependent on the E/S score (Blum et al., 2019). LncRNA such as NEAT1, PCT6, HOTAIR, GAS5 were identified as potential biomarkers.RNA editing patterns of PM have been studied in human according to E/S score of the tumors in untranslated regions (UTR) of transcripts and in introns . Results showed that PM of high Escore had RNA frequency editing at the 3'UTR, and in introns in PM of high S-score. Then, the regions vary with the EMT, consistent with epigenetic regulation of EMT . In asbestos-exposed Nf2 +/-mice a RNA-editing signature, mediated by adenosine-deaminase-acting-on dsRNA (ADAR), was found in inflamed tissues, 33 weeks after exposure, with a higher number of RNAediting events in tumors (Rehrauer et al., 2018).Research on PM microenvironment aims at identifying the different cell components including immune cells with the goal to reactivate the immune defense. Single-cell transcriptomic identified sarcomatoid enriched phenotype associated with foetal-like endothelial cells, CXCL9/10/11+ macrophages and cytotoxic, regulatory and exhausted T lymphocytes (Giotti B, et al, 2024), in line with bulk-RNA studies (Alcala et al., 2019) (Mangiante et al., 2023). Detailed analysis of tumor cell populations will permit novel immunotherapy to increase mesothelioma cell susceptibility to immune cell killing. Research on PM concerning the role and mechanism of action of asbestos fibers have demonstrated the role of asbestos as major etiological factor and its multifactorial mechanism of action. Asbestos specificities are related to the mechanism of fiber-cell interaction (phagocytosis) and genomic damages. One of the key mechanisms of cancer involves gene mutations, which are not at high level in PM, but chromosomal damages are significant. As PM is an unfrequent cancer among the whole population, largely dependent on asbestos exposure in diverse populations, it is likely that the DNA repair polymorphism plays a significant role in PM induction. Additionally, a background of persistent inflammation can act at different levels (ROS production, increased proliferation of pre-malignant cells, immunosuppression), to elicit the neoplastic progression and modify the tumor microenvironment. The different tumor microenvironment according to PM histology and E and S components of the tumor is remarkable, in line with tumoral/EMT evolution posing a significant challenge for effective therapeutic intervention.While mesothelioma genomics provided us footprints on the link between established disease and asbestos, early imprints are poorly identified. Investigations of early effects of asbestos on mesothelial cells and pleura have shown activation of inflammatory pathways and apoptosis, and permitted identifying early genetic and epigenetic impacts at the onset of exposure (Sayan and Mossman, 2015) (Huang et al., 2011) (Chernova et al., 2017) (Rehrauer et al., 2018). Studies of asbestos-exposed mice heterozygous on key TSGs confirmed the importance of these genes in the neoplastic progression of mesothelial cells under asbestosexposure (reviewed in (Blanquart et al., 2020) and (Testa and Berns, 2020)). Although NF2 is frequently altered, its alteration is a late event (reviewed in (Sekido and Sato, 2023)) and the application of novel technologies will reveal cell environment cues driving that alteration.Presently, we may propose that PM appears as a double paradigm, toxicologic for approaches on further researches on EMPs, and genomic of asbestos diseases due to some specific molecular changes. Nevertheless, progress have to be made to distinguish the asbestos mechanism of action from neoplastic progression of mesothelial cells.