6–8 weeks-old C57BL/6 mice lung tissue was harvested as per institutional guidelines at UAB. single-cell suspension preparation involved the following steps. Mice lungs were perfused with 2.5 mL PBS and washed with cold PBS solution. Bronchi were carefully avoided, and tissue was sectioned and minced before adding 5 mL of collagenase A. This was incubated for 30 min at 37 °C with gentle agitation every 10 min. After the incubation, 10 mL of PBS was added and suspension were passed through 100µ cell strainer. The filtered suspension was centrifuged for 5 min at 300 g and washed with complete DMEM consisting of 10% FBS, penicillin and streptomycin, and Glutamax. The cell pellet was then resuspended in ACK lysis buffer to lyse red blood cells and was incubated at room temperature for 5 min with occasional shaking. The reaction was stopped by diluting the ACK lysis buffer with 30 mL of PBS, and the cells were centrifuged at 300 g for 5 min. The supernatant was removed carefully, and the pellet was resuspended in minimal essential media (MEM, Gibco) supplemented with non-essential amino acids (Gibco) and 1% antibiotic-anti-mycotic solution (Gibco).

Cell suspension obtained was assessed for cell viability upon drug treatment. Drug concentration leading to 80% viability of cells was selected for drug exposure related experiments. Cell viability assay was performed using MTT based Cell Proliferation Kit I, following the manufacturer’s protocol (11 465 007 001; Roche). Briefly, Cells were seeded in 96-well flat-bottom plates and treated as indicated for 24 h. MTT labeling reagent was then added to each well (final concentration 0.5 mg/mL), and plates were incubated for 4 h at 37 °C to allow formation of intracellular formazan crystals. The solubilization solution was then added and incubation was continued until complete dissolution of the formazan, after which absorbance was recorded at 570 nm using a microplate reader (Synergy HTX, Biotek). Cell viability for each condition was calculated after blank subtraction as 100 × [(A_treated−A_blank)/(A_control−A_blank)], with untreated controls defined as 100% viability, and drug concentrations displaying approximately 80% viability (i.e., 20% inhibition) were identified based on this calculation.

Anti-adherence rinsing solution was added to each well of a 96-well U-bottom plate, to ensure organoids were free floating and accessible to harvest from the wells without trypsinization. The plates were left at room temperature in a sterile laminar hood for 30 min. Solution was collected, wells were washed with PBS (twice) and air dried. These coated plates were then used for the preparation of MiLO. MiLO were prepared from cell suspension obtained from lung tissue. The total cell pellet was resuspended in 2 mL complete MEM medium. Cells were stained with trypan blue and counted to check for cell density and viability. Cell suspension was mixed with corning organoid matrigel matrix (356255, Corning, United States) in 1:1 ratio. 8,000–10,000 cells (depending upon total number of cells and assays) were added per well in the U-bottom 96 well plates. Plates were incubated for 30 min at 37 °C in CO₂ (5%) incubator. Corning Matrigel Matrix for Organoid Culture is a basement membrane extract from EHS mouse sarcoma, rich in ECM proteins like laminin, collagen IV, entactin, heparan sulfate proteoglycans, and growth factors. Once matrigel solidified complete media was added to each well and plates were incubated for 3 days and fresh media was replaced every other day. Cells take spheroid shape in 2 days. The resulting 3D mice lung organoids (MiLO) were treated with low dose of cadmium chloride (CdCl₂, 10 μM; CAS 10108-64-2, Sigma-Aldrich), amiodarone (AD, 20 μM; CAS J6045606, Thermo Scientific Chemicals) or nitrofurantoin (NF, 5 μM; CAS B2407914, Thermo Scientific Chemicals) for 24 h. When treatment was complete, MiLO were fixed and paraffinized to section to stain for fibrotic markers visualized using All-in-One Fluorescence Microscope BZ-X810 (Keyence, United States).

Animals were purchased from the Jackson Laboratory and all procedures were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. Mice (6–8 weeks old; 25–30 g) of both sexes were included, with equal numbers of females and males per group. Animals were acclimatized for at least 1 week before experimentation and then randomly allocated to treatment or control groups. Mice were treated once by intratracheal instillation with cadmium chloride (CdCl₂; Sigma-Aldrich, CAS 10108-64-2, 0.458 mg/kg) or saline vehicle as previously described for Cd-associated mouse models of lung injury and remodeling. Lung tissues were collected on days 0, 3, 6, 9, 12, 15, 18, and 21 (n = 3 per time point per treatment group) for biochemical and histological analyses. Hydroxyproline content was quantified according to the manufacturer’s instructions (Abcam) as an indicator of collagen deposition and fibrosis. A separate cohort of mice (n = 3 per group) was assessed for respiratory mechanics at day 21 post-instillation. Following anesthesia, airway resistance (Rrs) was measured using the single-compartment model as previously described (Li et al., 2021; Li et al., 2022).

For collagen quantification, the right lung from mice was removed, weighed, and homogenized. Collagen content in mouse lung tissue and MiLO was assessed by the Hydroxyproline assay (ab222941, Abcam) following the manufacturer’s instructions. The soluble collagen levels in supernatants from Cd treatment cells were also evaluated.

MiLO were washed and fixed in formalin for 24 h at room temperature. Formalin was pipetted out, and 100 μL of warmed (at 80 °C) histogel (Corning, United States) was added to the MiLO. The histogel- MiLO suspension was refrigerated until the gel became firm. The gel was taken out from the tube with the help of a spatula and kept in a cassette. The cassette was incubated in formalin solution overnight. These cassettes were processed for paraffin sectioning to obtain 5 µ thin sections.

After blocking in PBST (0.1% Triton X-100 in PBS) containing 3% Bovine Serum Albumin (Sigma-Aldrich) overnight at 4 °C and washing in PBST (twice for 15 min), MiLO were incubated with primary antibodies diluted in PBST on a gently rocking rotator at 4 °C for 48 h and rinsed in PBST (4 times for 30 min). When necessary, MiLO were then incubated in appropriate AlexaFluor-conjugated secondary antibodies (Molecular Probes, Life technologies) for 24 h. Cell nuclei were counterstained by DAPI (Invitrogen) diluted 1:500 in PBS for 40 min at room temperature. Immunolabeled MiLO were imaged using confocal microscopy. For paraffin sections, immunofluorescent staining was performed for SPC (ab32575, Abcam), CD31 (558737, Thermo Fisher), F4/80 (123116, Abcam), α-SMA (NB300-978, Novus Bio.), Fibronectin (FN1; ab54477, Abcam), Ki67 (sc15402, Santa Cruz), COL1A1 (ab260043, Abcam) and Ig G isotype (sc 2005, Santa Cruz). All immunofluorescence-stained slides were co-stained with DAPI for visualization of the nucleus using All-in-One Fluorescence Microscope BZ-X810 (Keyence, United States).

Lung tissue and MiLO were processed for total RNA isolation using TRIzol reagent (Invitrogen, United States) and cDNA was synthesized to perform qRT-PCR for gene expression analysis. Briefly, the manual extraction of total RNA TRIzol reagent was added to the sample, followed by chloroform and isopropanol for RNA precipitation. Ethanol gradient washing provided total RNA, which was then dissolved in water. Concentrations were recorded and 2 µg of each RNA was mixed with cDNA synthesis master mix (1708891, iScript cDNA synthesis kit, BioRad, United States) PCR was run as per the manufacturer’s protocol. cDNA synthesized was used as a template for amplification and quantification of target genes by quantitative real-time PCR (StepONE, Applied Biosystems, United States) using TaqMan fast master mix (Thermo Fisher Scientific, United States). ß-actin was co-amplified for semi-quantitative comparison. TaqMan primer assay mix with forward and reverse sequence. Relative expression was calculated by using the 2^−ΔΔCT method. The reactions were performed in duplicates for each sample and mean value of 2^−ΔΔCT was plotted on heat map.

MiLO were collected from 96-well plates and suspended in media. Rat tail collagen type I (3.98 mg/mL, CLS354236 Corning) was mixed with neutralization solution as per the manufacturer’s instructions to prepare collagen hydrogels. MiLO were incorporated into a final collagen concentration of 2 mg/mL for media alone group without a test drug in a single well. For treatment groups, single MiLO were suspended in 100 μL of the collagen-DMEM (DMEM with 10% FBS and 1% antibiotic-antimycotic solution) solution containing Cd (10 μM) or amiodarone (20 μM) or nitrofurantoin (5 μM). The suspension was then poured into a ultra-low attachment 96-well clear flat bottom plates (corning) and gelled for 30 min at 37 °C. Fresh medium was added, and samples were imaged on Day 0. After imaging, samples were incubated at 37 °C with 5% CO₂ for 24 h to allow cell invasion into the matrix. In total, n = 5 MiLO were seeded for each treatment. The plates were incubated at 37 °C in a CO₂ incubator overnight. Images were captured with Keyence microscope, the invasive cells’ area was delineated and calculated (H). Additionally, the center of the MiLO was outlined and measured (R). The percentage of the total invaded area (ZOI%) was determined by (H - R)/R x 100. The percentage invasion area was calculated. The ZOI fold change was measured as the ratio of the ZOI% with treatment to the ZOI% without treatment in vitro with an antifibrotic drug.

Lipid peroxidation assay (MDA assay, ab233471, Abcam) and Superoxide dismutase (SOD) activity (ab65354, Abcam) were performed to estimate oxidative stress in mice lung tissue or MiLO as per the manufacturer’s protocol. Experiments were performed in triplicates.

Data was analyzed by one-way analysis of variance (ANOVA) followed by Dunnett multiple comparison post hoc test and two-way ANOVA followed by Sidak multiple comparison post hoc test (only if p < 0.05). The response rate to each treatment was calculated, and an exact 95% confidence interval around the response rate was calculated using the Clopper-Pearson method. Differences were considered statistically significant at p < 0.05. The data is presented as the means ± standard error unless otherwise stated.

To prepare mice lung tissue derived organoids, mice lungs were perfused, minced (excluding primary bronchi), and digested with collagenase. Cells were filtered through a 100 µm strainer, pelleted, and treated with ACK lysis buffer to remove RBCs. The cell suspension was mixed 1:1 with Matrigel, and 50 µL (0.5 × 10⁴ cells) was dispensed into each well of a round-bottom 96-well plate. After 30 min incubation at 37 °C, culture media was added, with media replacement every 2 days over a 6-day incubation period. organoids were harvested using blunt end tips in 2 mL tubes (n = 10/tube), fixed, paraffin embeded for sectioning and stained for cellular markers. These 3D organoids (Figure 1a) generated from mouse lung tissue formed multicellular structure mimicking mouse lung tissue architecture (Figure 1b). These MiLO contained various lung cell types, including epithelial type II cells expressing surfactant protein C (SPC), myofibroblasts or vascular smooth muscle cells positive for α-smooth muscle actin (α-SMA), endothelial cells labeled by CD31 and macrophages identified by F4/80 (Figure 1c). These diverse cells were embedded within extracellular matrix components such as collagen I (COL1A1) and fibronectin-I (FN1), closely replicating the native lung microenvironment and architectural organization (Figure 1c). Lung tissue and MiLO were further compared to predicting the gene expression pattern (Figure 1d). A comparable expression level was observed for fibrosis related markers such as Tgfb1, Acta2, Col1a1, Col3a1, Ctgf, Lox2, Pdgfb and Fn1, which were selected due to their critical roles in collagen synthesis, ECM remodeling, cross-linking, and fibrotic progression. To ensure the physiological relevance of the model, apoptosis-related genes including Bax, Fas, Mapk8, Casp3, and Casp8 were analyzed to monitor programmed cell death and confirm cell viability at the time of assessment. Other genes evaluated were also evaluated here. The inclusion of basal cells expressing Krt5, important progenitors for airway repair, further highlights the diversity of progenitor populations. These diverse cells reside within extracellular matrix components such as collagen I and fibronectin 1, replicating the native lung microenvironment and organization. Additionally, genes involved in cell survival (Akt1), matrix remodeling (Mmp9, Mmp13, Timp1, Serpine2), oxidative stress (Txnip) (Lin et al., 2025), and inflammation regulation (Nfkb1) were assessed to capture the complex signaling and proteolytic landscape within the organoids (Cabral-Pacheco et al., 2020; Craig et al., 2015). Together, these genes provide insights into progenitor cell populations, matrix remodeling dynamics, antioxidant status, and epithelial cell function, thereby validating the organoid as a multi-cellular model reflecting lung tissue complexity and fibrosis-relevant pathways.

The pulmonary toxicity of cadmium (Cd) is highly dose- and exposure-dependent (Li et al., 2022). Our laboratory has established well-characterized mouse models demonstrating Cd-induced lung toxicity (Li et al., 2021; Li et al., 2017). Mice (n = 3) were exposed to a pre-evaluated Cd dose (0.458 mg/kg), reflecting concentrations found in human lung tissue exposed to Cd. Histological analysis revealed increased expression of α-SMA, a myofibroblast marker, in lung tissue following Cd exposure (Figure 2a). Functional assessment via methacholine challenge demonstrated a dose-dependent increase in airway resistance in Cd-treated mice compared to controls (Figure 2b), indicative of fibrosis-associated airflow obstruction. Elevated extracellular matrix deposition was quantitatively confirmed by hydroxyproline assay (Figure 2c). These results confirm that low-dose Cd exposure, relevant to environmental, occupational, and behavioral sources such as smoking, induces pulmonary fibrosis characterized by myofibroblast activation and collagen accumulation, impairing lung function. This mouse model thus provides a critical benchmark for evaluating pulmonary toxicity mechanisms and supports the translational utility of lung organoid systems in toxicology research.

To evaluate the relevance of mouse lung organoids in toxicity assessments, we compared outcomes from a well-established Cd-induced lung fibrosis mouse model with murine lung organoids (MiLO). MiLO generated from C57BL/6 mouse, widely used in environmental toxicology studies, were exposed to 10 μM Cd for 24 h and analyzed for extracellular matrix protein changes analogous to in vivo lung fibrosis. Immunostaining revealed increased collagen I (COL1A1) deposition and presence of alveolar type 2 cells (SPC) in Cd-treated MiLO (Figure 2d). To assess myofibroblast activation and invasiveness, MiLO were cultured on collagen-coated 96-well plates with Cd, and the percentage zone of invasion (ZOI%) was quantified, showing a significant increase in invasiveness (p < 0.0001, Figure 2e). Hydroxyproline assays performed on MiLO (n = 3) and Cd-exposed mouse lung tissue (day 21) confirmed comparable elevations in collagen content after normalizing for dry weight (Figure 2f). These results demonstrate that Cd exposure induces extracellular matrix remodeling and myofibroblast invasiveness in MiLO, mirroring pathological changes observed in vivo, thereby supporting the utility of MiLO as an in vitro platform for pulmonary toxicity studies.

Lipid peroxidation is a key marker of oxidative stress and contributes to the development of fibrotic responses in lung tissue (Ayala et al., 2014; Zhou et al., 2025). Malondialdehyde (MDA), a byproduct of lipid peroxidation, was significantly elevated in lung tissue from Cd-exposed mice (n = 3), indicating increased oxidative damage (Figure 2g). Similarly, murine lung organoids (MiLO) exposed to 10 µM Cd for 24 h showed a marked increase in MDA levels (p = 0.0001), confirming enhanced oxidative stress in vitro (Figure 2g). Additionally, superoxide dismutase (SOD) activity, an essential antioxidant defense enzyme, was significantly reduced in both Cd-exposed mice lung tissue (p = 0.0343) and Cd-treated MiLO (p < 0.0001) (Figure 2h). This decrease in SOD activity indicates impaired antioxidant capacity, thereby exacerbating oxidative stress. Together, these findings demonstrate that Cd exposure induces oxidative damage via increased lipid peroxidation and diminished antioxidant defenses, mechanisms known to promote myofibroblast activation and extracellular matrix remodeling in pulmonary fibrosis.

Fibrosis invasion assays are essential for elucidating the progression and development of lung fibrosis. In this study, we evaluated two known pulmonary toxicants, nitrofurantoin (NF) (Cameron et al., 2000; Kaye et al., 2025; Suliman et al., 2023) and amiodarone (AD) (Terzo et al., 2020; Wolkove and Baltzan, 2009), both reported to induce pulmonary fibrosis as a side effect. Cell viability assays established 80% cell viability (or 20% cell inhibitory) concentrations for NF (5 µM) and AD (20 µM), which were used for subsequent experiments (Figure 3a). MDA levels, a marker of lipid peroxidation and oxidative stress, were significantly elevated in mice lung tissue derived cells exposed to increasing concentrations of these drugs (Figure 3b), correlating with increased MDA in MiLO exposed to NF (5 µM) and AD (20 µM) (Figure 3c). Both NF and AD triggered enhanced invasive behavior of MiLO cultured on collagen-coated plates, as demonstrated by increased invasion areas (Figures 3d,e). Consistent with their known fibrotic effects in human and murine lungs, NF and AD treatments induced upregulation of fibrosis markers in MiLO, including Tgfb1, fibronectin (Fn1), collagen 1 (Col1a1), and α-smooth muscle actin (Acta2) (n = 3, Figures 3f,g). These findings validate the use of MiLO for modeling drug-induced pulmonary fibrosis and highlight NF and AD as potent inducers of fibrotic responses via oxidative stress and myofibroblast activation. Additionally, increased expression of Akt1, Ctgf, Nfkb1, Mmp9, and Mapk8 were observed, emphasizing the activation of signaling pathways associated with myofibroblast survival, matrix remodeling, inflammation, and stress responses (El-Tanbouly et al., 2017; Hsu et al., 2017; Larson-Casey et al., 2016; Nyp et al., 2014; Redente et al., 2021; Sinha et al., 2025). These findings support the use of MiLO for modeling drug-induced pulmonary fibrosis and highlight NF and AD as potent inducers of fibrotic responses via oxidative stress and myofibroblast activation.