Characterization of tri-culture alveolar-like organoids to study fibrotic lung diseases

Rationale: Fibrotic interstitial lung diseases are progressive disorders characterized by lung scarring and declining respiratory function. Repeated injury and dysregulated epithelial-mesenchymal crosstalk are implicated in disease pathogenesis but remain incompletely understood. Three-dimensional (3D) organoids incorporating epithelial-mesenchymal crosstalk provide a physiologically relevant platform for investigating these mechanisms. Here, we present a 3D tri-co-culture alveolar organoid model and evaluate its responses to two profibrotic stimuli, TGF-{beta} and bleomycin. Methods: CI-huArlo, NCI-H441, and MRC-5 cells were co-cultured for 14 days to generate alveolar-like organoids. Cell marker expression was assessed by immunofluorescence (IF). To test injury response, organoids were exposed to TGF-{beta} (50 ng/mL) or bleomycin (20 g/mL) for 48 hours and characterized. Supernatants were collected to assess interleukin 8 (IL-8) and procollagen I by ELISA. qPCR assessed expression of CDKN1A and COL1A1 following treatments. Bulk RNA sequencing (RNA-seq) evaluated the transcriptomic responses to fibrotic stimulus. Results: Cellular marker expression was confirmed using IF for aquaporin-5 (alveolar type I cell marker) and TE-7 (fibroblast marker). Bleomycin exposure reduced viability and significantly increased IL-8 (126.3 {+/-} 16.86 vs. 48.88 {+/-} 4.470 pg/mL; p = 0.0002; N=6), with no change in secreted procollagen I compared to control. TGF-{beta} stimulation significantly increased secreted procollagen I (149.6 {+/-} 27.07 vs. 53.38 {+/-} 5.672 pg/mL; p <0.0001; N=6) without affecting viability or IL-8 release. qPCR resulted in no change in CDKN1A expression, while COL1A1 expression was increased with TGF-{beta} treatment compared with control and bleomycin. RNA sequencing demonstrated distinct and reproducible transcriptional responses to TGF-{beta} and bleomycin. TGF-{beta} induced 750 significantly upregulated and 641 downregulated genes, including increased COL1A1, COL4A1, FN1, and TGFB1, with enrichment of epithelial-mesenchymal transition and TGF-{beta} signalling programs. In contrast, bleomycin induced 953 significantly upregulated and 552 downregulated genes relative to untreated controls and was characterized by p53 signalling, DNA-damage responses, and reduced cell-cycle progression. Reference-state analysis further indicated reduced normal alveolar epithelial signatures following both treatments, with TGF-{beta} producing the strongest aberrant basaloid and myofibroblast-associated signatures. Conclusions: Tri-culture alveolar-like organoids exhibited stimulus-specific responses in a 3D multicellular system, supporting their use for mechanistic studies of epithelial-mesenchymal crosstalk, environmental exposures, and therapeutic responses.