Evaluating Alveolar Regenerative Properties of Caveolin Scaffolding Peptides (CSD) in Three Dimensional (3D) Alveolospheres From IPF and Normal Donor Lung Samples

Abstract Rationale: Idiopathic pulmonary fibrosis is a progressive disease characterized by unrelenting lung remodeling ultimately leading to lung failure and death. Single cell RNAseq studies have revealed that there is marked decrease in the abundance of alveolar type 2 cells (AT2) in lung samples from IPF patients. Reduction of AT2 viability impairs alveolar regeneration since these cells are a source of alveolar type 1 cells (AT1). In this study, we optimized a 3D alveolosphere model generated with isolated and cultured alveolar type 2 cells from IPF and donor, non-IPF control patients and evaluated the effect of CSD peptides LTI-03 and LTI-2355.Methods: Cryo-preserved IPF and normal donor lung explants were processed using enzymatic and mechanical digestion to obtain lung epithelial cell preparations. Primary human AT2 cells (EPCAM+ HTII-280+) were isolated using fluorescence-activated cell sorting (FACS). FACS-sorted AT2 cells were subsequently resuspended in Matrigel (1:1 Matrigel to media). A 30 μL drop containing 5 x104 AT2 cells was applied to the center of each well in a 24 well plate. Either vehicle control, CSD peptide (LTI-03 or LTI-2355 at 0.5, 3, or 10 μM) or Nintedanib (80 nM) were added into each drop, and to the overlying tissue culture media. Treatments were changed every other day for 28 days. Results: Both LTI-03 and LTI-2355 CSD peptides at the 10 μM dose increased the number of IPF alveolosperes compared with the control treatment of IPF alveolospheres at day 28 in culture. CSD peptide treatment did not alter the average size of IPF alveolospheres. Quantitative PCR analysis of SFTPC levels in IPF and normal donor alveolospheres revealed that CSD peptides increase SFTPC transcripts in both groups of alvelospheres. Finally, Nintedanib treatment of normal donor alveolospheres significantly reduced the number and size of alveolospheres at day 28 of culture compared with vehicle or CSD treated normal donor alveolospheres.Conclusion: LTI-03 and LTI-2355 increased the outgrowth of IPF alveolospheres in 3D tissue culture indicating that LTI CSD peptides exert a putative pro-regenerative effect on IPF AT2 cells. In contrast, nintedanib exhibited inhibitory effects on the outgrowth of normal donor alveolospheres. Ethical approval: Institutional Review Boards both at Cedars-Sinai Medical Center and Aileron Therapeutics approved all experiments with human tissue.