Public Health Weekly Report 2025; 18(4): 197-221
Published online December 17, 2024
https://doi.org/10.56786/PHWR.2025.18.4.3
© The Korea Disease Control and Prevention Agency
Ryeo-Eun Go , Kyoungin Na
, Su-Min Seong
, Ye-Ji Kang
, Yong Ae Jeong
, Younjhin Ahn *
Division of Climate Change and Health Hazard, Department of Health Hazard Response, Korea Disease Control and Prevention Agency, Cheongju, Korea
*Corresponding author: Younjhin Ahn, Tel: +82-43-219-2950, E-mail: carotene@korea.kr
This is an Open Access aritcle distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) which permits unrestricted distribution, and reproduction in any medium, provided the original work is properly cited.
Cigarette use is a health hazard that has been reported to reduce life expectancy, increase the risk of death, and result in high socioeconomic costs. New cigarettes increase the ratio of users and reduce the perception of harm to cigarettes by adding liquid nicotine, flavor, and taste. However, aerosols from cigarettes are presumed to be non harmless vapors. Because health effects greatly depend on smokers’ behaviors, the risk of smoking cannot be used to assess the content of harmful chemicals. Accordingly, we conducted an experiment based on behavioral surveys of smokers to evaluate the health effects of exposure to harmful substances. Following this global trend, alternative animal testing methods are proposed to ban animal testing. Therefore, we reviewed the necessity of alternative animal testing for smoking hazards. Organoids are three-dimensional organized cell-derived organs that can be studied in tissues. Therefore, lung organoids can be used as biological tools for smoking hazard research. Six research papers on the current situation of smoking hazard using lung-organoids have been confirmed. These papers discuss the effects of smoking on lung organoids production or pathway study of lung disease by smoking based on the chronic obstructive pulmonary disease. We examined lung organoid-related studies on lung diseases. Eventually, we will apply the results of exposure to harmful substances following smokers’ behaviors from experiments.
Key words Smoking hazard; Cigarette; Electronic nicotine delivery systems; Lung-organoids; Lung disease model
Previously, experiments on smoking hazards have been conducted using cell and animal models for inhalation exposure. Organoids are known to overcome the limitations of cell-organ interactions and ethical issues in animal experiments.
Lung organoids can reproduce parts of organ, depending on their constituent cells. Disease-specific lung organoids can be generated using chemicals, thereby inducing changes in gene expression and stem cells.
The lung organoid model can regenerate complex cellular structures and interactions, thereby mimicking the major diseases caused by smoking, may be used in smoking research.
Smoking and second-hand smoke are major causes of health issues (Table 1). Continued smoking among lung cancer patients results in a poor prognosis, including an increased risk of recurrence and reduced treatment effectiveness. According to the “Comprehensive Report on the Harms of Tobacco” by the Korea Disease Control and Prevention Agency (KDCA), smoking directly causes approximately 58,000 deaths annually, with associated socioeconomic costs amounting to 12 trillion won [1]. Republic of Korea (ROK) has implemented tobacco control measures, including a national smoking cessation policy, which successfully reduced the smoking rate among adult males from 66% in 1998 to 31% in 2021 [2]. However, with the introduction of new tobacco products such as electronic nicotine delivery systems (ENDS) (2008) and heated tobacco products (HTPs) (2017), the behaviors of cigarette users have been changing significantly. ENDS allow users to customize their nicotine levels, scents, and flavors, which enhances their appeal and potential for abuse (dependence). Additionally, sweet flavors and aromas are known to diminish users’ perceptions of tobacco-related harm [3]. According to the “Report on the Harms of Tobacco: New Tobacco Products” by the KDCA, the usage rate of ENDS among individuals in their 20s rose from 1% in 2013 to 8% in 2021. Moreover, HTPs sales increased from 440 million packs in 2021 to 540 million packs in 2022, marking a 21.3% rise [4]. However, aerosols released by new tobacco products are not simply harmless water vapor; they contain nicotine, carbonyl compounds, volatile organic compounds, propylene glycol, and flavoring additives. These substances differ from those found in traditional cigarettes and can be harmful [4]. Additionally, recent ENDS contain synthetic nicotine, which is produced through an artificial process [5]. However, the impact of smoking on health varies significantly depending on an individual’s smoking habits, and the only presence of harmful chemicals, such as carcinogens, is not, by itself, sufficient to fully assess the extent of harm caused by smoking to the human body. Therefore, behavioral surveys are essential to understand changes in smoking behaviors and habits, as well as experiment-based research to gather scientific evidence on the effects of smoking on health at various levels of exposure.
Class | Disease causing by directly smoking | Disease causing by indirectly smoking |
---|---|---|
Cancer | Lung cancer, esophageal cancer, head and neck cancer, pancreatic cancer, stomach cancer, colon cancer, cervical cancer, etc. | Lung cancer, etc. |
Cardiovascular disease | Myocardial infarction, ischemic heart disease, aortic aneurysm, heart failure, disease-related heart attack, stroke, cerebral aneurysm, etc. | Coronary artery disease, stroke, worsening of heart disease, etc. |
Respiratory disease | Chronic obstructive pulmonary disease, asthma, tuberculosis | Aggravation of asthma and lung disease, adolescent lung dysfunction, cold, pneumonia, acute lower respiratory disease, etc. |
Digestive diseases | Crohn's disease, irritable bowel syndrome, gastroesophageal reflux disease, etc. | - |
Reproductive diseases | Congenital malformations, fetal development disorders, hypertensive diseases during pregnancy, male sexual dysfunction, etc. | Reduced female reproductive function, increased risk of sudden infant death syndrome |
Eye disease | Increased risk of cataracts, macular degeneration, etc. | - |
Other disease | Periodontal disease, diabetes, rheumatoid arthritis | Increased risk of otitis media |
To date, studies on the harmful effects of smoking have mostly been cell studies or animal experiments that focus on inhalation toxicity testing. However, the United States and Europe have revised their laws to mandate the assessment of the effects of smoking using in vitro alternatives to animal testing. In the ROK, the legislative process for the “Revision of Alternatives to Animal Experiments” is also in progress. Therefore, a thorough review of the laws concerning alternatives to animal testing is needed. Proposed alternatives to animal testing include organoids, which are three-dimensional (3D) structures of cells derived from organs and stem cells. Organoids have been used to study various aspects of cellular processes, such as cell-to-cell interactions, cell development, and homeostasis, and have been applied in disease modeling [6]. The current literature review summarizes the existing literature on lung organoid models for studying the harms of smoking and lung diseases.
Studies on the harmful effects of smoking on lung organoids were searched using relevant keywords in PubMed, a major medical database. Studies published in the last 10 years on lung organoids and smoking-related terms (e.g., smoke or cigarette) were included in the search. For the search method, a ‘systemic literature review’ was used [7]. A search using ‘lung organoids’ as a keyword identified 1,327 studies; however, when ‘smoke’ or ‘cigarette’ were added as additional keywords, the number of relevant studies decreased to 26. After reviewing the titles, abstracts, and full texts, six papers that conducted experiments examining the harmful effects of smoking using lung organoids were identified (Figure 1). Studies examining lung organoids in relation to smoking focused on lung cancer and chronic obstructive pulmonary disease (COPD). These studies explored the effects of smoking on lung organoid development and the underlying mechanisms of smoking-induced lung disease.
In a study on lung cancer, three months of intermittent exposure to cigarette smoke induced lung adenocarcinoma and severe emphysema in mice. After dissecting the lung tissue from the mice to produce lung organoids, it was found that the organoids from the group intermittently exposed to cigarette smoke were more efficiently developed than those from the group that was continuously exposed. A study reported that fatty acid oxidation is induced in type 2 alveolar cells derived from mice directly exposed to mainstream smoke, leading to increased stemness1) in lung cells. These observations indicate a potential positive correlation between stemness and tumor malignancy [8,9]. Furthermore, a study identified a mechanism of the lung emphysema development based on lung organoids derived from mice exposed to cigarette smoke [10]. It has also been found that cigarette smoke exposure inhibits the normal proliferation and recovery of lung epithelial cells, as well as the differentiation of type 2 alveolar cells and the formation of organoids [11].
A study on COPD found that cigarette smoke exposure induces COPD and related diseases in lung organoids derived from mouse fetuses, demonstrating a relationship between prenatal cigarette smoke exposure and COPD development [12]. Another study investigating the mechanisms involved in lung repair following cigarette smoke exposure revealed a significant reduction in both the size and number of alveolar cell organoids after 14 days of exposure to cigarette smoke extract in human and mouse lung organoids [13]. A study reported a reduction in the expression of liver kinase B1 (LKB1), a tumor suppressor gene, in the lungs of COPD patients and mice exposed to cigarette smoke. This lung organoid culture study demonstrated that suppressing LKB1 expression promotes cell differentiation in the airway and the expression of genes that induce macrophages. The study also confirmed that cigarette smoke is associated with mechanisms that increase mucus secretion in the airways and promote excessive cell proliferation, suggesting a correlation between LKB1 gene expression, smoking, and COPD [14].
Current research on lung organoids and smoking has primarily focused on lung cancer and COPD, as approximately 90% of individuals who die from these diseases are smokers [15]. These studies used lung organoids to investigate the mechanisms underlying the development of COPD and lung cancer, thereby elucidating the root causes of smoking-induced lung damage.
Given the limitations of cell models with short life cycles, air-liquid interface cultures with restricted microenvironment control, organ-on-a-chip models that require specialists and advanced technology, spheroid models that cannot replicate organ and vascular functions, and the ethical, economic, and interspecies heterogeneity issues in animal experiments, organoids have emerged as an ideal model for assessing human toxicity (Table 2) [16]. They can replicate organ structure and function and house a diverse range of cell types. Lung organoids utilize cells from various regions of the respiratory system and can replicate the structure, mucus secretion, ciliary movement, and regeneration of alveolar cells (Figure 2). Therefore, researchers have focused on lung organoids as a model for studying the lung, which is the first organ to be damaged by smoking [17]. In this context, the current study conducted a systematic literature review of research utilizing lung organoids to investigate major lung diseases, such as lung cancer, COPD, and pulmonary fibrosis (PF), aiming to evaluate the potential of lung organoids in studies on the harmful effects of smoking.
Class | Advantage | Disadvantage |
---|---|---|
2D cell lines | Economical test cost Easy reproducibility Simple and easy test method Test of a short time Easy high-throughput screening (HTS) | Limitation of the completely mimic primary cell Unstable gene Limitation of the test by short cell division and death cycle Limited number of models or types Low sensitivity to entering and replicating of viruses Limitation of interaction confirmation due to absence of stroma, blood vessels, inflammatory cells, etc. |
Air-liquid interface | Mimicking the structure and function of human tissue barriers Possibility of an alternative to animal testing such as skin toxicity Possibility of realistic exposure conditions | Non-standardization of culture and testing methods Limitations of in vivo structures reproduction Limitations of microenvironment control during culture and testing |
Organ-on-a-chip | Control of microenvironment during culture and testing Mimicking the action of organ-to-capillary | Non-standardization of culture and testing methods Requires experts, advanced technology, and long-term time Expensive cost due to complex design and manufacturing Impossible HTS |
Spheroids | Possibility to check cell-to-cell interactions and physiological effect Easy reproducibility Easy HTS | Difficulty of control the size Impossible mimicking the action of organ-to-capillary Complex of formation method |
Animal models | Possibility of understanding disease by physiological similarity to humans Confirmation of interaction due to stroma, blood vessels, inflammatory cells, etc. | High cost and labor Test of a long time Bioethical issues Genetic and anatomical differences on humans and animals |
Organoids | Reproduction of the structural and functional of major organs Possibility of diseases modeling Possibility of various cells complex Stable gene Long-term storage in Biobank Maintain the characteristics of the original species Useful of HTS | Non-standardization of culture and testing methods Lack of reproduction of blood vessels and immune system Limitations of microenvironment control Limitations on diffusion of nutrients and metabolites into organoids |
2D=two-dimensional.
Lung cancer is a leading cause of cancer-related deaths worldwide, and smoking is a major risk factor. Carcinogens and toxic substances in cigarette smoke affect all regions of the lungs. Numerous studies have confirmed the harmful effects of carcinogens in cigarette smoke, including nicotine, which induces addiction and affects genetic toxicity and the immune system; nitrosamines, cigarette-specific substances derived from nicotine that reduce reproductive function; polycyclic aromatic hydrocarbons, which induce gene mutations; benzene, which damages bone and impairs reproductive function; and acetaldehyde, which irritates the airway and skin [18-21]. However, given the complexity and variability of the mechanisms underlying the progression and treatment of lung cancer, further research is required. This study summarizes research using lung organoids through in vitro methods, which can serve as alternatives to in vivo models.
As presented in Figure 1, a systematic literature review was conducted to identify studies published in the past 10 years that focused on lung organoids in cancer research. A total of 611 studies were initially identified. After excluding literature reviews and carefully reviewing the titles, abstracts, and full texts, 42 studies were found to be suitable for inclusion, and their details are as follows.
Studies on lung cancer and organoids have employed lung cancer cells or tissues from patients with non-small cell lung cancer [22,23], lung adenocarcinoma [24,25], and squamous cell carcinoma [26] and have proposed efficient methods for producing and culturing these organoids [27-31]. Most studies have used lung cancer organoid models for high-throughput screening to select drug candidates for treating lung cancer and have proposed methods for this process [32-39]. Additionally, some studies have examined the underlying mechanisms of action of various anti-cancer drugs, including cisplatin [40], pyrotinib [41], alectinib [42], and halofuginone [43]. Other studies have used lung cancer organoids derived from patients to confirm their potential in identifying optimized treatments and to suggest their clinical applicability [44,45]. Other studies have also examined the general characteristics of lung cancer organoids [46,47], confirmed their feasibility as disease models [48,49], and explored their association with the immune system [50].
Lung cancer organoids preserve the genetic expression information of the originating source and can replicate the structure and microenvironment of lung cancer tissues. Therefore, they are actively used in research on the complexities of lung cancer and personalized treatment. Leveraging the characteristics of patient-derived lung cancer organoids facilitates the investigation of the impact of smoking on lung cancer development across diverse genetic profiles. These organoids offer a promising model for uncovering the underlying mechanisms by which smoking drives lung cancer progression.
COPD is a complex lung disorder accompanied by chronic bronchitis, emphysema, and mucorrhea, which results in damage to the alveolar parenchyma. New treatments and drug development for COPD depend heavily on preclinical models; however, these models have limitations, including high costs and an inability to accurately replicate the structural complexity of the human lung, which poses a challenge for research [51]. To address these limitations, there is ongoing research on the use of lung organoids to investigate the pathogenesis of COPD. This systematic literature review includes studies published in the past 10 years related to lung organoids and COPD. A total of 57 studies were reviewed, 22 of which were deemed suitable for inclusion. Details of these studies are summarized below.
Most studies on COPD have used type 2 alveolar cells to generate lung organoids. In these studies, inhibiting the expression of specific genes in lung organoids (such as rho-kinase 1/2 [52], frizzled receptor 4 [11], mitogenactivated protein kinase 13 [53,54], interleukin-1β [55], interleukin-11 [56], receptor-interacting protein kinase 3 [57], WNT-5A/5B [58], and LL-37 [59]) resulted in impaired fibroblast function, reduced regenerative capacity of damaged lung cells, and increased lung cell deformation. In one study, COPD lung organoids were derived from cells isolated from the nasopharynx and bronchi of patients with COPD. This model successfully reproduced key features of COPD, including goblet cell hyperplasia, reduced ciliary movement, and increased susceptibility to viruses and bacteria [56]. In another study, lung progenitor cells2) were isolated from cystic fibrosis patients and used to generate a COPD lung organoid model [60]. Additionally, it was reported that plastics inhaled from fibrous materials inhibit the differentiation of airway epithelial cells, impairing the repair of lung cells and potentially leading to COPD [61].
Most studies included in this systematic literature review suggested that diverse structural abnormalities in lung tissues, resembling those observed in fibrosis and indicating lung cell deformation and dysfunction, may contribute to the development of COPD. The exposure of organoids to mainstream/sidestream smoke, resulting in cell deformation and dysfunction, could provide valuable insights into various aspects of smoking-induced COPD pathogenesis.
PF is a lung disease characterized by repeated damage to the lung epithelium, leading to the hardening of lung tissue (fibrosis) and resulting in severe breathing difficulties. Drugs can delay fibrosis but cannot completely restore damaged lung cells. Smoking is a major cause of PF [62]. This systematic literature review searched for studies published in the last 10 years on lung organoids and PF. A total of 137 studies were reviewed, 42 of which were deemed suitable for inclusion. Given the similar mechanisms underlying COPD and PF, many studies grouped them together, and duplicate studies were excluded during the review. Key findings from these studies are summarized below.
Similar to studies on COPD, studies on PF have also used type 2 alveolar cells to generate lung organoids [63]. However, PF differs from COPD in that it is a chronic, progressive disease characterized by widespread accumulation of extracellular matrix, irreversible damage to alveolar cells, and cellular aging [64]. Studies in which PF was induced have primarily employed anti-cancer antibiotics, such as bleomycin and transforming growth factor-beta (TGF-β). Lung organoids treated with bleomycin replicate the pathogenic characteristics of PF, including epithelial cell-mediated activation of fibroblasts and cellular aging [65]. Additionally, exposing alveolar epithelial cells to TGF-β induces epithelial-mesenchymal transition, triggering the onset of PF [66]. The PF models are distinguished from others that they employed bioprinting to create 3D. Using bioprinting to create a 3D alveolar cell wall model and exposing it to fibrosis-inducing cytokines, a previous study induced structural changes, impaired alveolar cell function, and triggered epithelial-mesenchymal transition, thus validating the model as a suitable fibrosis model [67].
Studies developing PF models with lung organoids have employed various approaches, notably bioprinting, to create consistent PF models that overcome the limitations of traditional organoids. However, as no study has thoroughly investigated the mechanism by which smoking causes PF to be a chronic progressive disease, further research is needed.
Smoking damages type 2 alveolar cells. As viruses typically target airway epithelial and type 2 alveolar cells, the damage caused by smoking may be further exacerbated. Cigarette smoke disrupts the normal functioning of the immune system, weakening its antiviral defense [68]. This systematic literature review searched for studies published in the last 10 years related to lung organoids and viral infections. A total of 156 studies were reviewed, 63 of which were deemed eligible for inclusion.
Recent studies on respiratory viral infections using lung organoids have predominantly focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These studies used lung organoid models infected with SARS-CoV-2 to create a viral infection model, examining cellular structural changes along with alterations in protein and gene expression. Benzo[a]pyrene, a component of cigarette smoke, has been shown to increase the susceptibility of lung organoids and type 2 alveolar cells to SARS-CoV-2 infection [69]. Additionally, human lung organoids infected with respiratory syncytial virus (RSV) were used to replicate pathological changes, including alterations in the lung epithelium, cell structures, and syncytium formation, enabling the investigation of the underlying mechanisms [70]. A study examining the correlation between RSV infection and particulate matter (PM) reported increased DNA damage and apoptosis in lung organoids infected with RSV after PM exposure [71]. This finding suggests that external stress applied to virus-infected lung tissues can lead to severe lung damage. A study supports the finding that lung organoids, owing to their ability to replicate lung structures, are well suited for studying cellular responses resulting from interactions between the virus and the host [72].
As both smoking and viral infections target type 2 alveolar cells, this commonality highlights the value of using lung organoid models derived from these cells. Studies indicate that smokers are exposed to high levels of PM, which can trigger varying toxicity responses depending on particle size [73]. Additionally, PM exposure has been shown to increase apoptosis in lung organoids infected with RSV, highlighting the need for further research on the correlation between smoking and respiratory viral infection. This systematic literature review underscores the need for additional research on the relationship between smoking and respiratory viral infections using lung organoids.
Although smoking is known to induce various diseases, research investigating its mechanisms through animal experiments and cell models encounters physical, ethical, physiological, and biological limitations. To overcome these limitations, organoids have been proposed as a model, with the lung—the first organ damaged by smoking—being examined as the primary target. This study, which investigated the current state of research on the harms of smoking using lung organoids, found that most studies focused on organoid models related to lung cancer and COPD but did not incorporate animal experiments or utilize a diverse range of lung disease organoids. Given the high mortality rates from lung cancer and COPD, the studies lacked diversity in their approaches. Thus, this systematic literature review explored the applicability of lung organoids in research examining the harmful effects of smoking by reviewing existing studies on lung cancer, COPD, PF, and infectious respiratory diseases that utilized lung organoids.
The majority of studies have used lung cancer cells to develop organoids that replicate the structure of lung tissues or employed lung cancer organoids derived from the lung tissues of cancer patients. These organoids have been used in anti-cancer drug screening and to assess their clinical applicability in developing effective treatments. A COPD organoid model mimicking patient lung tissues was developed and used to identify genes contributing to COPD induction and to examine cell functional characteristics. PF has often been studied alongside COPD, but unlike COPD, it is characterized by extracellular matrix accumulation, irreversible damage to alveolar cells, and cellular aging, rather than changes in cell function. Studies have identified key chemicals that induce PF and have proposed 3D bioprinting as a solution to overcome the limitations of organoids. Research on lung organoids in response to the global SARS-CoV-2 pandemic is particularly active. One study examined the correlation between RSV infection and PM, confirming the need for further research on PM (fine PM, including tar) produced in large quantities during smoking. The findings from studies modeling lung cancer, COPD, PF, and viral respiratory infections using lung organoids highlight the potential of lung organoids for various applications in research on the harmful effects of smoking, including traditional cigarettes and new tobacco products.
The Smoking Harms Cell Laboratory of the KDCA has conducted research to identify biological markers in lung organoids derived from lung cancer patients and to develop lung organoids that replicate smoking-related lung diseases. In the study, organoids were isolated from surgical tissues of lung cancer patients, and culture conditions were optimized to confirm that the organoids originated from primary cancerous tumors. The cilia, a characteristic feature of lung cells, were examined, and next-generation sequencing was conducted to validate the lung organoid and lung cancer organoid models. Based on this study, research using lung organoids to overcome the limitations of traditional cell and animal models is expected to continue. Since the impact of smoking on health can vary based on personal use patterns, such as frequency, product type, and overall smoking habits, long-term follow-up studies on changes in smokers’ behaviors and the levels of harmful substances present in their bodies will be incorporated into experimental models of smoking-related harm.
1) Although most tissues have a limited capacity for repair following damage, excessive stemness can promote unlimited self-renewal. These findings suggest that when stemness is induced in cells other than cancer or stem cells, there is a high likelihood of these cells transforming into cancerous cells.
2) Cells in the stage prior to differentiating into specialized lung cells with their final function.
Ethics Statement: Not applicable.
Funding Source: None.
Acknowledgments: None.
Conflict of Interest: The authors have no conflicts of interest to declare.
Author Contributions: Conceptualization: REG. Data curation: SMS, YJK, YAJ. Supervision: YJA. Writing – original draft: REG. Writing – review & editing: KIN, YJA, REG.
Public Health Weekly Report 2025; 18(4): 197-221
Published online January 23, 2025 https://doi.org/10.56786/PHWR.2025.18.4.3
Copyright © The Korea Disease Control and Prevention Agency.
Ryeo-Eun Go , Kyoungin Na
, Su-Min Seong
, Ye-Ji Kang
, Yong Ae Jeong
, Younjhin Ahn *
Division of Climate Change and Health Hazard, Department of Health Hazard Response, Korea Disease Control and Prevention Agency, Cheongju, Korea
Correspondence to:*Corresponding author: Younjhin Ahn, Tel: +82-43-219-2950, E-mail: carotene@korea.kr
This is an Open Access aritcle distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) which permits unrestricted distribution, and reproduction in any medium, provided the original work is properly cited.
Cigarette use is a health hazard that has been reported to reduce life expectancy, increase the risk of death, and result in high socioeconomic costs. New cigarettes increase the ratio of users and reduce the perception of harm to cigarettes by adding liquid nicotine, flavor, and taste. However, aerosols from cigarettes are presumed to be non harmless vapors. Because health effects greatly depend on smokers’ behaviors, the risk of smoking cannot be used to assess the content of harmful chemicals. Accordingly, we conducted an experiment based on behavioral surveys of smokers to evaluate the health effects of exposure to harmful substances. Following this global trend, alternative animal testing methods are proposed to ban animal testing. Therefore, we reviewed the necessity of alternative animal testing for smoking hazards. Organoids are three-dimensional organized cell-derived organs that can be studied in tissues. Therefore, lung organoids can be used as biological tools for smoking hazard research. Six research papers on the current situation of smoking hazard using lung-organoids have been confirmed. These papers discuss the effects of smoking on lung organoids production or pathway study of lung disease by smoking based on the chronic obstructive pulmonary disease. We examined lung organoid-related studies on lung diseases. Eventually, we will apply the results of exposure to harmful substances following smokers’ behaviors from experiments.
Keywords: Smoking hazard, Cigarette, Electronic nicotine delivery systems, Lung-organoids, Lung disease model
Previously, experiments on smoking hazards have been conducted using cell and animal models for inhalation exposure. Organoids are known to overcome the limitations of cell-organ interactions and ethical issues in animal experiments.
Lung organoids can reproduce parts of organ, depending on their constituent cells. Disease-specific lung organoids can be generated using chemicals, thereby inducing changes in gene expression and stem cells.
The lung organoid model can regenerate complex cellular structures and interactions, thereby mimicking the major diseases caused by smoking, may be used in smoking research.
Smoking and second-hand smoke are major causes of health issues (Table 1). Continued smoking among lung cancer patients results in a poor prognosis, including an increased risk of recurrence and reduced treatment effectiveness. According to the “Comprehensive Report on the Harms of Tobacco” by the Korea Disease Control and Prevention Agency (KDCA), smoking directly causes approximately 58,000 deaths annually, with associated socioeconomic costs amounting to 12 trillion won [1]. Republic of Korea (ROK) has implemented tobacco control measures, including a national smoking cessation policy, which successfully reduced the smoking rate among adult males from 66% in 1998 to 31% in 2021 [2]. However, with the introduction of new tobacco products such as electronic nicotine delivery systems (ENDS) (2008) and heated tobacco products (HTPs) (2017), the behaviors of cigarette users have been changing significantly. ENDS allow users to customize their nicotine levels, scents, and flavors, which enhances their appeal and potential for abuse (dependence). Additionally, sweet flavors and aromas are known to diminish users’ perceptions of tobacco-related harm [3]. According to the “Report on the Harms of Tobacco: New Tobacco Products” by the KDCA, the usage rate of ENDS among individuals in their 20s rose from 1% in 2013 to 8% in 2021. Moreover, HTPs sales increased from 440 million packs in 2021 to 540 million packs in 2022, marking a 21.3% rise [4]. However, aerosols released by new tobacco products are not simply harmless water vapor; they contain nicotine, carbonyl compounds, volatile organic compounds, propylene glycol, and flavoring additives. These substances differ from those found in traditional cigarettes and can be harmful [4]. Additionally, recent ENDS contain synthetic nicotine, which is produced through an artificial process [5]. However, the impact of smoking on health varies significantly depending on an individual’s smoking habits, and the only presence of harmful chemicals, such as carcinogens, is not, by itself, sufficient to fully assess the extent of harm caused by smoking to the human body. Therefore, behavioral surveys are essential to understand changes in smoking behaviors and habits, as well as experiment-based research to gather scientific evidence on the effects of smoking on health at various levels of exposure.
Class | Disease causing by directly smoking | Disease causing by indirectly smoking |
---|---|---|
Cancer | Lung cancer, esophageal cancer, head and neck cancer, pancreatic cancer, stomach cancer, colon cancer, cervical cancer, etc. | Lung cancer, etc. |
Cardiovascular disease | Myocardial infarction, ischemic heart disease, aortic aneurysm, heart failure, disease-related heart attack, stroke, cerebral aneurysm, etc. | Coronary artery disease, stroke, worsening of heart disease, etc. |
Respiratory disease | Chronic obstructive pulmonary disease, asthma, tuberculosis | Aggravation of asthma and lung disease, adolescent lung dysfunction, cold, pneumonia, acute lower respiratory disease, etc. |
Digestive diseases | Crohn's disease, irritable bowel syndrome, gastroesophageal reflux disease, etc. | - |
Reproductive diseases | Congenital malformations, fetal development disorders, hypertensive diseases during pregnancy, male sexual dysfunction, etc. | Reduced female reproductive function, increased risk of sudden infant death syndrome |
Eye disease | Increased risk of cataracts, macular degeneration, etc. | - |
Other disease | Periodontal disease, diabetes, rheumatoid arthritis | Increased risk of otitis media |
To date, studies on the harmful effects of smoking have mostly been cell studies or animal experiments that focus on inhalation toxicity testing. However, the United States and Europe have revised their laws to mandate the assessment of the effects of smoking using in vitro alternatives to animal testing. In the ROK, the legislative process for the “Revision of Alternatives to Animal Experiments” is also in progress. Therefore, a thorough review of the laws concerning alternatives to animal testing is needed. Proposed alternatives to animal testing include organoids, which are three-dimensional (3D) structures of cells derived from organs and stem cells. Organoids have been used to study various aspects of cellular processes, such as cell-to-cell interactions, cell development, and homeostasis, and have been applied in disease modeling [6]. The current literature review summarizes the existing literature on lung organoid models for studying the harms of smoking and lung diseases.
Studies on the harmful effects of smoking on lung organoids were searched using relevant keywords in PubMed, a major medical database. Studies published in the last 10 years on lung organoids and smoking-related terms (e.g., smoke or cigarette) were included in the search. For the search method, a ‘systemic literature review’ was used [7]. A search using ‘lung organoids’ as a keyword identified 1,327 studies; however, when ‘smoke’ or ‘cigarette’ were added as additional keywords, the number of relevant studies decreased to 26. After reviewing the titles, abstracts, and full texts, six papers that conducted experiments examining the harmful effects of smoking using lung organoids were identified (Figure 1). Studies examining lung organoids in relation to smoking focused on lung cancer and chronic obstructive pulmonary disease (COPD). These studies explored the effects of smoking on lung organoid development and the underlying mechanisms of smoking-induced lung disease.
In a study on lung cancer, three months of intermittent exposure to cigarette smoke induced lung adenocarcinoma and severe emphysema in mice. After dissecting the lung tissue from the mice to produce lung organoids, it was found that the organoids from the group intermittently exposed to cigarette smoke were more efficiently developed than those from the group that was continuously exposed. A study reported that fatty acid oxidation is induced in type 2 alveolar cells derived from mice directly exposed to mainstream smoke, leading to increased stemness1) in lung cells. These observations indicate a potential positive correlation between stemness and tumor malignancy [8,9]. Furthermore, a study identified a mechanism of the lung emphysema development based on lung organoids derived from mice exposed to cigarette smoke [10]. It has also been found that cigarette smoke exposure inhibits the normal proliferation and recovery of lung epithelial cells, as well as the differentiation of type 2 alveolar cells and the formation of organoids [11].
A study on COPD found that cigarette smoke exposure induces COPD and related diseases in lung organoids derived from mouse fetuses, demonstrating a relationship between prenatal cigarette smoke exposure and COPD development [12]. Another study investigating the mechanisms involved in lung repair following cigarette smoke exposure revealed a significant reduction in both the size and number of alveolar cell organoids after 14 days of exposure to cigarette smoke extract in human and mouse lung organoids [13]. A study reported a reduction in the expression of liver kinase B1 (LKB1), a tumor suppressor gene, in the lungs of COPD patients and mice exposed to cigarette smoke. This lung organoid culture study demonstrated that suppressing LKB1 expression promotes cell differentiation in the airway and the expression of genes that induce macrophages. The study also confirmed that cigarette smoke is associated with mechanisms that increase mucus secretion in the airways and promote excessive cell proliferation, suggesting a correlation between LKB1 gene expression, smoking, and COPD [14].
Current research on lung organoids and smoking has primarily focused on lung cancer and COPD, as approximately 90% of individuals who die from these diseases are smokers [15]. These studies used lung organoids to investigate the mechanisms underlying the development of COPD and lung cancer, thereby elucidating the root causes of smoking-induced lung damage.
Given the limitations of cell models with short life cycles, air-liquid interface cultures with restricted microenvironment control, organ-on-a-chip models that require specialists and advanced technology, spheroid models that cannot replicate organ and vascular functions, and the ethical, economic, and interspecies heterogeneity issues in animal experiments, organoids have emerged as an ideal model for assessing human toxicity (Table 2) [16]. They can replicate organ structure and function and house a diverse range of cell types. Lung organoids utilize cells from various regions of the respiratory system and can replicate the structure, mucus secretion, ciliary movement, and regeneration of alveolar cells (Figure 2). Therefore, researchers have focused on lung organoids as a model for studying the lung, which is the first organ to be damaged by smoking [17]. In this context, the current study conducted a systematic literature review of research utilizing lung organoids to investigate major lung diseases, such as lung cancer, COPD, and pulmonary fibrosis (PF), aiming to evaluate the potential of lung organoids in studies on the harmful effects of smoking.
Class | Advantage | Disadvantage |
---|---|---|
2D cell lines | Economical test cost. Easy reproducibility. Simple and easy test method. Test of a short time. Easy high-throughput screening (HTS). | Limitation of the completely mimic primary cell. Unstable gene. Limitation of the test by short cell division and death cycle. Limited number of models or types. Low sensitivity to entering and replicating of viruses. Limitation of interaction confirmation due to absence of stroma, blood vessels, inflammatory cells, etc.. |
Air-liquid interface | Mimicking the structure and function of human tissue barriers. Possibility of an alternative to animal testing such as skin toxicity. Possibility of realistic exposure conditions. | Non-standardization of culture and testing methods. Limitations of in vivo structures reproduction. Limitations of microenvironment control during culture and testing. |
Organ-on-a-chip | Control of microenvironment during culture and testing. Mimicking the action of organ-to-capillary. | Non-standardization of culture and testing methods. Requires experts, advanced technology, and long-term time. Expensive cost due to complex design and manufacturing. Impossible HTS. |
Spheroids | Possibility to check cell-to-cell interactions and physiological effect. Easy reproducibility. Easy HTS. | Difficulty of control the size. Impossible mimicking the action of organ-to-capillary. Complex of formation method. |
Animal models | Possibility of understanding disease by physiological similarity to humans. Confirmation of interaction due to stroma, blood vessels, inflammatory cells, etc.. | High cost and labor. Test of a long time. Bioethical issues. Genetic and anatomical differences on humans and animals. |
Organoids | Reproduction of the structural and functional of major organs. Possibility of diseases modeling. Possibility of various cells complex. Stable gene. Long-term storage in Biobank. Maintain the characteristics of the original species. Useful of HTS. | Non-standardization of culture and testing methods. Lack of reproduction of blood vessels and immune system. Limitations of microenvironment control. Limitations on diffusion of nutrients and metabolites into organoids. |
2D=two-dimensional..
Lung cancer is a leading cause of cancer-related deaths worldwide, and smoking is a major risk factor. Carcinogens and toxic substances in cigarette smoke affect all regions of the lungs. Numerous studies have confirmed the harmful effects of carcinogens in cigarette smoke, including nicotine, which induces addiction and affects genetic toxicity and the immune system; nitrosamines, cigarette-specific substances derived from nicotine that reduce reproductive function; polycyclic aromatic hydrocarbons, which induce gene mutations; benzene, which damages bone and impairs reproductive function; and acetaldehyde, which irritates the airway and skin [18,,-21]. However, given the complexity and variability of the mechanisms underlying the progression and treatment of lung cancer, further research is required. This study summarizes research using lung organoids through in vitro methods, which can serve as alternatives to in vivo models.
As presented in Figure 1, a systematic literature review was conducted to identify studies published in the past 10 years that focused on lung organoids in cancer research. A total of 611 studies were initially identified. After excluding literature reviews and carefully reviewing the titles, abstracts, and full texts, 42 studies were found to be suitable for inclusion, and their details are as follows.
Studies on lung cancer and organoids have employed lung cancer cells or tissues from patients with non-small cell lung cancer [22,23], lung adenocarcinoma [24,25], and squamous cell carcinoma [26] and have proposed efficient methods for producing and culturing these organoids [27,,,-31]. Most studies have used lung cancer organoid models for high-throughput screening to select drug candidates for treating lung cancer and have proposed methods for this process [32,,,,,,-39]. Additionally, some studies have examined the underlying mechanisms of action of various anti-cancer drugs, including cisplatin [40], pyrotinib [41], alectinib [42], and halofuginone [43]. Other studies have used lung cancer organoids derived from patients to confirm their potential in identifying optimized treatments and to suggest their clinical applicability [44,45]. Other studies have also examined the general characteristics of lung cancer organoids [46,47], confirmed their feasibility as disease models [48,49], and explored their association with the immune system [50].
Lung cancer organoids preserve the genetic expression information of the originating source and can replicate the structure and microenvironment of lung cancer tissues. Therefore, they are actively used in research on the complexities of lung cancer and personalized treatment. Leveraging the characteristics of patient-derived lung cancer organoids facilitates the investigation of the impact of smoking on lung cancer development across diverse genetic profiles. These organoids offer a promising model for uncovering the underlying mechanisms by which smoking drives lung cancer progression.
COPD is a complex lung disorder accompanied by chronic bronchitis, emphysema, and mucorrhea, which results in damage to the alveolar parenchyma. New treatments and drug development for COPD depend heavily on preclinical models; however, these models have limitations, including high costs and an inability to accurately replicate the structural complexity of the human lung, which poses a challenge for research [51]. To address these limitations, there is ongoing research on the use of lung organoids to investigate the pathogenesis of COPD. This systematic literature review includes studies published in the past 10 years related to lung organoids and COPD. A total of 57 studies were reviewed, 22 of which were deemed suitable for inclusion. Details of these studies are summarized below.
Most studies on COPD have used type 2 alveolar cells to generate lung organoids. In these studies, inhibiting the expression of specific genes in lung organoids (such as rho-kinase 1/2 [52], frizzled receptor 4 [11], mitogenactivated protein kinase 13 [53,54], interleukin-1β [55], interleukin-11 [56], receptor-interacting protein kinase 3 [57], WNT-5A/5B [58], and LL-37 [59]) resulted in impaired fibroblast function, reduced regenerative capacity of damaged lung cells, and increased lung cell deformation. In one study, COPD lung organoids were derived from cells isolated from the nasopharynx and bronchi of patients with COPD. This model successfully reproduced key features of COPD, including goblet cell hyperplasia, reduced ciliary movement, and increased susceptibility to viruses and bacteria [56]. In another study, lung progenitor cells2) were isolated from cystic fibrosis patients and used to generate a COPD lung organoid model [60]. Additionally, it was reported that plastics inhaled from fibrous materials inhibit the differentiation of airway epithelial cells, impairing the repair of lung cells and potentially leading to COPD [61].
Most studies included in this systematic literature review suggested that diverse structural abnormalities in lung tissues, resembling those observed in fibrosis and indicating lung cell deformation and dysfunction, may contribute to the development of COPD. The exposure of organoids to mainstream/sidestream smoke, resulting in cell deformation and dysfunction, could provide valuable insights into various aspects of smoking-induced COPD pathogenesis.
PF is a lung disease characterized by repeated damage to the lung epithelium, leading to the hardening of lung tissue (fibrosis) and resulting in severe breathing difficulties. Drugs can delay fibrosis but cannot completely restore damaged lung cells. Smoking is a major cause of PF [62]. This systematic literature review searched for studies published in the last 10 years on lung organoids and PF. A total of 137 studies were reviewed, 42 of which were deemed suitable for inclusion. Given the similar mechanisms underlying COPD and PF, many studies grouped them together, and duplicate studies were excluded during the review. Key findings from these studies are summarized below.
Similar to studies on COPD, studies on PF have also used type 2 alveolar cells to generate lung organoids [63]. However, PF differs from COPD in that it is a chronic, progressive disease characterized by widespread accumulation of extracellular matrix, irreversible damage to alveolar cells, and cellular aging [64]. Studies in which PF was induced have primarily employed anti-cancer antibiotics, such as bleomycin and transforming growth factor-beta (TGF-β). Lung organoids treated with bleomycin replicate the pathogenic characteristics of PF, including epithelial cell-mediated activation of fibroblasts and cellular aging [65]. Additionally, exposing alveolar epithelial cells to TGF-β induces epithelial-mesenchymal transition, triggering the onset of PF [66]. The PF models are distinguished from others that they employed bioprinting to create 3D. Using bioprinting to create a 3D alveolar cell wall model and exposing it to fibrosis-inducing cytokines, a previous study induced structural changes, impaired alveolar cell function, and triggered epithelial-mesenchymal transition, thus validating the model as a suitable fibrosis model [67].
Studies developing PF models with lung organoids have employed various approaches, notably bioprinting, to create consistent PF models that overcome the limitations of traditional organoids. However, as no study has thoroughly investigated the mechanism by which smoking causes PF to be a chronic progressive disease, further research is needed.
Smoking damages type 2 alveolar cells. As viruses typically target airway epithelial and type 2 alveolar cells, the damage caused by smoking may be further exacerbated. Cigarette smoke disrupts the normal functioning of the immune system, weakening its antiviral defense [68]. This systematic literature review searched for studies published in the last 10 years related to lung organoids and viral infections. A total of 156 studies were reviewed, 63 of which were deemed eligible for inclusion.
Recent studies on respiratory viral infections using lung organoids have predominantly focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These studies used lung organoid models infected with SARS-CoV-2 to create a viral infection model, examining cellular structural changes along with alterations in protein and gene expression. Benzo[a]pyrene, a component of cigarette smoke, has been shown to increase the susceptibility of lung organoids and type 2 alveolar cells to SARS-CoV-2 infection [69]. Additionally, human lung organoids infected with respiratory syncytial virus (RSV) were used to replicate pathological changes, including alterations in the lung epithelium, cell structures, and syncytium formation, enabling the investigation of the underlying mechanisms [70]. A study examining the correlation between RSV infection and particulate matter (PM) reported increased DNA damage and apoptosis in lung organoids infected with RSV after PM exposure [71]. This finding suggests that external stress applied to virus-infected lung tissues can lead to severe lung damage. A study supports the finding that lung organoids, owing to their ability to replicate lung structures, are well suited for studying cellular responses resulting from interactions between the virus and the host [72].
As both smoking and viral infections target type 2 alveolar cells, this commonality highlights the value of using lung organoid models derived from these cells. Studies indicate that smokers are exposed to high levels of PM, which can trigger varying toxicity responses depending on particle size [73]. Additionally, PM exposure has been shown to increase apoptosis in lung organoids infected with RSV, highlighting the need for further research on the correlation between smoking and respiratory viral infection. This systematic literature review underscores the need for additional research on the relationship between smoking and respiratory viral infections using lung organoids.
Although smoking is known to induce various diseases, research investigating its mechanisms through animal experiments and cell models encounters physical, ethical, physiological, and biological limitations. To overcome these limitations, organoids have been proposed as a model, with the lung—the first organ damaged by smoking—being examined as the primary target. This study, which investigated the current state of research on the harms of smoking using lung organoids, found that most studies focused on organoid models related to lung cancer and COPD but did not incorporate animal experiments or utilize a diverse range of lung disease organoids. Given the high mortality rates from lung cancer and COPD, the studies lacked diversity in their approaches. Thus, this systematic literature review explored the applicability of lung organoids in research examining the harmful effects of smoking by reviewing existing studies on lung cancer, COPD, PF, and infectious respiratory diseases that utilized lung organoids.
The majority of studies have used lung cancer cells to develop organoids that replicate the structure of lung tissues or employed lung cancer organoids derived from the lung tissues of cancer patients. These organoids have been used in anti-cancer drug screening and to assess their clinical applicability in developing effective treatments. A COPD organoid model mimicking patient lung tissues was developed and used to identify genes contributing to COPD induction and to examine cell functional characteristics. PF has often been studied alongside COPD, but unlike COPD, it is characterized by extracellular matrix accumulation, irreversible damage to alveolar cells, and cellular aging, rather than changes in cell function. Studies have identified key chemicals that induce PF and have proposed 3D bioprinting as a solution to overcome the limitations of organoids. Research on lung organoids in response to the global SARS-CoV-2 pandemic is particularly active. One study examined the correlation between RSV infection and PM, confirming the need for further research on PM (fine PM, including tar) produced in large quantities during smoking. The findings from studies modeling lung cancer, COPD, PF, and viral respiratory infections using lung organoids highlight the potential of lung organoids for various applications in research on the harmful effects of smoking, including traditional cigarettes and new tobacco products.
The Smoking Harms Cell Laboratory of the KDCA has conducted research to identify biological markers in lung organoids derived from lung cancer patients and to develop lung organoids that replicate smoking-related lung diseases. In the study, organoids were isolated from surgical tissues of lung cancer patients, and culture conditions were optimized to confirm that the organoids originated from primary cancerous tumors. The cilia, a characteristic feature of lung cells, were examined, and next-generation sequencing was conducted to validate the lung organoid and lung cancer organoid models. Based on this study, research using lung organoids to overcome the limitations of traditional cell and animal models is expected to continue. Since the impact of smoking on health can vary based on personal use patterns, such as frequency, product type, and overall smoking habits, long-term follow-up studies on changes in smokers’ behaviors and the levels of harmful substances present in their bodies will be incorporated into experimental models of smoking-related harm.
1) Although most tissues have a limited capacity for repair following damage, excessive stemness can promote unlimited self-renewal. These findings suggest that when stemness is induced in cells other than cancer or stem cells, there is a high likelihood of these cells transforming into cancerous cells.
2) Cells in the stage prior to differentiating into specialized lung cells with their final function.
Ethics Statement: Not applicable.
Funding Source: None.
Acknowledgments: None.
Conflict of Interest: The authors have no conflicts of interest to declare.
Author Contributions: Conceptualization: REG. Data curation: SMS, YJK, YAJ. Supervision: YJA. Writing – original draft: REG. Writing – review & editing: KIN, YJA, REG.
Class | Disease causing by directly smoking | Disease causing by indirectly smoking |
---|---|---|
Cancer | Lung cancer, esophageal cancer, head and neck cancer, pancreatic cancer, stomach cancer, colon cancer, cervical cancer, etc. | Lung cancer, etc. |
Cardiovascular disease | Myocardial infarction, ischemic heart disease, aortic aneurysm, heart failure, disease-related heart attack, stroke, cerebral aneurysm, etc. | Coronary artery disease, stroke, worsening of heart disease, etc. |
Respiratory disease | Chronic obstructive pulmonary disease, asthma, tuberculosis | Aggravation of asthma and lung disease, adolescent lung dysfunction, cold, pneumonia, acute lower respiratory disease, etc. |
Digestive diseases | Crohn's disease, irritable bowel syndrome, gastroesophageal reflux disease, etc. | - |
Reproductive diseases | Congenital malformations, fetal development disorders, hypertensive diseases during pregnancy, male sexual dysfunction, etc. | Reduced female reproductive function, increased risk of sudden infant death syndrome |
Eye disease | Increased risk of cataracts, macular degeneration, etc. | - |
Other disease | Periodontal disease, diabetes, rheumatoid arthritis | Increased risk of otitis media |
Class | Advantage | Disadvantage |
---|---|---|
2D cell lines | Economical test cost. Easy reproducibility. Simple and easy test method. Test of a short time. Easy high-throughput screening (HTS). | Limitation of the completely mimic primary cell. Unstable gene. Limitation of the test by short cell division and death cycle. Limited number of models or types. Low sensitivity to entering and replicating of viruses. Limitation of interaction confirmation due to absence of stroma, blood vessels, inflammatory cells, etc.. |
Air-liquid interface | Mimicking the structure and function of human tissue barriers. Possibility of an alternative to animal testing such as skin toxicity. Possibility of realistic exposure conditions. | Non-standardization of culture and testing methods. Limitations of in vivo structures reproduction. Limitations of microenvironment control during culture and testing. |
Organ-on-a-chip | Control of microenvironment during culture and testing. Mimicking the action of organ-to-capillary. | Non-standardization of culture and testing methods. Requires experts, advanced technology, and long-term time. Expensive cost due to complex design and manufacturing. Impossible HTS. |
Spheroids | Possibility to check cell-to-cell interactions and physiological effect. Easy reproducibility. Easy HTS. | Difficulty of control the size. Impossible mimicking the action of organ-to-capillary. Complex of formation method. |
Animal models | Possibility of understanding disease by physiological similarity to humans. Confirmation of interaction due to stroma, blood vessels, inflammatory cells, etc.. | High cost and labor. Test of a long time. Bioethical issues. Genetic and anatomical differences on humans and animals. |
Organoids | Reproduction of the structural and functional of major organs. Possibility of diseases modeling. Possibility of various cells complex. Stable gene. Long-term storage in Biobank. Maintain the characteristics of the original species. Useful of HTS. | Non-standardization of culture and testing methods. Lack of reproduction of blood vessels and immune system. Limitations of microenvironment control. Limitations on diffusion of nutrients and metabolites into organoids. |
2D=two-dimensional..