Development of an integrated testing strategy using in vitro models to predict lung carcinogenesis

Min-Ju Kim; Cho Hee Park; Seung Min Oh

doi:10.5620/eaht.2025s04

Environ Anal Health Toxicol > Volume 40:2025 > Article

Kim, Park, and Oh: Development of an integrated testing strategy using in vitro models to predict lung carcinogenesis

Original Article

Environ Anal Health Toxicol 2025; 40: e2025s04.

Published online: April 4, 2025

DOI: https://doi.org/10.5620/eaht.2025s04

Development of an integrated testing strategy using in vitro models to predict lung carcinogenesis

Min-Ju Kim¹

, Cho Hee Park²

, Seung Min Oh^1,^3,^*

¹Department of Bio-application Toxicity, Hoseo University, Asan, Republic of Korea

²Nonclinical Research Institute, Orient Genia Incorporated, Seongnam, Republic of Korea

³Department of Animal Health and Welfare, Hoseo University, Asan, Republic of Korea

^*Correspondence: ohsm0403@hoseo.edu

Recommended by: Prof. Yong Joo Park

Received December 30, 2024 Accepted March 20, 2025

This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Carcinogenicity testing has traditionally been conducted using long-term animal studies, as specified in OECD TG 451 and 453 guidelines. These studies typically use rats and last for two years, requiring significant time and resources. Consequently, there is a pressing need to develop alternative toxicity testing methods that can efficiently predict lung cancer risks caused by chronic chemical exposure. In this study, we designed integrated testing strategies (ITS) to assess carcinogenesis by focusing on cell survival, clonal growth, and metastasis using the BEAS-2B cell model. Non-tumorigenic BEAS-2B cells were exposed to Benzo(a)pyrene (B(a)P), Ethyl carbamate (EC), Epichlorohydrin (ECH), and chloromethyl methyl ether (CMME) for 4 months (#40 passages). After treatment, the BEAS-2B cells showed enhanced anchorage-dependent and anchorage-independent colony formation. Furthermore, cell migration and invasion assays using transwell chambers revealed a significant increase in these malignant characteristics in treated BEAS-2B cells. Collectively, our findings demonstrate that prolonged exposure of non-tumorigenic BEAS-2B cells to B(a)P, EC, ECH, and CMME can lead to the acquisition of metastatic potential and multiple malignant characteristics. These integrated testing strategies for assessing carcinogenic potential could serve as a valuable tool for identifying unknown carcinogens.

Keywords: BEAS-2B cells, carcinogenesis, Integrated Testing Strategy (ITS), in vitro model

Introduction

Lung cancer mainly occurs in people over the age of 65 and is known as a cancer with a high mortality rate worldwide. In Korea, the 5-year survival rate is improving due to advances in diagnosis and treatment, but it is still low at 38.5% [1]. Although the major causes (80-90%) of lung cancer are due to long-term exposure to tobacco smoke, environmental pollutants and occupational exposure could be pivotal causes in lung cancer [2]. Carcinogenicity testing has been conducted through animal testing for a period corresponding to chronic toxicity such as OECD TG453 [3] and 451 [4]. When rats are used, experiments are conducted for two years and involve a lot of effort, especially in case of inhalation exposure. Therefore, there is a great need to develop alternative toxicity testing methods that can efficiently predict lung cancer toxicity that may occur when chronically exposed to chemicals.

Lung cancers can start in the cells lining the bronchi and parts of the lung such as the bronchioles or alveoli [5]. The lung epithelium plays an important role as a barrier, protecting the respiratory tract from toxic materials. Normal human bronchial epithelial BEAS-2B cell line is immortalized but not tumorigenic. Thus, BEAS-2B cell line has been used as a useful cell for predicting the carcinogenic effects for lung-targeting in humans [6, 7, 8, 9].

In this study, we designed integrated testing strategies (ITS) as alternative methods for assessing carcinogenesis, focusing on major characteristics such as cell survival, clonal growth, and metastasis using BEAS-2B cell model.

Benzo(a)pyrene (BaP), Ethyl carbamate (EC), epichlorohydrin (ECH), and chloromethyl methyl ether (CMME) are established lung carcinogen. BaP and CMME are classified as group 1 carcinogen by International Agency for Research on Cancer (IARC), while EC and ECH are categorized as Group 2A. Although these chemicals have been identified as lung carcinogens through various animal models and epidemiological studies, their carcinogenic mechanisms have not been fully elucidated through in vitro tests. Therefore, the in vitro carcinogenic potential of these four carcinogens was evaluated using the ITS designed in this study.

Materials and Methods

Chemicals

BaP (CAS# 50-32-8), EC (CAS# 51-79-6), ECH (1-chloro-2,3-epoxypropane, CAS# 106-89-8), CMME (CAS# 107-30-2) (Table 1), and nitroblue tetrazolium (NBT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All chemicals were dissolved in 100% dimethyl sulfoxide (DMSO) and stored at -20°C until use.

Cell culture conditions and chemical treatment

BEAS-2B cells (human normal bronchial epithelial cells), obtained from the American Type Culture Collection (Manassas, VA, USA), were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRL, NY, USA) supplemented with 5% fetal bovine serum (FBS, HyClone, TU, USA), penicillin (100 units/mL), and streptomycin (100 mg/mL). The cells were maintained in a humidified incubator at 37°C with 5% CO₂ and 95% air and subcultured approximately 10 times (#10 passage) per month. After confirming that the cells were fully attached following subculture, they were continuously exposed to the culture medium containing chemicals. Specifically, the cells were exposed to a positive control [benzo(a)pyrene, B(a)P] and three known lung carcinogens (EC, ECH, and CMME) for four months (up to passage #40) in the growth medium. No significant differences in carcinogenesis endpoints were observed in passages less than 40 (not shown data).

Cell viability test by crystal violet assay (SOP 1) and colony forming efficacy (SOP 2)

BEAS-2B cells at approximately 80% confluence were trypsinized and suspended in DMEM complete medium (DMEM containing 10% FBS). Crystal violet (CV) and colony-forming efficacy (CFE) assays [15] were commonly used to evaluate cytotoxicity resulting from chemical exposure. Additionally, these results were used to determine appropriate exposure concentrations for low-dose and long-term exposure studies.

Anchorage dependent colony formation assay (SOP 3)

BEAS-2B cells exposed to the test compounds were seeded in a 6-well culture plate at a density of 300 cells per 4 mL of 10% FBS DMEM complete medium. After 9 days, the cells were fixed with methanol and stained with a 0.04% Giemsa solution. Colonies containing more than 50 cells or with a diameter greater than 2 mm were scored through microscopic examination using an Olympus CX31 microscope (Japan) equipped with a CCD digital camera (IMTscan Cooled Model, Germany).

Anchorage independent colony formation assay (SOP 4)

The exposed BEAS-2B cells were harvested, and 3 × 10³ cells were suspended in 1 mL of 5% FBS DMEM medium containing 0.6% top agar. The cells were layered over a 0.5% agar base layer in a 12-well culture plate and maintained at 37°C in a 5% CO₂/95% air atmosphere incubator. After four weeks, the cells were stained overnight with 1 mg/mL NBT, and colonies with a diameter greater than 2 mm were scored through a microscopic examination using an Olympus SZX7×1.0 magnification.

Cell migration (SOP 5) and invasion assay (SOP 6)

In the treated BEAS-2B cells (#40 passages), migration/invasion assay was performed to evaluate whether the cells were metastatic. The test was performed using an uncoated or coated transwell chamber (6.5 mm diameter, 8 μm pore size, BD Biosciences, MA, USA). For the migration assay, cells (5×10⁵ cells/300 μL per well) were seeded in a serum-free medium in the upper chamber, with 1% FBS in the lower chamber. After 24 hours, migrated cells were stained and analyzed. For the invasion assay, Matrigel (Corning, MA, USA)-coated chambers were used, with cells seeded at the same density and 5% FBS in the lower chamber. After 48 hours, invaded cells were analyzed. Each stained cell was extracted using an ethanol-HCl solution for optical density measurement at 540 nm. The detailed experimental method was performed according to the report by Choo et al. (2016) [15].

Statistical methods

Each assay was performed at least three times. Data analysis was conducted using SigmaPlot 12.0 (Systat Software Inc., IL, USA) and Excel 2019 (Microsoft, Redmond, WA). Results were expressed as the mean ± SD. Statistical analysis was performed using PASW 18.0 (IBM-SPSS Inc., IL, USA), and differences between group were assessed using a two-sided Student’s test. Statistical significance was recognized at *p < 0.05.

Results

Design of Integrated Testing Strategy for lung carcinogenesis

We developed an ITS as an alternative to animal testing for evaluating carcinogenic potential. This ITS comprises various in vitro bioassays targeting key characteristics, including cell viability, clonal growth, and metastasis (Fig. 1).

The first assay assesses cell viability using the CFE assay and the CV assay. Second, clonal assays evaluate anchorage-dependent colony formation and anchorage-independent agar colony formation. Finally, cell migration and invasion assays are utilized to assess metastasis, a hallmark of malignant characteristics. By integrating these assays, the predictivity of carcinogenic potential can be significantly enhanced.

Cell viability by the Crystal Violet assay and Colony Forming Efficacy

The cell viability of the test compounds was evaluated using the CV and CFE assays (Fig. 2A). Additionally, appropriate exposure doses for the in vitro chronic toxicity test were determined based on the no observable adverse effects concentration (NOAEC), low observable adverse effects concentration (LOAEC), Inhibitory concentration (IC) 50, and IC90 values measured through the CV and the CFE assays for each test compound. As shown in Fig. 2B, B(a)P and EC did not exhibit significant changes in cell viability, with NOAEC values exceeding 1 and 2000 μg/ml, respectively, as determined by the CV and CFE assays. In contrast, ECH and CMME showed a significant decrease of cell viability in a dose-dependent manner. For ECH, the NOAEC derived from CFE assay was 31.25 μg/ml, while the LOAEC values determined by the CV and CFE assays were 7.82 and 62.5 μg/ml, respectively. For CMME, the NOAEC and LOAEC values obtained through both of the CV and CFE assays were 1.56 and 3.12 μg/ml, respectively. According to these results, the chronic exposure dose of test compounds was selected as follows (Fig. 2B): B(a)P (0.25, 1 μg/mL), EC (500, 2000 μg/mL), ECH (1.95, 7.81 μg/mL), and CMME (0.78, 3.12 μg/mL). These doses are considered appropriate for low-dose, long-term exposure studies.

Anchorage-dependent colony formation assay

To evaluate clonal cell growth through cell transformation, an anchorage-dependent colony formation assay was performed using BEAS-2B cells (#40 passages) chronically exposed to the test compounds. As shown in Fig. 3, all test compounds significantly enhanced clonal cell growth compared to the control at both low and high concentrations. In particular, clonal cell growth increased by 47-fold, 121-fold, 87-fold, and 83-fold in cells exposed to B(a)P (0.25 μg/ml), EC (2000 μg/ml), ECH (1.95 μg/ml), and CMME (0.78 μg/ml), respectively.

Anchorage independent agar colony formation

Anchorage-independent growth, characterized by the ability of transformed cells to proliferate without attachment to a solid surface (extracellular matrix), is a hallmark of carcinogenesis [16]. In this study, an anchorage-independent colony formation assay was used to evaluate malignant cell transformation in BEAS-2B cells (#40 passages) chronically exposed to the test compounds. As shown in Fig. 4, the test compounds significantly enhanced colony formation in agar compared to the control. Colony formation in soft agar increased by 12-fold, 38-fold, 11-fold, and 40-fold in the B(a)P (0.25 μg/ml), EC (500 μg/ml), ECH (7.81 μg/ml), and CMME (0.78 μg/ml) exposure groups, respectively.

Cell migration and invasion

Cell migration and invasion are critical processes in the physiological progression of tumor metastasis. To evaluate the effects of the test compounds on these processes, a transwell chamber assay was conducted using BEAS-2B cells (#40 passages). As shown in Fig. 5, the transwell chamber assay revealed a significant increase in the migration of BEAS-2B cells chronically exposed to the test compounds. Specifically, cells exposed to B(a)P (0.25 μg/ml), EC (2000 μg/ml), ECH (1.95 μg/ml), and CMME (0.78 μg/ml) demonstrated enhanced cell migration. In the cell invasion assay (Fig. 6), which employed matrigel-coated transwells, the test compounds significantly increased cell invasion in a dose-dependent manner compared to the control. B(a)P, EC, ECH, and CMME significantly increased cell invasion by up to 1.84-fold, 1.83-fold, 2.42-fold, and 2.98-fold, respectively.

Discussion

Carcinogenesis occurs through a multistep process involving initiation, promotion, and progression [17, 18]. Initiation represents the first step in carcinogenesis, characterized by sequential genetic changes caused by DNA damage in a single target cell. In contrast, promotion and progression are non-genotoxic mechanisms that lead to the formation of malignant tumors [19].

The traditional two-year inhalation carcinogenicity test in rats is labor-intensive, costly, and time-consuming, making it impractical for evaluating the carcinogenicity of numerous chemicals. Consequently, there is a pressing need to develop alternative methods to the two-year chronic carcinogenicity test that can accurately predict carcinogenicity while efficiently screening many substances.

In this study, we developed an ITS as an alternative to animal testing for evaluating carcinogenic potential. The ITS is based on mechanisms for multistage carcinogenesis, with a focus on key characteristics such as cell viability, clonal growth, and metastasis (Fig. 1). This ITS incorporates six in vitro bioassays, and integrating these assays may significantly enhance the predictivity of carcinogenic potential. To validate this approach, four carcinogenic substances - B(a)P, CMME, ECH, and EC - were tested. B(a)P, CMME [20, 21], ECH [22, 23], and EC [24] have been shown to induce DNA damage, suggesting their role as initiators in the multistep carcinogenesis process.

Increased cell proliferation can be achieved through direct mitosis or cytotoxicity accompanied by regenerative proliferation [25], leading to clonal expansion. Clonal expansion plays an important role in the carcinogenic potential induced by promotion and progression. Clonogenic assays under either anchorage-dependent or -independent conditions are very useful for testing the sensitivity of tumor cells to cytotoxic drugs [26]. To evaluate the role of these four substances as promoter / progressor, a cell viability test and a clonal expansion assay were carried out. In the cell viability test, B(a)P and EC did not show significant changes in cell viability, whereas ECH and CMME caused a significant decrease in cell viability. The four carcinogens demonstrated inconsistent results on cell proliferation (Fig. 2).

Anchorage to extracellular matrix (ECM) is crucial for execution of the mitotic program in non-transformed cells as they require concurrent signals starting from mitogenic molecules, such as growth factors (GFs), and adhesive agents within ECM [27]. Accordingly, the anchorage dependent clonal formation assay has been utilized to evaluate the impact of specific agents on cell survival and proliferation [28]. Chronic exposure (40# passage) of BEAS-2B cells to B(a)P, EC, CMME, and ECH increased anchorage-dependent colony formation (Fig. 3). Additionally, anchorage-independent colony formation in agar demonstrated a significant increase (Fig. 4), aligning with the results of the anchorage dependent colony formation assay. Anchorage-independent growth in soft agar, a hallmark of cellular transformation, is considered the most accurate and stringent in vitro assay for assessing malignant transformation [29]. These findings suggest that chronic exposure to the test compounds enhances the malignant transformation of non-tumorigenic BEAS-2B cells.

The Metastasis characteristics of lung cancer cells are a major contributor to the high mortality rate among lung cancer patients [30]. Cell mobility is a key indicator of metastatic potential in cancer cells. Specially, cell migration refers to the movement of cells from one location to another, whereas cell invasion involves three-dimensional migration of cells through the extracellular matrices (ECM) [31]. In this study, chronic exposure to four lung carcinogens resulted in a significant increase in cell migration and invasion in BEAS-2B cells (Fig. 5 and Fig. 6). While invasive migration plays a fundamental role in physiological processes such as angiogenesis, embryonic development, and immune response, it is also closely linked to cancer metastasis [32]. Based on these findings, we suggest that the test compounds enhanced malignant characteristics, including metastasis.

The results obtained using the ITS developed in this study suggest that chronic exposure of non-tumorigenic BEAS-2B cells to B(a)P, EC, ECH, and CMME, can induce malignant cell transformation and enhance multiple characteristics associated with tumor metastasis. Ultimately, the ITS, which incorporates six in vitro bioassays, could serve as an effective alternative to animal testing for evaluating carcinogenic potential.

Notes

Acknowledgement

This work was supported by the Korea Environmental Industry & Technology Institute (KEITI) through Core Technology Development Project for Environmental Diseases Prevention and Management Program, funded by the Korea Ministry of Environment (MOE) (RS-2021-KE00142) and through the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1F1A1077028).~

Conflict of interest

The authors declare that they have no conflict of interest..

CRediT author statement

MJK: Conceptualization, Visualization, Writing-Original draft preparation, CHP: Methodology, Writing-Original draft preparation, SMO: Supervision, Project administration, Writing-Reviewing and Editing.

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Figure 1.

The Integrated Testing Strategy (ITS) using in vitro alternative methods to predict lung carcinogenesis. The characteristics for carcinogenesis were based on clonal growth and metastasis. Each testing method was described in the standard operating procedure (SOP).

Figure 2.

Cell viability of test compounds in BEAS-2B cells by crystal violet assay (CV) and colony formation assay (CFE). (a) The cells were seeded in 6-well plates (black circle) or a 60 mm dish (white circle) and treated with test compounds (BaP, EC, ECH, CMME) for 72 h. Cells were changed to fresh medium, followed by incubation for 3 days (CV) or 5 days (CFE). The cells were then stained with crystal violet (CV) or Giemsa stain (CFE). The cell viability of the compounds was represented as the relative of control (% of control). (b) NOAEC/LOAEC/IC50/IC90 values for test chemicals are presented. The results are expressed as the mean ± SD of three separate experiments. Values are significantly different from the control at * p < 0.05, ** p < 0.01.

Figure 3.

Anchorage dependent colony formation in BEAS-2B cells chronically exposed to test compounds. BEAS- 2B cells were exposed to test compounds for 4 months (#40 passages). The cells were seeded in 6-well plates. Cells were changed to fresh medium, followed by incubation for 9 days. (a) The cells were then stained with Giemsa stain. (b) The anchorage dependent colony formation of the compounds was expressed as a relative value compared to the control. The results are expressed as the mean ± SD of three separate experiments. Values are significantly different from the control at * p < 0.05, ** p < 0.01.

Figure 4.

Anchorage independent agar colony formation in BEAS-2B cells chronically exposed to test compounds. The cells were exposed to test compounds for 4 months (#40 passages) and assessed for anchorage independent growth using a soft agar assay. Exposed cells were seeded in a 12-well culture plate (3,000 cells/well). Four weeks later, (a) cell colonies were stained with NBT and (b) colonies of > 50μm in diameter were counted by microscopic examination (Olympus SZX7, magnification ×0.8). The results are expressed as the mean ± SD of three separate experiments. Values are significantly different from the control at ** p <0.01.

Figure 5.

Cell migration in BEAS-2B cells chronically exposed to test compounds. BEAS-2B cells exposed to test compounds for 4 months (#40 passages) were seeded in 8 μm pore size transwell chamber of 24-well plates. After 24 h incubation, the migrated cells were stained with crystal violet. (a) The migrated cells were investigated by microscope examination (magnification × 100) and then (b) the stained cell was extracted by extraction buffer as indicated in Material and Methods. The results were expressed as the mean ± SD of three separate experiments. Values are significantly different from the control at ** p < 0.01.

Figure 6.

Cell invasion in BEAS-2B cells chronically exposed to test compounds. BEAS-2B cells exposed to test compounds for 4 months (#40 passages) were seeded in an 8 μm pore size transwell chamber coated with Matrigel in 24 well plates. After 48 h incubation, the invaded cells were stained with crystal violet. (a) The migrated cells were investigated by microscope examination (magnification × 100) and then (b) the stained cell was extracted by extraction buffer as indicated in Material and Methods. The results were expressed as the mean ± SD of three separate experiments. Values are significantly different from the control at ** p < 0.01.

Table 1.

Characteristics of test compounds.