Hydrogel‐Embedded Precision‐Cut Lung Slices Model Lung Cancer Premalignancy Ex Vivo

Abstract

Lung cancer is the leading global cause of cancer‐related deaths. Although smoking cessation is the best prevention, 50% of lung cancer diagnoses occur in people who have quit smoking. Research into treatment options for high‐risk patients has been constrained to rodent models, which are time‐consuming, expensive, and require large cohorts. Embedding precision‐cut lung slices (PCLS) within an engineered hydrogel and exposing this tissue to vinyl carbamate, a carcinogen from cigarette smoke, creates an in vitro model of lung cancer premalignancy. Hydrogel formulations are selected to promote early lung cancer cellular phenotypes and extend PCLS viability to six weeks. Hydrogel‐embedded PCLS are exposed to vinyl carbamate, which induces adenocarcinoma in mice. Analysis of proliferation, gene expression, histology, tissue stiffness, and cellular content after six weeks reveal that vinyl carbamate induces premalignant lesions with a mixed adenoma/squamous phenotype. Putative chemoprevention agents diffuse through the hydrogel and induce tissue‐level changes. The design parameters selected using murine tissue are validated with hydrogel‐embedded human PCLS and results show increased proliferation and premalignant lesion gene expression patterns. This tissue‐engineered model of human lung cancer premalignancy is the foundation for more sophisticated ex vivo models that enable study of carcinogenesis and chemoprevention strategies.This article is protected by copyright. All rights reserved

Hydrogel-embedded precision-cut lung slices supportex vivoculture ofin vivo-induced premalignant lung lesions

“…55, 56 We have explored using human tissue in hydrogel-embedded PCLS for initiating PMLs ex vivo , but accessing human PMLs in tissue suitable for long-term PCLS is challenging. 13 We continue to investigate the possibilities for human relevant hydrogel-embedded PCLS, including designing hydrogels to mimic human microenvironment conditions in mouse PCLS.…”

“…PPARγ activity was assessed using a PPARγ response element (PPRE) luciferase assay as described previously. 10, 13 Immortalized Human Bronchial Epithelial Cells (HBEC; a gift from John Minna) were transfected using TransIT-X2 reagent (Mirus Bio). Conditions included 45 ng PPRE luciferase (a gift from Bruce Spiegelman; Addgene plasmid #1015) and 5 ng renilla control reporter vector (Promega), mock luciferase, and empty vector.…”

“…An 8-mm diameter silicone mold facilitated hydrogel embedding as previously described. 13, 27 First, 25 μl of the hydrogel precursor solution was added to the silicone mold, and polymerized by exposure to 365 nm UV light at 10 mW/cm 2 for 5 min. Hydrogel formulations were designed to be slightly off-stoichiometry (thiol:ene = 0.9) to leave free reactive groups within this base layer after polymerization that enables the second hydrogel layer to covalently bond to the first.…”

“…18 In a subsequent study, we maintained the health of carcinogen-exposed lung tissue slices for an unprecedented six weeks in culture with hydrogel embedding and promoted cellular proliferation and altered gene expression. 13 Here, our collaborative group presents a study investigating the capacity of hydrogel-embedded PCLS to maintain lung PMLs generated by an in vivo urethane adenocarcinoma carcinogenesis model. In the present study, the non-degradable hydrogel that best supported initiation of premalignant cellular responses was compared to a hydrogel degradable by matrix metalloproteinase-9 (MMP-9).…”

Precision cut lung slices: an integrated ex vivo model for studying lung physiology, pharmacology, disease pathogenesis and drug discovery

“…Some studies have utilized lung cancer explants into murine systems and analyzed the PCLS derived from these mice, where others have used cancerous tissue derived from the lungs of patients to generate PCLS. A few studies have utilized PCLS as a preclinical model to test therapeutics in both human and mouse tissues [55][56][57][58]. Others have examined lymphocyte migration into lung tumors to try to understand lymphocyte accumulation in the tumors [59], while another study examined changes in macrophage activation and the contribution to lung cancer growth [60].…”