International Journal of Molecular Sciences Review In Vitro Approaches to Determine the Potential Carcinogenic Risk of Environmental Pollutants Irene Barguilla 1,2 , Veronique Maguer-Satta 2 , Boris Guyot 2 , Susana Pastor 1 , Ricard Marcos 1, * and Alba Hernández 1, * 1 Group of Mutagenesis, Department of Genetics and Microbiology, Faculty of Biosciences, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain 2 CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon, 69008 Lyon, France * Correspondence: ricard.marcos@uab.es (R.M.); alba.hernandez@uab.es (A.H.) Abstract: One important environmental/health challenge is to determine, in a feasible way, the potential carcinogenic risk associated with environmental agents/exposures. Since a significant proportion of tumors have an environmental origin, detecting the potential carcinogenic risk of environmental agents is mandatory, as regulated by national and international agencies. The challenge mainly implies finding a way of how to overcome the inefficiencies of long-term trials with rodents when thousands of agents/exposures need to be tested. To such an end, the use of in vitro cell transformation assays (CTAs) was proposed, but the existing prevalidated CTAs do not cover the complexity associated with carcinogenesis processes and present serious limitations. To overcome such limitations, we propose to use a battery of assays covering most of the hallmarks of the carcinogenesis process. For the first time, we grouped such assays as early, intermediate, or advanced biomarkers which allow for the identification of the cells in the initiation, promotion or aggressive stages of tumorigenesis. Our proposal, as a novelty, points out that using a battery containing assays from all three groups can identify if a certain agent/exposure can pose a carcinogenic risk; furthermore, it can gather mechanistic insights into the mode of the action of a specific carcinogen. This structured battery could be very useful for any type of in vitro study, containing human cell lines aiming to detect the potential carcinogenic risks of environmental agents/exposures. In fact, Citation: Barguilla, I.; Maguer-Satta, here, we include examples in which these approaches were successfully applied. Finally, we provide V.; Guyot, B.; Pastor, S.; Marcos, R.; a series of advantages that, we believe, contribute to the suitability of our proposed approach for Hernández, A. In Vitro Approaches the evaluation of exposure-induced carcinogenic effects and for the development of an alternative to Determine the Potential strategy for conducting an exposure risk assessment. Carcinogenic Risk of Environmental Pollutants. Int. J. Mol. Sci. 2023, 24, Keywords: in vitro cell transformation; oncogenic phenotype; cancer hallmarks; long-term exposure 7851. https://doi.org/10.3390/ ijms24097851 Academic Editor: Hansruedi Glatt 1. Introduction Received: 20 March 2023 Revised: 22 April 2023 Chemical safety and exposure science have largely advanced by following a material- Accepted: 23 April 2023 by-material approach and using conventional and robust assays established to assess Published: 25 April 2023 well-known toxicity endpoints. However, the number of chemicals in the environment and in consumer products is overwhelmingly large, and some traditional assays are inefficient in evaluating the wide diversity of human exposures across individuals and throughout their lifetimes [1]. Most of these methodologies are animal-based, which are too resource- Copyright: © 2023 by the authors. consuming to address all exposures and, further, present some limitations, such as the Licensee MDPI, Basel, Switzerland. sometimes-conflictive extrapolation of results from the animal models to human popula- This article is an open access article tions and, most importantly, the ethical concerns that they present [2–4]. Moreover, many distributed under the terms and traditional approaches lack mechanistic insight and fail to mimic real-world exposure sce- conditions of the Creative Commons narios in which chemical mixtures and long-term/low-dose exposures are contemplated [5]. Attribution (CC BY) license (https:// Thus, although the data gathered have great value, large knowledge gaps remain. As a creativecommons.org/licenses/by/ result, the development of new approach methodologies (NAMs) is a field in need of 4.0/). Int. J. Mol. Sci. 2023, 24, 7851. https://doi.org/10.3390/ijms24097851 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 7851 2 of 26 expansion. NAMs comprise in silico, in chemico and in vitro approaches aiming to fill in those gaps in current exposure science to perform better-informed risk assessments and support regulatory decisions [6]. As defined by the ECHA (European Chemical Agency), NAMs include diverse and innovative high-throughput screening tools or high-content methods, such as genomics, proteomics and metabolomics, but also other more conven- tional methods able to inform on the toxicokinetic or toxicodynamics of substances in the context of hazard characterization [7]. The paradigm shift towards these new approaches is promoted by policies that call for the use of NAMs when suitable as a response to animal welfare issues, while also aiming to improve the speed and accuracy of the data necessary for safety evaluations. However, NAMs still exhibit several uncertainties and, thus, it is not currently possible to fully replace in vivo studies with alternative approaches [8]. Nonetheless, it is believed that applying an evaluation strategy that integrates data from several NAMs would efficiently allow us to prioritize data needed for the preliminary determination of the risks posed by exposures [9,10]. The development of NAMs becomes particularly important for the evaluation of the potential carcinogenic effects related to environmental exposures. Human populations are exposed daily to an ever-growing number of environmental pollutants. These environmen- tal exposures are mainly characterized by two central aspects: (i) being long-term/low-dose exposures and (ii) being highly diverse [11]. Therefore, in the long term, there is a very likely scenario in which an agent—or a mixture—can initiate the carcinogenic process that develops over time. Up till now, in vivo studies have been the gold standard tool for carcinogenesis assessments, with rodents being the most popular model to evaluate the genotoxic and carcinogenic effects of exposures [4]. The two-year rodent bioassays are the accepted tests in standard OECD regulations to study the carcinogenic potential of chemicals [12]. In these assays, groups of animals are continuously exposed for most of their lifespan and are closely monitored to track any signs of toxicity or the development of neoplastic lesions. As previously mentioned, this approach presents important limi- tations: in vivo assays are too time-consuming, costly, and require many animals, which makes them impractical for the large testing programs desirable for carcinogenicity studies, despite efforts to implement the 3Rs principle (replacement, refinement and reduction). Therefore, approaches based on in vitro models are urgently required [13]. Among the in vitro alternative approaches to studying carcinogenicity, cell transforma- tion assays (CTAs) are the most advanced in terms of standardization and validation. OECD (Organisation for Economic Co-operation and Development) test guidelines have been recommended for three CTAs able to identify agents with tumor-initiating and promoting activities [14–16]. They are: BALB/c3T3 CTA: This assay predicts the tumor-initiating activity of an exposure. Briefly, following a standardized concentration range-finding experiment, A31-1-1 mouse fibroblasts are exposed for 72 h to the compound and are thereafter cultured in a fresh compound-free medium for 31 days. Then, the cells are fixed and foci are scored. Only the malignantly transformed BALB/c 3T3 cells form these foci, characterized by the multilay- ering of cells randomly orientated and morphologically divergent from the monolayer in the culture [17]. Bhas 42 CTA: This system was implemented to evaluate the tumor-initiating and promoting ability of the chemicals using the Bhas 42 cell line. This cell clone is prone to transformations, as it was isolated after transfecting BALB/c 3T3 mouse fibroblasts with an activated ras oncogene. Subsequent to the experiment carried out to find the optimal concentrations to be applied, the tumor-initiation capability of chemicals was determined by scoring the foci formed after an 18-day culture, following a 3-day exposure to the compound. In a modified version of this test, Bhas 42 cells were cultured for 3 days in a compound-free culture medium. Then, the medium was replaced with a compound- containing medium every 3 days until day 14, at which point the fresh compound-free medium was again added. On day 21, the cells were fixed and foci were scored to assess the tumor promotion ability of the exposure [18]. Int. J. Mol. Sci. 2023, 24, 7851 3 of 26 SHE CTA: The Syrian hamster embryo assay is based on primary cells isolated from Syrian hamster embryos, which form morphologically transformed colonies when exposed to an agent with carcinogenic potential. After choosing the concentration range, SHE cells were exposed to the test chemical from the third day of culturing and the exposure was continued until day 10. Then, the transformed cell colony frequency was quantified to determine the tumor-initiating potential of the exposures [19]. All the above-indicated tools are valuable as a first approach in carcinogenicity screen- ing, contributing to a significant reduction in the number of agents that must undergo in vivo testing [15]. Nevertheless, CTAs do not completely mimic the whole carcinogenesis process as it happens in vivo and, in addition, they present important limitations [20], such as: Performing single-endpoint analysis: Despite the multistep nature of carcinogenesis, these systems rely on the evaluation of a single endpoint related to cell transformation. Thus, information on other carcinogenic-related effects, mechanistic insights and potential differences in the transformed status of the exposed cells is missed. Functioning with short-term periods of exposure: Although these approaches do not strictly belong to acute exposure systems, they far from mimic a real-life scenario, as only 3–10 days of exposure are contemplated and environmental exposures often last for long periods, even a lifetime. Using animal cell models: The use of rodent cell clones can be very efficient in de- tecting rodent carcinogens; however, these systems can fail to identify specific human carcinogens [21]. Further, concerns regarding the extrapolation results to humans could arise as is the case for in vivo studies [22,23]. Despite the prevalidation efforts carried out by the European Center for Validation of Alternative Methods (ECVAM) [24], these limitations have hampered the extended use of CTAs. Overcoming these limitations by developing human-cell-line-based CTAs, for which different carcinogenic-related effects can be analyzed under a long-term exposure scenario, would greatly contribute to further improving the relevance of these in vitro approaches and increase the interest in incorporating CTAs into carcinogenicity testing strategies. Such efforts are ongoing as scientists aim to broaden the scope of CTAs by improving their prediction ability and including mechanistic explanations [25]. According to such expectations, this review aims to outline an in vitro long-term exposure approach and a battery of alternative CTAs that would fall under the umbrella of NAMs. In this scenario, not only the novel method of exposure is remarkable, but also the multistep endpoint analysis of the transformation biomarkers based on different hallmarks of carcinogenesis. Additionally, we include relevant studies in which these techniques were successfully used in the framework of exposure safety evaluations. Finally, we provide a series of advantages that show the suitability of our proposed approach for the evaluation of exposure-induced carcinogenic effects and for the development of an alternative strategy for assessing exposure risks. 2. Long-Term Exposures as a Novel Approach for Carcinogenesis Evaluation Due to the progressive nature of cancer, the carcinogenic effects induced by envi- ronmental pollutants (chemicals or other agents) appear because of sustained exposures throughout extended periods of time. Accordingly, these types of exposures are especially relevant for in vitro scenarios, where the long-term accumulative effects under chronic exposure settings should be evaluated. Long-term in vitro studies have successfully been used by different groups to de- termine the exposure-derived induction of cell transformation, the progression and the promotion of tumoral cells, and to establish potential mechanisms of action involved in the process at different time points (as discussed later). Following this approach, and as schematized in Figure 1, relevant target cells are chronically exposed to subtoxic concentra- tions of the tested agent for long periods of time, ranging from 6 to 30 weeks on average. Exposed cells are generally passaged weekly, and the agent-containing medium is changed Int. J. Mol. Sci. 2023, 24, 7851 4 of 26 Int. J. Mol. Sci. 2023, 24, 7851 4 of 26 concentrations of the tested agent for long periods of time, ranging from 6 to 30 weeks on average. Exposed cells are generally passaged weekly, and the agent-containing medium is changed once/twice a week to continuously maintain the selected exposure level, mim- once/twice a week to continuously maintain the selected exposure level, mimicking real-life icking real-life scenarios. More importantly, nonexposed control cells must also be main- scenarios. More importantly, nonexposed control cells must also be maintained in parallel tained in parallel for the selected number of weeks to discriminate the effects induced by for the selected number of weeks to discriminate the effects induced by the continuous the continuous passaging from those caused by the sustained exposure. passaging from those caused by the sustained exposure. Figure1.1.In Figure Invitro vitrolong-term/low-concentration long-term/low-concentration studies. studies. Cells Cells progressively progressivelydevelop developaatransformed transformed phenotype when cultured for long periods of time whilst chronically exposed to low concentrations phenotype when cultured for long periods of time whilst chronically exposed to low concentrations of a carcinogenic agent. of a carcinogenic agent. Atdifferent At differenttime time points points during during thethe long-term long-term in vitro in vitro studies, studies, severalseveral hallmarkhallmark biomark- bi- omarkers of the cell transformation process are evaluated (as developed ers of the cell transformation process are evaluated (as developed later). This allows for later). This allows for identification the the identification and description and description of theofcarcinogenic the carcinogenic phenotypephenotype reached reached by theby theupon cells cells upon exposure exposure over time, over time, as wellas well as theas the comparison comparison of the of the transformed transformed statusatatthe status theinitial, initial, middle and end stages of the exposure period. More importantly, middle and end stages of the exposure period. More importantly, several authors have several authors have demonstrated that subsequent to prolonged exposure, the cells demonstrated that subsequent to prolonged exposure, the cells obtained in the in vitro cell obtained in the in vitro cell transformation transformation models models induceinduce tumorigenesis tumorigenesis in micein[26–29], mice [27–30], which iswhich is a conclusive a conclusive marker marker of of the identification the identification of an agent of an ableagent able toa induce to induce neoplastica neoplastic transformation transformation [30]. [31]. Long-termexposures Long-term exposuresreservereserve further further uses. uses. Thus, Thus, throughout throughout the entire the entire exposure exposure time, time, cell cell samples samples can be collected can be collected to constitute to constitute a biobank a biobank for furthering for furthering the understand- the understanding of the ing of the mechanisms mechanisms involved in involved in the transformation the transformation process. Onceprocess. the cell Once the cell transfor- transformation occurs mation occurs after long-term exposure to a specific agent, diverse after long-term exposure to a specific agent, diverse tools, mainly molecular, can be used to tools, mainly molecu- lar, can the identify be used precise to moment identify thewhereprecise moment different where different biomarkers started tobiomarkers be significantlystarted to be altered. significantly Moreover, thisaltered. approach Moreover, permitsthis theapproach use of cells permits the useinofthe maintained cells maintained biobanks even in the after biobanks the even of completion after thethe completion study, until a timeof the newstudy, toolsuntil becomea time new tools available becomeunsolved to answer available to answer In questions. unsolved this way,questions. In this way, DNA methylation DNA have changes methylation changes with been associated havemultiwalled been associ- carbon ated withnanotube (MWCNT) multiwalled carbon long-term nanotubeexposures (MWCNT)[31], and alterations long-term exposures in [32], the expression and altera- levels of specific genes (as Mth1) were observed in cells chronically exposed tions in the expression levels of specific genes (as Mth1) were observed in cells chronically to ZnONPs and CoNPs [32], as well as changes in a panel of microRNAs being exposed to ZnONPs and CoNPs [33], as well as changes in a panel of microRNAs being evaluated in cells previously long-term evaluatedexposed to TiO2 NPs long-term in cells previously and MWCNTs [33]. to exposed Further, TiO2NPs the and collection MWCNTs of samples for the [34]. Further, application the collection of omics analyses of samples for can the greatly contribute application of omicsto identifying analyses can keygreatly events contribute and adverse to outcome identifyingpathways, key events allowing to unravel and adverse the mechanisms outcome pathways,of action. to unravel the mecha- allowing nisms of action. 3. Alternative Cell Transformation Assays Based on the Hallmarks of Carcinogenesis The hallmarks 3. Alternative Cell of carcinogenesisAssays Transformation have been Baseddeeply characterized on the Hallmarks after they were first of Carcinogenesis defined The[34] and extended hallmarks [35]. Interestingly, of carcinogenesis a new have been paper deeply on this topicafter characterized has they recently werebeen first published pointing out the hallmarks of cancer as an integrative concept to explain the defined [35] and extended [36]. Interestingly, a new paper on this topic has recently been complexity of the cancer process and to understand the mechanisms of cancer development published pointing out the hallmarks of cancer as an integrative concept to explain the and malignant progression more fully [36]. These hallmarks describe distinctive events of complexity of the cancer process and to understand the mechanisms of cancer develop- cancer development, namely, two enabling capabilities—(i) genomic instability and muta- ment and malignant progression more fully [37]. These hallmarks describe distinctive tion and (ii) tumor-promoting inflammation—as well as eight hallmark capabilities—(i) events of cancer development, namely, two enabling capabilities—(i) genomic instability sustaining proliferative signaling, (ii) enabling replicative immortality, (iii) evading growth suppressors, (iv) resisting cell death, (v) deregulating cellular energetics, (vi) avoiding im- mune destruction, (vii) inducing angiogenesis and (viii) activating invasion and metastasis. and mutation and (ii) tumor-promoting inflammation—as well as eight hallmark capabil- ities—(i) sustaining proliferative signaling, (ii) enabling replicative immortality, (iii) evad- Int. J. Mol. Sci. 2023, 24, 7851 ing growth suppressors, (iv) resisting cell death, (v) deregulating cellular energetics,5 of 26 (vi) avoiding immune destruction, (vii) inducing angiogenesis and (viii) activating invasion and metastasis. Mimicking the situation that takes place in vivo, the in vitro cell transfor- mation Mimickingis a the process where situation cells that acquire takes placespecific in vivo,features thatcell the in vitro are transformation representative of is adifferent process stages during where the progression cells acquire of the tumoral specific features that are phenotype representative andof that differ from different stagesthose duringof non- the transformedof progression cells. the These tumoral phenotypic phenotype manifestations and that differ can be explored from with different in vitro those of nontransformed cells. systems These and, therefore, phenotypic they are presented manifestations here as can be explored a battery with of oncogenic different biomarkers in vitro systems that and, there- fore, theyinformation provide are presented onhere the as a battery ofstatus transformed oncogenic of the biomarkers that align cells, as they providewithinformation different on the transformed hallmarks of cancer.status of the cells, as they align with different hallmarks of cancer. Due totothe theprogressive progressivenature natureof of thethe cellcell transformation, transformation, somesomeof theofproposed the proposedbiomark- bi- ers are informative omarkers of the specific are informative status ofstatus of the specific the cells during of the cells the acquisition during of the tumoral the acquisition of the phenotype. Accordingly, tumoral phenotype. we grouped Accordingly, we the different grouped the biomarkers in early, intermediate different biomarkers in early, interme-or ad- vanced diate orstages, advanced which allowed stages, which forallowed the identification of the cells of for the identification in the cells initiation, in thepromotion initiation, or aggressive promotion stages of tumorigenesis, or aggressive as schematized stages of tumorigenesis, in Figure in as schematized 2. Figure Keeping 2. this Keeping in mind, this rather than focusing on single endpoints, the proposal was to use a in mind, rather than focusing on single endpoints, the proposal was to use a battery of battery of biomarkers under long-term biomarkers underexposure long-term settings. exposure In this way,In settings. the whole this way,analysis the whole would provide analysis would a more pro- comprehensive vide view of the changes a more comprehensive view of taking place during the changes the carcinogenesis taking place process. It is during the carcinogenesis essentialIttoisselect process. diverse essential endpoints to select diversefrom all categories endpoints from allofcategories the battery of of thebiomarkers battery of bi- to accurately characterize the in vitro transformation process at different omarkers to accurately characterize the in vitro transformation process at different time time points to gather mechanistic points to gatherinsight and, ultimately, mechanistic insight and, contribute ultimately,robust information contribute robust for risk assessment information for risk strategies. assessment strategies. Figure 2. A battery of oncogenic biomarkers. The proposed biomarkers were grouped into early, Figure 2. A battery of oncogenic biomarkers. The proposed biomarkers were grouped into early, intermediate and advanced stages, as they were representative of different stages during the pro- intermediate and advanced stages, as they were representative of different stages during the pro- gression of the transformed phenotype. Each biomarker is associated with one or more hallmarks gression of cancerof theenabling and transformed phenotype. Each biomarker is associated with one or more hallmarks of capabilities. cancer and enabling capabilities. 3.1. 3.1. In In Vitro Vitro Cell Cell Transformation Biomarkers and Transformation Biomarkers and Methods Methods In the following In the following sections, sections, we we described described the rationale behind the rationale behind the different oncogenic the different oncogenic biomarkers biomarkers selected for our proposed battery. In addition, some of the available method- selected for our proposed battery. In addition, some of the available method- ologies ologies for their evaluation for their evaluation using in vitro using in vitro approaches approaches were were also also presented. presented. 3.1.1. Early Biomarkers DNA Damage DNA Damage and and Genotoxicity Genotoxicity Genetic instability is defined as a status of an increased propensity for genomic alterations, and it is one of the defining features of cancer. During the carcinogenic pro- cess, tumor cells accumulate both genetic and epigenetic alterations; thus, genetic and epigenetic instabilities are mechanisms intimately associated with carcinogenesis. DNA methylation processes prevent transcription factors from binding to DNA and, in addi- tion, post-transcriptional histone modifications regulate the chromatin structure, having Int. J. Mol. Sci. 2023, 24, 7851 6 of 26 an important impact on biological processes such as gene expression, DNA repair and chromosomal condensation. It was estimated that between 40 and 60 mutations occur in most solid tumors. How- ever, not all occur at tumor initiation, but also later during the progression stage [37,38]. Therefore, assessing the genetic instability of cells during the in vitro transformation pro- cess is informative both at early and intermediate time points. Given that monitoring mutations in a nontargeted way is unrealistic, the evaluation of DNA damage and geno- toxicity levels is an indirect biomarker of genetic instability that can eventually result in mutations [39]. Further, incorporating this endpoint into our proposed battery of assays allowed us to identify both genotoxic and nongenotoxic carcinogens, partly contributing to the mechanistic insight. There are two gold standard methodologies for the assessment of DNA damage and genotoxicity: the comet assay and the micronucleus assay. OECD guidelines exist for both techniques [40,41], given their usefulness in detecting genotoxic agents and being surrogate biomarkers of carcinogenic risk. The comet assay is based on the migration of DNA strands of individual cells embedded in agarose. Briefly, the cells were fixed in agarose and placed on a support slide. Then, DNA was denaturized using alkaline lysis and a brief electrophoresis step was performed. If the DNA contained breaks, uncoiled DNA loops would migrate toward the anode, while migration would be prevented if the DNA was undamaged. Therefore, after fluorescent staining, the cells with damaged DNA would appear as “comets” with a bright head and a tail of variable intensities depending on the number of DNA breaks. From the initial protocol proposal [42], different modifications of the assay have been published, incorporating enzymes to detect oxidized DNA bases [43] or to improve the high throughput potential of the methodology and scoring [44,45]. On the other hand, the micronucleus assay measures the incidence of micronuclei (MNs), which are small chromatin bodies generated during cell division due to chromoso- mal fragmentation or chromosomal loss. Chromosomal fragmentation, more specifically double-strand breaks, leads to a structural chromosomal instability, characterized by on- going errors in chromosomal segregation throughout successive cell divisions, providing a proliferative advantage to cancer cells. Consequently, this mechanism is particularly important in cancer progression. Classically, MNs are manually scored through microscopy after inducing a cell cycle arrest [46]. Due to the labor-intensive nature of the MN analysis through the use of microscopy, alternatives based on the flow cytometry analysis have been proposed. This approach involves the lysis of outer membranes and the use of dyes to discriminate the MNs from the nuclei, based on the fluorescence intensity, which allows for a high-throughput analysis [47]. New variations of the MN assay are being proposed, aiming to further automatize the process and increment the high-throughput aspect of the technique [48]. Uncontrolled Proliferation The proliferation of normal cell populations is controlled by a balance between growth- promoting and growth-suppressing signals, which allows for normal tissue homeosta- sis [49]. The dysregulation of these signals through diverse mechanisms leads to sustained cell growth, which is arguably the most identifying trait of cancer cells. Not for nothing, four out of the eight hallmark capabilities are related to uncontrolled cell proliferation, in- cluding sustained proliferative signaling, enabling replicative immortality, evading growth suppressors and resisting cell death [35]. Early in the tumorigenic process, cancer cells acquire the ability to constitutively activate proliferative signaling through autocrine sig- naling to their own growth factors, increased levels of mitogen receptors and the induction of ligand-independent or stroma signaling [50]. Moreover, transformed cells can evade cell cycle arrest signals and escape from cell death through the interaction of diverse signaling pathways at multiple levels, such as the overexpression of antiapoptotic molecules [51]. As a result, increased cell proliferation can be identified as an early cell transformation biomarker. lation of the population-doubling time, which estimates the time it takes for a cell popu- lation to double in size based on the number of cells in the population at two or more time points [55]. The population size can be estimated either through direct cell counting using a hemocytometer counting chamber or automated cell counters. However, aiming to Int. J. Mol. Sci. 2023, 24, 7851 move to high-throughput formats, quantitative methods compatible with multiwell plates 7 of 26 have been developed. On the one hand, metabolic assays rely on the detection of a color- imetric signal proportional to the cells’ metabolic activity, allowing for the measurement of viable cells. MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) [56], Diverse methodologies allow for the evaluation of cell proliferation, and many of them resazurin [57] or sulforhodamine [58] assays, among others, fall within this category of have been exhaustively reviewed [52,53]. The most common approach is the calculation metabolic proliferation assays. Moreover, assays based on the DNA content and DNA of the population-doubling time, which estimates the time it takes for a cell population synthesis are also direct and simple approaches to measuring cell proliferation. Within to double in size based on the number of cells in the population at two or more time this category, the incorporation of BrdU (5-bromo-2′-deoxy-uridine) in DNA can be de- points [54]. The population size can be estimated either through direct cell counting using a tected through the use of antibody-based techniques, in what is called the BrdU assay [59]. hemocytometer counting chamber or automated cell counters. However, aiming to move to Additionally, in the EdU (5-ethynyl-2′-deoxyuridine) proliferation assay, EdU is incorpo- high-throughput formats, quantitative methods compatible with multiwell plates have been rated into replicating developed. DNA,metabolic On the one hand, providingassays a method tothe rely on quantify andofmonitor detection proliferating a colorimetric signal cells throughtoflow proportional cytometry the cells’ or microscopic metabolic approaches activity, allowing for the[60]. Further, inofrecent measurement viableyears, cells. there(3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium MTT has been an increment in the development of noninvasive technologies bromide) that allow [55], resazurin [56] forsulforhodamine or the real-time monitoring [57] assays,ofamong cell proliferation others, fallin an incubator within basedofonmetabolic this category imaging prolif- [61] or measuring eration ultrasonic assays. signals[62]. Moreover, assays based on the DNA content and DNA synthesis are also direct and simple approaches to measuring cell proliferation. Within this category, the Morphological incorporation of Changes BrdU (5-bromo-20 -deoxy-uridine) in DNA can be detected through the use In culture populations, of antibody-based techniques, tracking in what theis cell morphology called the BrdU assayis a very simple [58]. but informative Additionally, in the biomarker EdU (5-ethynyl-2 0 of in vitro cell transformation. -deoxyuridine) Earlyassay, proliferation in theEdUoncogenesis process, is incorporated epithelial into cells replicating activate DNA, the epithelial-to-mesenchymal providing a method to quantify and transition monitor (EMT) program, proliferating cellswhich through is characterized flow cytome- by or try major transcriptional microscopic and [59]. approaches signaling Further,alterations in recent[63]. Eventually, years, thesean there has been lead to pheno- increment in the development typical changes in of epithelial noninvasive technologies cells, including the thatloss allow of for the real-time polarity monitoring of and morphological cell modi- proliferation in anpolygonal-shaped fications; normal incubator based on imaging cells change, [60] or measuring acquiring ultrasonic an elongated signals [61]. spindle-like mor- phology (Figure 3) [64]. Morphological Changes In fibroblasts, morphological alterations indicate their activation into myofibroblasts, In culture typically populations, enriched tracking in the tumor the cell morphology microenvironment is a very simple [65]. Through but informative the simple microscopic biomarker scoring of of theincell vitro cell transformation. population presentingEarly in the oncogenesis an abnormal morphology, process, epithelial we could easilycells esti- activate mate the the epithelial-to-mesenchymal proportion of cells transitioning transition from a(EMT) program, stationary statewhich is characterized to becoming by able to dis- major transcriptional and signaling alterations [62]. Eventually, these seminate towards other tissues, or to promote the invasive potential of surrounding cells. lead to phenotypical changes in epithelial The growing body ofcells, including studies the loss ofable in the literature polarity andthe to infer morphological modifications; metastatic potential of cells normal polygonal-shaped from diverse morphological cells change, acquiring parameters such as the an elongated spindle-like angle, diameter or size morphology supports the (Figure 3) [63]. suitability of using cell morphologies as an in vitro transformation biomarker [66–68]. Figure 3. Comparison between the cell morphology of non-transformed cells and the spindle-like shape of transformed cells. In fibroblasts, morphological alterations indicate their activation into myofibroblasts, typically enriched in the tumor microenvironment [64]. Through the simple microscopic scoring of the cell population presenting an abnormal morphology, we could easily estimate the proportion of cells transitioning from a stationary state to becoming able to disseminate towards other tissues, or to promote the invasive potential of surrounding cells. The growing body of studies in the literature able to infer the metastatic potential of cells from diverse morphological parameters such as the angle, diameter or size supports the suitability of using cell morphologies as an in vitro transformation biomarker [65–67]. At an ultrastructural level, diverse changes in the mitochondrial structure have been observed as associated with tumoral cells [68,69]. Mitochondrial swelling is a characteristic feature of most cancer cells, but other alterations, such as the acquisition of an irregu- lar shape or the disorganization and reduction in the cristae number, are also frequently reported. These morphological alterations have been correlated with mitochondrial dys- Int. J. Mol. Sci. 2023, 24, 7851 8 of 26 function [70] and, therefore, are closely linked to a cancer hallmark capability, as it pertains to the deregulation of cellular energetics. Hence, we proposed the analysis of ultrastructural morphological changes as part of the battery of in vitro assays. Furthermore, the use of imaging techniques, such as transmission electron microscopy (TEM) or scanning electron microscopy (SEM), has also been long-used to evaluate ultrastructural alterations in very different cell types, and are still of great use [71,72]. Secretome Alterations Tumor cells are not isolated, but rather subsist surrounded by a highly rich microenvi- ronment of stromal cells formed by fibroblasts, pericytes, leukocytes, endothelial cells and other cell types [73]. The complexity of events that lead to the transformation of normal cells into tumoral cells is parallel to the evolution of the microenvironment into an activated state, triggered by the continuous crosstalk between the tumor and stromal cells [74]. This paracrine communication is based on the release of inflammatory cytokines (e.g., IL-6, IL-8 and IL-1β), growth factors, extracellular matrix (ECM)-remodeling enzymes and other soluble factors that constitute the cells’ secretome. During the process of oncogenic trans- formation, the secretome varies in composition, and can influence several hallmark abilities of tumoral cells, such as immune response modulation, angiogenesis, inflammation and invasiveness induction, therefore, influencing tumor promotion from the early stages of the transformation process [75]. Further, specific components of the secretome can actively drive cell transformation. This is especially important for the stem cell compartment, as regulation through soluble factors plays a major role in the cell fate determinant and in con- trolling their proliferation/differentiation balance. Bone morphogenetic proteins (BMPs) are representative of this role, given that they have been identified as key components of a permissive microenvironment that triggers stem cell transformation into cancer stem cells (CSCs) and allows for transformed cell persistence, as well as tumor progression [76]. Therefore, studying the microenvironment has become an important focus of interest in cancer research and, accordingly, we included it in the proposed battery of assays for the characterization of the oncogenic phenotype. The use of 3D (co)cultures is arguably the most suitable in vitro approach for determining the role of the microenvironment in tumor progression, as it allows for the stimulation of the tumor and stroma context by including ECM and different cell types in the model [77]. Nonetheless, here, we presented different alternatives for the study of the secretome in a 2D culture context, which is more adaptable for long-term exposure settings. The conditioned media (CM) of starved cells is representative of the set of factors composing the secretome. Different approaches can be followed to analyze the CM and its potential influence on surrounding cells. The indirect soft-agar assay (Figure 4) allows for a global analysis of the secretome at the functional level. To proceed, the CM of the long- term exposed cells is mixed with the soft-agar substrate—a semisolid matrix containing agar and cell culture media [78]—to grow a model tumoral cell line (e.g., HCT-116 or HeLa cells) for 15 days. These cells are prone to colony formation on soft-agar; therefore, changes in colony number or size indicate an effect of the CM in promoting surrounding cell growth [79]. At a molecular level, changes in the levels of inflammatory mediators, such as IL-6, IL-10, TFG-β or TNF-α, among others, are indicative of an inflammatory microenvironment [80], which potentially contributes to cell transformation and tumor promotion, given the influence of chronic inflammation on cancer onset [81]. Therefore, using arrays for the evaluation of large sets of cytokine and soluble factors present in the CM is an informative approach (e.g., the human XL cytokine array kit from R&D Systems). The matrix metalloproteinases (MMPs) are ECM-remodeling enzymes sometimes represented in this kind of array. Increased MMP secretion and activity have been described in several types of cancers associated with poor prognosis, given their key role in ECM degradation, leading to angiogenesis and invasiveness promotion from the early stages of tumor progression [82,83]. The MMP activity in the cells’ CM can be specifically analyzed with substrate zymography. In that assay, proteins are run in an SDS-PAGE gel containing CM is an informative approach (e.g., the human XL cytokine array kit from R&D Systems). The matrix metalloproteinases (MMPs) are ECM-remodeling enzymes sometimes repre- sented in this kind of array. Increased MMP secretion and activity have been described in several types of cancers associated with poor prognosis, given their key role in ECM deg- Int. J. Mol. Sci. 2023, 24, 7851 radation, leading to angiogenesis and invasiveness promotion from the early stages 9of of 26 tumor progression [83,84]. The MMP activity in the cells’ CM can be specifically analyzed with substrate zymography. In that assay, proteins are run in an SDS-PAGE gel containing a specific substrate, a specific namely, substrate, gelatin, namely, casein gelatin, or collagen. casein In this or collagen. Instep, the proenzyme this step, is ac- is the proenzyme tivated, triggering the degradation of the substrate. Then, following a simple process activated, triggering the degradation of the substrate. Then, following a simple process of renaturation, development and staining, the areas of high protease activity can of renaturation, development and staining, the areas of high protease activity can be be identi- fied as clear bands identified against as clear bandsa against dark background [85]. Although a dark background [84]. MMP-2 Although andMMP-2 9 are the andmost 9 are typically the most used markers, typically usedmodifications in the zymography markers, modifications techniquestechniques in the zymography have beenhave imple-been mented to increase implemented the resolution to increase for the for the resolution analysis of other the analysis ofMMPs such as other MMPs MMP-1, such 8 or 13 as MMP-1, 8 or [86]. 13 [85]. Figure 4. Growth Figure 4. Growthof of tumoral cells tumoral (Hela cells oror (Hela HCT116) HCT116)ininthe theindirect indirectsoft-agar soft-agarassay assayin inthe the presence presence of of CM collected from the culture of non-transformed or transformed CM collected from the culture of non-transformed or transformed cells. cells. 3.1.2. 3.1.2. Intermediate Intermediate Biomarkers Biomarkers Anchorage-Independent Growth Anchorage-Independent Growth In healthy tissues, cell-to-cell and cell–ECM adhesions are essential for maintaining In healthy tissues, cell-to-cell and cell–ECM adhesions are essential for maintaining homeostasis and development. However, during the transformation process, cells with an homeostasis and development. However, during the transformation process, cells with an active EMT program adopt a more mesenchymal state by losing E-cadherin, increasing the active EMT program adopt a more mesenchymal state by losing E-cadherin, increasing expression of N-cadherin and altering the integrin expression profile [86]. Due to these the expression of N-cadherin and altering the integrin expression profile [87]. Due to these changes, transformed cells acquire the ability to grow independently of anchorage. In changes, transformed cells acquire the ability to grow independently of anchorage. In ep- epithelial tissue, this means that cells attach to the collagen in the ECM rather than to ithelial tissue, this means that cells attach to the collagen in the ECM rather than to the the basement membrane, contributing to ECM remodeling, which, eventually, facilitates basement membrane, contributing to ECM remodeling, which, eventually, facilitates tu- tumoral cell dissemination [87]. This scenario can be reliably mimicked by the soft-agar moral assay,cell dissemination considered [88]. This a stringent scenario marker of in can vitrobemalignant reliably mimicked by the To transformation. soft-agar perform as- this say, considered a stringent marker of in vitro malignant transformation. assay, cells are seeded in a semisolid matrix composed of two layers produced by mixing To perform this assay, cells aremedia cell culture seededand in aagar semisolid matrixconcentrations. at different composed of two Thelayers produced base layer has aby mixing higher agar cellconcentration, culture mediawhile and agar at different concentrations. The base layer has a the top layer has less agar and contains the cells (Figure 5). After an higher agar concentration, while of incubation period theapproximately top layer has less agar only 21 days, and contains cells withthe cells (Figurephenotype a transformed 5). After ancan incubation grow and expand, forming colonies [78,88]. The usefulness of the soft-agar assay hascan period of approximately 21 days, only cells with a transformed phenotype been grow and in proved expand, forming multiple studiescolonies [79,89]. testing the The usefulness potential carcinogenic of the soft-agar effects assayagents, of different has beensuch proved in multiple as tobacco smoke studies components,testing the potential where carcinogenic transforming biomarkers effects haveofbeen different agents, associated with such as tobacco several smoke of components components, where transforming the gene signature, biomarkers have such as the AKT/mTOR been pathway signaling associated that with wasseveral components dramatically of the[89]. activated geneArsenic signature, such asas exposure, thea AKT/mTOR well-known signaling pathway human carcinogenic that was dramatically inductor, activated was also shown [90]. Arsenic to induce alterationsexposure, as a well-known in the ability human carcino- to grow independent of anchor- genic inductor, was also shown to induce alterations in the ability age in different cell lines [79,90], and this ability grew in parallel with the to grow independent accumulation of of anchorage in different cell oxidatively damaged DNA [91]. lines [80,91], and this ability grew in parallel with the accumu- lation of oxidatively damaged DNA [92]. Int. Int.J.J.Mol. Mol.Sci. 2023,24, Sci.2023, 24,7851 7851 10 of 10 of 26 26 Figure5.5.Representation Figure Representation of ofcell cellcolony colonygrowth growthin inthe thedirect directsoft-agar soft-agarassay assaycomparing comparingthe theresult result before and after transformation. before and after transformation. MigrationPotential Migration Potential Inan In anintermediate intermediate stage of malignant malignanttransformation, transformation,cells cellsthat thatundergo undergoEMT EMT activa- acti- vation, morphologicalchanges tion, morphological changesand andthetheloss loss ofof substrate attachment initiate the the invasion– invasion– metastasis metastasiscascade. cascade.This Thisinvolves involvesaasuccession successionof ofevents eventsbeginning beginningwith withaalocal localinvasion invasionatat the thetumor tumorsite, site,followed followedby byextravasation extravasationinto intodistant distanttissues tissuesandandthe theeventual eventualformation formation of ofmetastatic metastaticlesions lesionsthat thatcancangenerate generatesecondary secondary tumors tumors [92]. [93]. Therefore, Therefore, in in the the progres- progres- sive activation sive activation of the cell metastatic potential, two distinct processes can metastatic potential, two distinct processes can be identified: (i) be identified: (i) cell cell migration, migration, defined defined as as thethe ability ability of cells of cells to move to move on aon a substrate, substrate, suchsuch as the asintact the intact basal basal membrane membrane or theorECM, the ECM, andcell and (ii) (ii) invasion, cell invasion, which which involves involves the degradation the degradation of theof sub- the substrate infiltrated strate infiltrated by bythethe cells cells [93]. [94]. In Invitro-transformed vitro-transformedcells cellsalso alsoprogressively progressivelyacquireacquiremetastatic metastaticpotential. potential.The The migra- migra- tion ability of cells can be evaluated with previously reviewed diverse tion ability of cells can be evaluated with previously reviewed diverse approaches [95,96]. approaches [94,95]. One Oneof ofthe themost mostused usedmethodologies methodologiesto toassess assessthethecell cellmigration migrationpotential potential isis the the wound- wound- healing healing assay or scratch assay, consisting of creating a scratch in the confluentmonolayer assay or scratch assay, consisting of creating a scratch in the confluent cell cell mono- and layerperiodically capturing and periodically images images capturing to monitor cell movement to monitor into the scratched cell movement area [96–98]. into the scratched area Different modifications of this protocol have been proposed to increase [97–99]. Different modifications of this protocol have been proposed to increase the repro- the reproducibility of the assay,ofsuch ducibility as using the assay, suchsilicone inserts as using to increment silicone inserts tothe consistency increment the of the scratchof[99] consistency the and using automated imaging systems [100] or video scratch [100] and using automated imaging systems [101] or video microscopy microscopy [101], which ensure [102], the continuous which ensure the monitoring continuous of cell migration monitoring ofincell identical migration sections. Another in identical typically sections. used Another approach is the Boyden chamber assay or transwell migration typically used approach is the Boyden chamber assay or transwell migration assay, where assay, where transwell inserts are placed in the culture wells to create a chamber with an apical part separated transwell inserts are placed in the culture wells to create a chamber with an apical part from the basolateral compartment by a porous membrane. Cells are generally seeded on separated from the basolateral compartment by a porous membrane. Cells are generally the apical side in a starvation medium, while a medium containing a chemoattractant seeded on the apical side in a starvation medium, while a medium containing a chemoat- or a higher serum concentration is added to the basal part (Figure 6). Therefore, cells tractant or a higher serum concentration is added to the basal part (Figure 6). Therefore, with an active migration potential translocate vertically through the porous membrane. cells with an active migration potential translocate vertically through the porous mem- To determine the migration rate, cells attached to the basal side of the membrane and brane. To determine the migration rate, cells attached to the basal side of the membrane those that detached and reached the basal compartment should be quantified, for which and those that detached and reached the basal compartment should be quantified, for colorimetric or cell-counting methods can be applied [97]. Using these approaches, the which colorimetric or cell-counting methods can be applied [98]. Using these approaches, migration rate of cells can be quantified at a low cost and with an easy setup and readout, the migration rate of cells can be quantified at a low cost and with an easy setup and although plenty of alternative methodologies exist, such as the impedance-based real-time readout, although migration measurement plenty[102] of alternative or single-cellmethodologies tracking [103]. exist, such as the impedance-based real-time migration measurement [103] or single-cell tracking [104]. Int. J. Mol. Sci. 2023, 24, 7851 11 of 26 Int. Int.J.J.Mol. Mol.Sci. 2023,24, Sci.2023, 24,7851 7851 11 of 11 of 26 26 Figure 6. Comparison of the Boyden chamber migration assay in transformed and non-transformed cells. Figure 6. Comparison Comparison 3.1.3. Advanced Figure 6. of the the Boyden Biomarkers of Boydenchamber chambermigration migrationassay assayin intransformed transformedand andnon-transformed non-transformedcells. cells. Invasion 3.1.3. 3.1.3.AdvancedPotential Advanced Biomarkers Biomarkers Invasion Potential mentioned, during the transformation process, cells progressively ac- As previously Invasion Potential quire Asmetastatic previously potential. mentioned, One of the advanced during stages of theprocess, the transformation invasion–metastasis cascade cells progressively and Asthe of previously carcinogenic mentioned, process during is the the transformation acquisition of invasion process, potential.cellsInvading progressively cells ac- can acquire metastatic potential. One of the advanced stages of the invasion–metastasis cascade quire metastatic penetrate potential. tissue barriers One of by degrading the advanced stages basal membranes of the and invasion–metastasis the ECM, which allows cascadefor and of the carcinogenic process is the acquisition of invasion potential. Invading cells can andtumor the of the carcinogenic cell infiltration process of is the acquisition secondary tissues of invasion [94]. Therefore, potential. the Invading invasion cells can potential can penetrate tissue barriers by degrading basal membranes and the ECM, which allows for penetrate be the considered tumor tissue cell barriers of a biomarker infiltration bysecondary ofdegrading an advanced basal tissues and membranes [93].aggressive Therefore, and the phenotype. the ECM, which invasion allows potential can befor the tumor cell Cell invasion considered infiltration a biomarker can be of secondary ofevaluated an advanced tissues following [94]. Therefore, similar inphenotype. and aggressive the invasion potential vitro approaches to those selected can be forconsidered the Cellmigration invasion a biomarker analysis, can of but be evaluated anincorporating advanced following and aggressive some similar kind in phenotype. of vitro ECM-like substrate approaches that the to those selected cells for Cell themust invasion degrade migration can analysis, be to move evaluated butthrough following the surface. incorporating similar some kind in To apply vitrotheapproaches of ECM-like Boyden to those chamber substrate selected invasion that the cells for the assay, migration the setup isanalysis, identical but to incorporating the transwell some migration must degrade to move through the surface. To apply the Boyden chamber invasion assay,kind of assay, ECM-like with the substrate exception that thea that cells layer must of a degrade synthetic to move matrix through mimicking the the surface. ECM is To apply added on the setup is identical to the transwell migration assay, with the exception that a layer of a the top Boyden of the chamber porous invasion membrane assay, (Figurethe synthetic 7). setup matrix is identical Onlymimicking invasive cellstodegrade the the transwell ECM the ECM is added migration on layer top ofand assay, the withmembrane translocate porous the exception through the that (Figure mem-7).a layer brane, Only of a synthetic while other invasive matrix cells arethe cells degrade mimicking blocked ECM layer the from andECM is migrating added translocate on top [98,105,106]. of through The the porous membrane impedance-based the membrane, while (Figure real-time other cells7).migration Only are invasive blocked fromcells measurement degrade migrating [103] the ECM layer or single-cell [97,104,105]. The and tracking translocate [104] can through impedance-based also themigra- be adapted real-time mem- to brane, measure tion while measurement other[102] cell invasion cellsbyorare blocked adding single-cellan ECMfrom tracking migrating coating [103]oncan [98,105,106]. thealso surface. The impedance-based Further, be adapted to for those cells measure cell real-time invasion able by to form migration adding spheroids, measurement an ECM severalcoating [103] on or variations single-cell theofsurface. spheroid tracking Further, invasion [104] for can cells those protocols alsocanbe adapted able be to form found to in measure spheroids, cell the literature. invasion several by variations Globally, adding an of spheroid to follow ECM coating invasioncells this approach, on the protocols surface. are grown can be Further, for those found in the literature. as multicellular cells spheroids able to form Globally, and embedded spheroids, to follow intothisECM several approach, variations cells are gels. After 72–96 ofhspheroid grown of as invasion multicellular incubation, protocols spheroids invading cellscan and be found embedded or cell clustersin the into literature. ECM spread outgels. Globally, on theAfter to follow 72–96 hbodies, spheroid this approach, of incubation, cells invading cells while noninvasive are grown cells or as multicellular cell clusters remain, spheroids spreadthe maintaining outcom- on andspheroid the pact embedded spheroid into ECM bodies, structurewhile gels. After noninvasive [107,108]. It is72–96cellsh remain, important of incubation, invading maintaining to point out thiscells that the compact kind orofcell clusters spheroid 3D model spread structure out better mimics on physiological the spheroid [106,107]. bodies, It is important tissue to while pointnoninvasive structures outthanthat2D thiscells kind remain, approaches,of 3D as maintaining model betterthe cell-to-cell com- mimics contacts pact spheroid physiological structure [107,108]. It is important to point are stronger and there is an irregular distribution of oxygen and nutrients. Moreover,and tissue structures than 2D approaches, as out cell-to-cell that this contacts kind are of 3D stronger model dif- better ferentismimics there an irregular cell types physiological candistribution tissue be incorporated of structures oxygen into the and than 2D approaches, nutrients. spheroids, Moreover, aiming as different cell-to-cell to reproduce a cell morecontacts types real- are can stronger isticbetumor and there incorporated is an into microenvironment theirregular spheroids, distribution and analyze aiming toofreproduce oxygen and the stromal/tumor nutrients. acell more Moreover, realistic interaction tumor during mi- dif- cell ferent cell[109]. croenvironment invasion typesandcan analyze be incorporated into the spheroids, the stromal/tumor aimingduring cell interaction to reproduce a more[108]. cell invasion real- istic tumor microenvironment and analyze the stromal/tumor cell interaction during cell invasion [109]. Figure 7.7. Representation Figure Representationofofthe theBoyden chamber Boyden invasion chamber assay invasion withwith assay transformed and non-trans- transformed and non- formed cells. transformed cells. Figure 7. Representation of the Boyden chamber invasion assay with transformed and non-trans- formed cells. Int. J. Mol. Sci. 2023, 24, 7851 12 of 26 Int. J. Mol. Sci. 2023, 24, 7851 12 of 26 Stem-like Features Stem-like Features Accumulating evidence supports the idea that cancer stem cells (CSCs) are a normal Accumulating evidence supports the idea that cancer stem cells (CSCs) are a normal constituent of many tumors, contributing to their heterogeneity. CSCs are a subpopula- constituent of many tumors, contributing to their heterogeneity. CSCs are a subpopulation tion of cells that present or acquire stem-cell-like properties, capable of self-renewal, but of cells that present or acquire stem-cell-like properties, capable of self-renewal, but also also divide asymmetrically, giving rise to the bulk of the tumor [110]. In the tumor micro- divide asymmetrically, giving rise to the bulk of the tumor [109]. In the tumor microenvi- environment, both nontumoral and differentiated tumoral cells interact with CSCs, mod- ronment, both nontumoral and differentiated tumoral cells interact with CSCs, modulating ulating key oncogenic events, such as the tumor growth, metastatic potential, treatment key oncogenic events, such as the tumor growth, metastatic potential, treatment resistance resistance and tumor and tumor [110]. recurrence recurrence [111]. During the in vitro carcinogenesisprocess, During the in vitro carcinogenesis process,tumoral tumoralstem-like stem-likecells cellscan canbebeenriched enriched andidentified and identifiedwith withthe thetumorsphere tumorsphereformation formationassay. assay.ToToreach reachsuch suchan anaim, aim,aalow lowdensity density of cells is seeded in ultra-low attachment plates under specific culture conditions of cells is seeded in ultra-low attachment plates under specific culture conditions achieved achieved byadding by addingseveral severalgrowth growthfactors factorsto tothe theserum-free serum-freemedia. media.Under Undersuchsuchconditions, conditions,tumor tumor spheres are formed [112,113]. These are solid, spherical structures easily spheres are formed [111,112]. These are solid, spherical structures easily distinguishable distinguishable fromcell from cellaggregates, aggregates,because becauseno noindividual individualcellscellscan canbebeidentified identified(Figure (Figure8). 8).The Thenumber number oftumorspheres of tumorspheresformed formedcan canbebeused usedto toestimate estimatethetheproportion proportionof ofstem-like stem-likecells cellspresent present in a population of transformed cells. Tumorspheres derived in vitro in a population of transformed cells. Tumorspheres derived in vitro from tumoral cells from tumoral cells have been described to be highly invasive and to initiate tumorigenesis have been described to be highly invasive and to initiate tumorigenesis when engrafted when engrafted invivo in vivo[113]. [114].Thus, Thus, thethe acquisition acquisition of thisof sphere-forming this sphere-forming capability capability is considered is considered a markera marker of aggressiveness and CSC enrichment in the in vitro cell-transformed of aggressiveness and CSC enrichment in the in vitro cell-transformed population. population. Figure8.8.Tumorsphere Figure Tumorsphereformation formation assay assay in ultra-low in ultra-low attachment attachment plates plates (ULA). (ULA). OnlyOnly transformed transformed cells cells form tumorspheres. form tumorspheres. Asaasummary As summaryof ofthe theprevious previoussections, sections, we we included included aa table table(Table (Table1) 1)showing showingthethe different described different described biomarkers biomarkers of of in invitro vitrotransformation, transformation,thetheproposed proposed assays assays and and the the referencescontaining references containingthe theprotocols protocolsexplaining explaininghow howthethedifferent differentassays assayscan canbe be carried carried out. out. Table1.1.Methodologies Table Methodologiesfor forthe theevaluation evaluationofofthe theoncogenic oncogenicbiomarkers. biomarkers. Biomarkers of In Vitro Trans- Biomarkers of In Vitro Transformation Assays Assays Protocols Protocols formation DNA damage and Comet assay [42–45] DNA damage and Comet assay [43–46] genotoxicity Micronucleus assay [46–48] genotoxicity Micronucleus assay [47–49] Population-doubling timePopulation-doubling time [55] [54] Uncontrolled pro- Metabolic assays Metabolic assays [56–58] [55–57] Uncontrolled proliferation liferation DNA synthesis and quantification assaysand quantification assays DNA synthesis [59,60] [58,59] Early bi- biomarkers Early Real-time monitoring of cell proliferation Real-time [61,62] monitoring of cell proliferation [60,61] omarkers Morphological Microscopic imaging Microscopic imaging [66–68] [65–67] changes Morphological changes Ultrastructural imaging (TEM and SEM) [72,73] Ultrastructural imaging (TEM and SEM) [71,72] Indirect soft-agar assay [79,80] Secretome altera- Indirect soft-agar assay [78,79] Cytokine arrays [115] tions Secretome alterations Cytokine arrays [114] Zymogram [85] Zymogram [84] Int. J. Mol. Sci. 2023, 24, 7851 13 of 26 Table 1. Cont. Biomarkers of In Vitro Transformation Assays Protocols Anchorage-independent Soft-agar assay [78,88] growth Wound-healing assay [96–101] Intermediate biomarkers Boyden chamber assay [97] Migration potential Impedance-based real-time migration assay [102] Single-cell tracking [103] Boyden chamber assay [97,104,105] Impedance-based real-time migration assay [102] Invasion potential Advanced biomarkers Single-cell tracking [103] Spheroid invasion assay [106–108] Stem-like feature acquisition Tumorsphere formation assay [111,112] 4. Representative Studies Using the Proposed Approach to Evaluate In Vitro Carcinogenesis In the literature, we could find several studies that successfully characterized the tumoral phenotype of cells by applying the in vitro long-term exposure approach together with the analysis of oncogenic biomarkers. In this section, we presented representative works developed in the context of the carcinogenic risk evaluation. The selected studies applied a low range of exposures lasting for at least 6 weeks on different in vitro systems to draw a conclusion on the cell-transforming potential of certain environmental exposures based (at least partially) on two or more of the assays previously presented here. Several of the chosen studies focused on chemicals. Interestingly, the use of in vitro systems to study carcinogenesis has allowed for characterizing the carcinogenic potential of classical environmental pollutants such as arsenic. Thus, a study carried out [115] showed that 26 weeks of low-concentration chronic arsenic exposure increased the proliferation and anchorage-independent growth of target pulmonary cells (A549) and suggested that cell transformation was mediated by increased levels of intracellular reactive oxygen species (ROS) and related signaling. The involvement of oxidative stress in the arsenic- induced transformation was evaluated by Bach et al. [79], who showed that oxidative DNA damage-sensitive mouse embryonic fibroblasts (MEF Ogg1−/− ) were more prone to transformation than their wild-type counterparts (MEFs Ogg1+/+ ) after 40 weeks of subtoxic arsenic exposure. MEF Ogg1−/− cells acquired a clear spindle-like morphology and increased the proliferation, while this effect was not observed in the exposed MEFs Ogg1+/+ . Although both cell types acquired anchorage-independent growth and tumor-promoting abilities through the increased secretion of MMP2 + 9, DNA damage-sensitive cells were more prone to the transformation being positive for the biomarkers analyzed 10 weeks earlier during the exposure period. That study highlighted the relevance of oxidative damage in arsenic-induced carcinogenesis and, further, allowed for the creation of a cell biobank with the established cell model, which was, subsequently, used to expand the mechanistic explanation by describing the role of FRA1 [32] and MTH1 [33] in the process. Additionally, in this context, the work by Weinmuellner et al. [116] reported that after 24 weeks of chronic arsenic trioxide (ATO) exposure, immortalized human keratinocytes acquired a spindle-like morphology, migration potential and an enhanced sphere-forming capacity. The acquisition of these oncogenic traits was observed in parallel to an increase in the ROS levels, which, again, was evidence of arsenic-induced oxidative stress. Further, the development of oncogenic features was confirmed by observing that the transformed cells were able to form tumors in SCID mice. However, tumor formation in the SCID mice was transient, which brought the authors to conclude that chronic exposure to low-level arsenic concentrations led to the development of a mild malignant phenotype that may Int. J. Mol. Sci. 2023, 24, 7851 14 of 26 require other factors (e.g., UV radiation or mechanic stress) for a full-blown malignant transformation. Interestingly, the in vitro long-term exposure approach was also applied to identify the chemopreventive ability of black tea extract (BTE) in a BTE and arsenic coexposure scenario [117]. In that study, skin keratinocyte cells (HaCaTs) exposed to arsenic for 34 weeks showed clear signs of transformation, while cells coexposed to arsenic and BTE for the same period did not develop such evident malignant features. Among the hallmarks altered by arsenic exposure, the authors described accelerated proliferation; modified cell morphology associated with the loss of contact inhibition; an altered expression of EMT markers (Vimentin, Snail, Slug, Twist, Zeb and N-cadherin); elevated MMP2 + 9 expression and activity, explored with Western blot and zymography, respectively; increased migration potential observed both through the wound-healing and transwell-based approaches; and high invasion potential evidenced by using the transwell invasion assay. However, all these transformation signs were ameliorated in the BTE + arsenic exposure conditions, which indicated the potential chemopreventive role of BTE in arsenic-induced carcinogenesis. The carcinogenic potential of other chemicals has also been evaluated following the long-term/low-concentration exposure approach. Here, we highlighted some studies that focused on chemicals with very diverse applications. As a first example, aluminum salts (AlCl3 ) are frequently present in industrial products of daily use, including food additives and cosmetics. In vitro studies using murine and human mammary cell models have shown that long-term exposure to AlCl3 induces alterations in hallmark traits in the cells. Mandriota et al. [28] showed that normal murine mammary gland epithelial cells suffered morphological changes and acquired the ability to grow independent of anchorage in the soft-agar assay, as well as the ability to form large metastatic tumors in immunodeficient mice. In the same direction, Sappino et al. [118] described that AlCl3 induced double-strand breaks and increased proliferation, the loss of contact inhibition and anchorage-independent growth of human mammary epithelial cells (MCF-10A). The use of mammary cells was previously found to be useful by Zou and Matsumura [119], who used MCF-7 cells to compare the effects of exposure to the pesticide contaminant β- hexachlorocyclohexane (β-HCH) with those induced by estrogen 17b-estradiol (E2 ), which was used as a positive control due to its protumorigenic effects in breast tissue. The authors evaluated the cellular phenotype at different time points. After 7 months of exposure, only E2 induced the alteration of oncogenic biomarkers. Nonetheless, the exposure to both E2 and β-HCH for 143 months triggered important phenotypical changes in the cells, including enhanced levels and activity of MMP-9 and increased anchorage-independent growth and invasiveness. Taken together, these studies evidenced that selecting relevant target models and time points allows to identify the potential detrimental effects that challenge the safety of widespread chemicals, in these cases regarding the pathogenesis and progression of breast cancer. Other interesting studies have focused on the long-term in vitro effects of tobacco components. As an example, two components of second-hand smoke, namely, nicotine (Nic) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), were shown to have an additive effect compared with the carcinogenicity of the compounds separately. Fararjeh and colleagues [120] exposed human breast epithelial cells (HBL-100) to very low concentrations of Nic and NNK alone or in combination for 9 weeks. The results showed that tumor progression features emerged earlier in coexposed cells, which also acquired a more marked tumoral phenotype with significant increases in cell proliferation, anchorage- independent growth, migration and stem-cell-like properties. Similarly, the compound 4-aminobiphenyl (4-ABP), which is another component of tobacco smoke, as well as of cooking oil and dyes, was found to induce liver carcinogenesis in an HBx Src (p53−/− ) transgenic zebrafish model after four months of exposure; nevertheless, when wild-type zebrafish were used, the exposure required seven months to show its tumoral potential [121]. Interestingly, when the authors carried out an in vitro study using hepatic cell lines (like HepG2 and L-02), significant increases in cell proliferation, the number of colonies formed in the soft-agar assay and the number of migrating cells in the transwell migration assay were observed. These effects were reported after exposures lasting for 8 weeks to 10 nM Int. J. Mol. Sci. 2023, 24, 7851 15 of 26 4-ABP. The enhancement of the oncogenic biomarkers was observed in a concentration- independent manner likely due to the stronger toxicity of 4-ABP concentrations above 10 nM. As another example, when L-02 liver cells were exposed for up to 20 passages to musk xylene, which is a common component of personal care products, high levels of cell proliferation, altered cell morphology and an increased anchorage-independent growth were observed. It is interesting to remark that when the clones obtained in the soft-agar assay were expanded, they showed very significant migration and invasion potentials. The authors also reported that these in vitro transformation effects were found in parallel to the repression of the TGF-β pathway [122]. It is of note that the application of the long-term exposure approach has also proven useful in identifying the impact of chemicals on the cells’ response to alterations in their surrounding microenvironment. Clément et al. [123] exposed a model of mammary stem cells (MCF10A) to very low concentrations of bisphenol A (BPA) or benzo(a)pyrene (B(a)P) for 8.5 weeks. The results showed that the exposure primed the cells’ response to BMPs, which, as previously mentioned, are key regulators of stem cells and their niche, and play a role in the initiation of stem cell transformation [124,125]. While BPA and B(a)P did not significantly change the phenotype of the cells, the long-term exposure enhanced the protumoral effects induced by high levels of BMPs, including a deregulated proliferation and tumorsphere formation ability [123]. Therefore, that work suggested that chemical-induced alterations both in the stem cells and their microenvironment could synergistically drive cell transformation. The use of in vitro models for a carcinogenic assessment has also contributed greatly to the field of nanoparticle safety evaluation. Nanoparticles (NPs) are becoming increas- ingly present in consumer products and medical applications due to their advantageous physicochemical properties. The diversity and complexity of these materials call for the development of novel methods with high-throughput potentials, such as the approach presented here. We were able to find several examples in the literature where a long- term/low-concentration approach was applied to evaluate the transforming potential of different NPs. Thus, Annangi and coworkers used the DNA damage-sensitive cells intro- duced previously (MEF Ogg1−/− ) to be chronically exposed to cobalt (CoNPs), zinc oxide (ZnONPs) and ZnCl2 (as the ionic form of zinc). After 12 weeks of exposure, CoNPs induced the malignant transformation of cells, changing their morphology, increasing MMP2 + 9 secretion and promoting colony formation in the soft-agar assay [126]. However, when cells were exposed to both ZnONPs and ZnCl2 under the same exposure scenario, no increases in the selected oncogenic biomarkers were observed. These negative effects were observed despite the toxic potential found upon acute exposure [127]. On the contrary, ZnONPs were reported to induce the malignant transformation of colonic mucosal cells (IMCEs), which showed hyperproliferation, morphological changes, an anchorage-independent growth ability in the soft-agar assay and migration potential (as shown with the wound-healing assay). In addition to the different biomarkers analyzed in the in vitro carcinogenesis study, the tumoral phenotype of the cells after 30 passages of exposure was confirmed, given their tumorigenic potential when subcutaneously administered to nude mice [128]. These conflicting results highlighted the importance of cell model selection, given that different cell types may present different levels of sensitivity to a specific exposure. Interestingly, lung cell models are often used in the nanotoxicology field, since in- halation is considered one of the main routes of NP exposure. Thus, bronchial epithelial cells (BEAS-2B) have previously been selected as models in several studies reporting on the in vitro carcinogenic potential of diverse NPs. Wang and collaborators [27] performed a comprehensive study of the carcinogenic potential of single-walled carbon nanotubes (SWCNTs), which have a resemblance to asbestos. In this work, BEAS-2B cells were chroni- cally exposed to a low concentration of SWCNTs for 6 months. The long-term exposure triggered the acceleration of cell doubling, induced morphological changes, increased the anchorage-independent growth in the soft-agar assay, enhanced the migration and inva- sion potential in transwell-based assays and, interestingly, promoted angiogenic activity. Once the cells had achieved a malignant in vitro phenotype, they were subcutaneously Int. J. Mol. Sci. 2023, 24, 7851 16 of 26 injected into nude mice, resulting in the formation of tumors that did not develop in the control mice. Further, the authors explored p53 signaling, which has been suggested as a potential mechanism driving the carcinogenic effect of SWCNTs. Regarding other NPs generally deemed as safe, it was found that 6 weeks of exposure to CeO2 NPs, cigarette smoke condensate (CSC) and the combination of both resulted in an altered cell transfor- mation status of BEAS-2B cells in a concentration-dependent manner. More importantly, the coexposure induced an enhanced malignant phenotype, as shown by the assessment of early, intermediate [129] and advanced in vitro transformation biomarkers [130]. Similarly, the same cell model was selected by Gliga et al. [131] to evaluate the transforming effects of NiNPs and their derivatives (NiONPs and NiCl2 , as the ionic form) after exposures lasting for 6 weeks. Although the exposure induced significant DNA damage and gene expression changes, only a tendency to increase the number of colonies and the proportion of invading cells was identified with the soft-agar and the transwell invasion assay, respectively. A more recent study reported on the transforming potential of NiNPs in the same cell line, but upon a longer period of exposure. Just two biomarkers of in vitro cell transforma- tion were analyzed after 21 weeks of exposure, and both were positive, namely, DNA damage and anchorage-independent growth. Nonetheless, a defect in the DNA repair (HIF- 1α/miR-210/Rad52 pathway) was identified as a contributing mechanism of Ni-induced carcinogenesis and confirmed with knock-out approaches (of the hypoxia-inducible fac- tor 1 (HIF) and mir-210), which significantly reduced the cell transformation effects [132]. Lastly, BEAS-2B cells have also been used to evaluate the protective effects of nanoparticle coatings. The work by Kornberg et al. [133] compared the transforming effects of a specific type of iron oxide NPs (Fe2 O3 NPs), the same particle, but coated with amorphous silica (SiO2 -Fe2 O3 NPs), and gas metal arc welding fumes generated from mild steel welding (GMA-MS), which contained insoluble iron. After 28 weeks of low-concentration exposure to Fe2 O3 NPs, the exposed cells presented a similar phenotype to that induced by GMA-MS. This was characterized by accelerated cell proliferation, the induction of DNA damage and anchorage-independent colony formation. These biomarkers were not significantly altered in BEAS-2B cells exposed to SiO2 -Fe2 O3 NPs, which highlighted the importance of the physicochemical properties of NPs in their associated risks. Regarding the potential of the in vitro carcinogenesis tools as a first approach to fill in current knowledge gaps, of note is their usefulness in determining potential health effects associated with exposures to emergent relevant pollutants, such as micro/nanoplastics (MNPLs). The field of MNPL risk assessments is currently in expansion, although most of the reported studies focus on short-term endpoints. Few publications using long-term exposures were found in the literature, and they mostly focused specifically on oxidative stress induction and the genotoxic aspect of the exposure [134–136]. Nonetheless, the carcinogenic potential of polystyrene nanoplastics (PSNPLs) has been explored using a sensitive pretransformed cell model called prone-to-transformation progress (PTP) cells, derived from the previously mentioned DNA damage-sensitive MEFs. This cell line is especially sensitive to oxidative damage induction. The results obtained using this cell line showed that a 12-week coexposure to PSNPLs and arsenic enhanced the malignant features of the phenotype, significantly increasing the proportion of spindle-like cells, the levels of DNA damage, the colony-formation ability in soft-agar and the percentage of migrating and invading cells. Interestingly, PSNPLs and arsenic alone did not induce the promotion of intermediate and advanced transformation biomarkers [137]. However, PSNPL exposure for an extended period (up to 24 weeks) did induce several cancer hallmarks, such as anchorage-independent cell growth, promoted cell migration and invasion ability, as well as an increase in the stem-cell-like population. Furthermore, several micro-RNAs, stress- related genes and pluripotency markers were also abnormally expressed in the same cell type [114]. Table 2 summarizes the main characteristics of the studies reported in this section. Int. J. Mol. Sci. 2023, 24, 7851 17 of 26 Table 2. Studies that applied the in vitro carcinogenesis approach for safety evaluation. Early Biomarkers Intermediate Biomarkers Advanced Biomarkers Time of DNA Uncontrolled Anchorage- Sem-Like Transformed Reference Agent Cell Line Concentration Morphological Secretome Migration Invasion Exposure Damage and Prolifera- Independent Features Phenotype Changes Alterations Potential Potential Genotoxicity tion Growth Acquisition DNA 12 weeks 0.05 µg/mL + + + Yes [126] CoNPs damage-sensitive (3 months) 0.1 µg/mL + + + Yes MEFs ZnONPs DNA - - - - No 12 weeks [127] damage-sensitive 1 µg/mL (3 months) ZnCl2 MEFs - - - - No MEFs - - + + Mild DNA 40 weeks [79] As 0.5–2 µM damage-sensitive (9 months) + + + + Yes MEFs CeO2 NPs + + Yes 2.5 µg/mL CSC 6 weeks CeO2 + + Yes [130] BEAS-2B (1.5 months) 5 µg/mL CeO2 NPs + CSC + + Yes CSC PSNPLs Prone-to- 25 µg/mL + - + - - - - Mild 12 weeks [137] As transformation PSNPLs + - + - - - - Mild (3 months) progress MEFs 2 µM As PSNPLs + As + - + + + + - Enhanced Prone-to- 24 weeks [114] PSNPLs transformation 25 µg/mL + + + + Yes (6 months) progress MEFs 0.25 µM + + Yes 26 weeks [115] As BEAS-2B 1 µM + + Yes (6.5 months) 5 µM + + Yes BPA +* + Enhanced 8.5 weeks 10−10 M BPA [123] MCF-10A (2 months) 10−10 M B(a)P B(a)P +* + Enhanced Nic - + + - + Yes 9 weeks 1 mM Nic [120] NKK HBL-100 100 femtM - + + + + Yes (2 months) NNK Nic + NKK + + + + + Enhanced As + + + + + Yes 34 weeks 100 nM As [117] HaCaT (8 months) 1 µM BTE As + BTE - - - - - Prevented Int. J. Mol. Sci. 2023, 24, 7851 18 of 26 Table 2. Cont. Early Biomarkers Intermediate Biomarkers Advanced Biomarkers Time of DNA Uncontrolled Anchorage- Sem-Like Transformed Reference Agent Cell Line Concentration Morphological Secretome Migration Invasion Exposure Damage and Prolifera- Independent Features Phenotype Changes Alterations Potential Potential Genotoxicity tion Growth Acquisition NiNPs + - +* - +* Mild 6 weeks [131] NiONPs BEAS-2B 0.5 µg/mL + - +* - +* Mild (1.5 months) NiCl2 + - +* - +* Mild Fe2 O3 NPs + + + Yes SiO2 - 28 weeks [133] BEAS-2B (6.5 months) 2.88 µg/mL - - - No Fe2 O3 NPs GMA-MS + + + Yes L-02 8 weeks + + + Yes [127] 4-ABP 10 nM HepG2 (2 months) + + + Yes NMuMG epithelial 16 weeks [126] AlCl3 100 µM - + + Yes cells (4 months) [128] ZnONPs IMCE 30 passages 1 µg/mL + + + + Yes 0.25 µg/mL + + Yes 21 weeks [132] NiNPs BEAS-2B (5 months) 0.5 µg/mL + + Yes CeO2 NPs - - - - - No 2.5 µg/mL CSC 6 weeks CeO2 - + + ** + + Yes [129] BEAS-2B (1.5 months) 5 µg/mL CeO2 NPs + CSC + + + + + Yes CSC 9 weeks [118] AlCl3 MCF-10A 10–300 µM + + + + Yes (2 months) 24 weeks [27] SWCNT BEAS-2B 0.02 µg/cm2 + + + + + Yes (6 months) 0.05 µM + - + Mild Immortalized human 24 weeks [116] ATO 0.1 µM + + + Mild keratinocytes (6 months) 0.25 µM + + + Mild Int. J. Mol. Sci. 2023, 24, 7851 19 of 26 Table 2. Cont. Early Biomarkers Intermediate Biomarkers Advanced Biomarkers Time of DNA Uncontrolled Anchorage- Sem-Like Transformed Reference Agent Cell Line Concentration Morphological Secretome Migration Invasion Exposure Damage and Prolifera- Independent Features Phenotype Changes Alterations Potential Potential Genotoxicity tion Growth Acquisition 10 µg/L + + + + + Yes [122] Musk xylene L-02 20 passages 100 µg/L + + + + + Yes 1000 µg/L + + + + + Yes E2 1 nM E2 + + + Yes [119] 56 weeks 100 nM MCF-7 (13 months) + + + Yes B-HCH β-HCH 1 µM B-HCH + + + Yes +: significantly increased result for the respective biomarker; -: unchanged result for the respective biomarker; *: no statistically significant result, but a clear tendency was described; **: disparity in the results of two different assays performed to evaluate the same oncogenic biomarker. Int. J. Mol. Sci. 2023, 24, 7851 20 of 26 5. Conclusions and Further Steps Our proposal solved the old topic of how to use in vitro approaches to determine the potential carcinogenic risk of environmental agents/exposures. Due to the complexity of the tumoral process, trying to identify it by using a simple endpoint (foci formation) seems to be a bit naïve; thus, the proposed/prevalidated CTAs have not yet received the general approval of the scientific community. Consequently, few studies have been carried out/published using these approaches. This is a serious issue, given that everybody agrees that detecting the potential genotoxic and/or carcinogenic risk of environmental agents, including those of emergent concern, is an urgent task. In this context, we consider that our proposal of using a structured battery of assays, detecting different biomarkers representative of known cancer hallmarks, is undoubtedly useful in covering the existing gaps. This proposal could pose a framework inspiring those researchers involved in hazard assessments, mainly focusing on cancer risk assessments. It is obvious that an in vitro approach does not completely mimic all the changes that occur in a complex in vivo model, as in mammals. Nevertheless, as a screening tool, our proposal could identify agents with carcinogenic potential, as well as their potential mechanisms of action. According to the reported data, further in vivo approaches could be planned if required. In addition to the well-recognized mechanisms underlying all the proposed biomark- ers of in vitro transformation, our proposal focused on the need of using long-term ap- proaches to determine such effects. In a good experimental approach, extending exposures for several weeks, ideally between 15 and 30 weeks, are necessary. It is important to main- tain constant exposure conditions and concurrent controls to distinguish if the observed effects were due to the exposure or to the long-term growth conditions. Moreover, it would be very useful to retain a biobank of cells at different time points (e.g., every 5 weeks) to identify the evolution of the different biomarkers or, ideally, to be used in the future when new and more sophisticated tools are available. To better simulate a real exposure scenario, long-term exposures should be associated with low-concentration (nontoxic) exposures. Thus, the low concentrations and the long-term exposures are a binomial that should preside over these types of experiments looking for the in vitro carcinogenicity potential of environmental agents/exposures. The selection of these concentrations should consider the cytotoxicity induced by the agent in the cell system of choice, and, if available, the data on the agent’s environmental and/or internal exposure levels. Thus, the selected range of concentrations should set a maximum limit at the concentration able to cause cytotoxicity, and should be coherent with a realistic human exposure scenario. Another advantage of our approach was the possibility to desist the use of rodent cells, such as those used in the prevalidated CTAs. For our proposal, any type of human cell can be selected, although generally aiming to use a cell line representative of the main target of the evaluated agent, if known (e.g., lungs for inhalation exposures). As ingestion and inhalation are the most important exposure routes, cells representative of such tissues can be selected as the first exposure targets. Nevertheless, if the final target (e.g., the kidneys) is known, representative cells from such organs can also be used. This flexibility permits the design of experimental approaches far from the corset of using only one type of cell line. Independent of the selected target cells, an interesting point to be highlighted is that to increase the sensitivity of the assays, cell lines deficient in specific pathways associated with the expected mechanisms of action of the evaluated agents could be very useful. In fact, this approach was used in the prevalidated CTAs. Thus, for example, since the induction of oxidative stress is a mechanism associated with the mode of the action of different carcinogens, the use of cell lines deficient in their ability to repair oxidative damage could be a good option. In addition, using models of human stem cells is of major importance for long-term effect assessments, as this rare subset of cells is the only one to persist in the body for many years, allowing abnormalities to be accumulated. In the field of cancer, this implies that stem cells are major targets from which transformed cells emerge. Outside this specific disease, stem cells are gatekeepers of tissue homeostasis, and perturbing their function likely impairs many aspects of human physiology. These Int. J. Mol. Sci. 2023, 24, 7851 21 of 26 are only examples of the many possibilities that this kind of in vitro approach provides. Following this approach, researchers can explore multiple options, including different agents or mixtures, concentrations, times of exposure, endpoints and mechanisms of action. All of this would ideally fill in urgent knowledge gaps in exposure science and contribute to the emergence of an alternative strategy for conducting exposure risk assessments. Author Contributions: R.M. and A.H. planned the overall content of the paper. I.B. carried out the writing of the first draft version. V.M.-S., B.G. and S.P. collaborated to create the final version. R.M. and A.H. edited the final version. All authors have read and agreed to the published version of the manuscript. Funding: This project received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no 965196. This work was partially supported by the Spanish Ministry of Science and Innovation (PID2020-116789, RB-C43) and the Generalitat de Catalunya (2021-SGR-00731). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data will be available on request. Acknowledgments: A. Hernández was granted an ICREA ACADEMIA award. S. Pastor is a Serra Húnter Fellow. Conflicts of Interest: The authors declare no conflict of interest. References 1. EPA/600/R-14/004Next Generation Risk Assessment: Incorporation of Recent Advances in Molecular, Computational, and Systems Biology; EPA: Washington, DC, USA, 2014. Available online: https://cfpub.epa.gov/si/si_public_record_report.cfm?Lab=NCEA& dirEntryId=286690 (accessed on 5 December 2022). 2. Rusche, B. 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