Preprint Separates Bleomycin Injury from TGF-β1 Matrix Responses in Lung Tri-Cultures
An alveolar-like tri-culture made from established human cell lines produced different responses to bleomycin and TGF-β1. Bleomycin reduced metabolic activity and increased IL-8 without increasing procollagen I, while TGF-β1 increased matrix-associated endpoints over 48 hours.
Comparing Two Common Fibrosis-Related Stimuli
A bioRxiv version-one preprint posted September 9, 2026, describes an alveolar-like tri-culture that separates an acute injury response from a matrix-producing response. University of British Columbia and collaborating researchers exposed the same three-dimensional model to bleomycin or transforming growth factor-β1, abbreviated TGF-β1.
The comparison is relevant to drug-induced lung injury and the design of antifibrotic assays. Bleomycin is a chemotherapy drug used experimentally to induce injury, whereas active TGF-β1 directly engages a major downstream fibrogenic pathway. Their effects are often grouped under fibrosis modeling, but they need not generate the same phenotype over the same interval.
A Defined Model from Three Established Human Cell Sources
The culture combines NCI-H441 lung adenocarcinoma cells, immortalized CI-huArlo alveolar epithelial cells and MRC-5 fetal lung fibroblasts at an 11:7:4 ratio. A shared medium supported each population, and the cells formed three-dimensional structures over fourteen days in a basement-membrane matrix.
Imaging showed increasing organoid diameter and fewer structures per dome over time. Aquaporin-5 staining and the fibroblast marker TE-7 supported the presence of epithelial and fibroblast compartments. Histology showed hollow structures with central debris. These findings characterize an alveolar-like architecture assembled from the specified cell sources.
The investigators then applied 50 ng/mL TGF-β1 or 20 μg/mL bleomycin for 48 hours. They combined resazurin reduction as a metabolic-activity readout with secreted proteins, targeted gene expression and bulk RNA sequencing. This design allowed injury and matrix production to be compared in the same experimental background.
Secreted Proteins Distinguished the Responses
Bleomycin lowered resazurin fluorescence relative to controls and increased interleukin-8, or IL-8, release. Secreted-protein values are reported as mean ± standard error of the mean (SEM). The abstract reports IL-8 concentrations of 126.3 ± 16.86 versus 48.88 ± 4.470 pg/mL, with P = 0.0002 and six replicates. Secreted procollagen I did not increase under this exposure.
TGF-β1 produced a different pattern. Procollagen I reached 149.6 ± 27.07 versus 53.38 ± 5.672 pg/mL in controls, with P < 0.0001 and six replicates. Metabolic activity and IL-8 did not differ significantly from controls. Targeted testing also found substantially greater COL1A1 expression after TGF-β1 than after bleomycin, while CDKN1A did not differ significantly between the two treatment groups in that assay.
The negative collagen result under bleomycin is central to the comparison. At this dose and time point, the model displayed acute injury and inflammatory signaling without the measured increase in matrix synthesis seen after TGF-β1.
Transcriptomics Showed Different Programs
Relative to controls, TGF-β1 changed 750 genes upward and 641 downward, with extracellular-matrix, adhesion and TGF-β-associated programs prominent. Bleomycin changed 953 genes upward and 552 downward, emphasizing DNA damage and p53 signaling while reducing cell-cycle programs. RNA sequencing used six biological replicates per condition.
A reference-based computational analysis compared these profiles with alveolar, basaloid and fibroblast states in a human pulmonary-fibrosis atlas. TGF-β1 produced the strongest myofibroblast and aberrant-basaloid resemblance scores. Bleomycin produced stronger fibroblast-associated scores and a basal injury module with nominal P = 0.048. Such scores describe transcriptional resemblance and do not measure cell fractions or demonstrate conversion into a new lineage.
Ligand–receptor analysis prioritized 30 candidate interactions: 25 associated only with TGF-β1, four shared and one specific to bleomycin. These are computational hypotheses about communication. A separate marker comparison with published primary type-II alveolar (AT2) spheroids showed partial concordance, with differences in composition, exposure and duration restricting the comparison.
Acute Phenotypes for Mechanistic Testing
The study did not directly establish cellular senescence, deposited mature collagen or persistent fibrosis after treatment withdrawal. Its established cell sources also lack the aging, genetic background and pre-existing abnormalities of patient lung tissue, and the model contains no immune or endothelial compartment.
Those features define useful next experiments: repeated or recovery-phase exposures, direct matrix and contraction measurements, cell-resolved validation and pathway-inhibitor tests. Within the present 48-hour design, the tri-culture supplies separable injury and matrix-associated endpoints for investigating epithelial–fibroblast responses.