Meredith A. Tennis

Supplementary Table S3 lists antibody combinations used for immune cell detection in Figure 5.

Supplementary Figure S2 shows a dot blot for Figure 4C.

Supplementary Figure S3 shows a representative immunofluorescence image for Figure 5.

Supplementary Figure S1 shows a dot blot for Figure 3D.

Supplementary Table S2 lists antibody information for immunofluorescence in Figure 5.

Supplementary Figure S5 shows a representative immunofluorescence image for Figure 5.

Supplementary Figure S4 shows a representative immunofluorescence image for Figure 5.

Supplementary Table S1 lists targets in the dot blots for Figures 3D and 4C.

Lung cancer is the leading cause of global cancer death, and prevention strategies are key to reducing mortality. Medical prevention of premalignant lesion (PML) progression may have a larger impact than treatment on mortality by targeting high‐risk populations and reducing their lung cancer risk. PMLs are difficult to study in humans but are easily accessible in murine preclinical carcinogenesis studies. Precision‐cut lung slices (PCLS) are underutilized as an ex vivo model for lung cancer studies due to limited culture time. Embedding PCLS within bioengineered hydrogels extends PCLS viability and functionality for up to 6 weeks. Here, we embedded PCLS with PMLs generated from urethane‐exposed mice in cell‐degradable and nondegradable hydrogels to study viability and responsiveness to PML interception over 6 weeks. PMLs in hydrogel‐embedded PCLS maintained viability, gene expression, and proliferation. Treatment of hydrogel‐embedded PCLS containing urethane‐induced PMLs with iloprost, a known lung cancer prevention agent, recapitulated in vivo gene expression and activity. Our studies also showed that iloprost reduced proliferation and PML size in PCLS embedded in degradable hydrogels. These results demonstrate that hydrogel‐embedded PCLS models support long‐term culture of in vivo generated PMLs. This modeling approach will improve preclinical studies of cancer biology and prevention across cancer types.

Lung cancer chemoprevention is critical to addressing cancer burden in high-risk populations. Chemoprevention clinical trials rely on data from pre-clinical models however, in vivo studies have high financial, technical, and staffing requirements. Precision cut lung slices (PCLS) provide an ex vivo model that maintains the structure and function of native tissues. This model can be used for mechanistic investigations and drug screenings and reduces the number of animals and time required to test hypotheses compared to in vivo studies. We tested use of PCLS for chemoprevention studies, demonstrating recapitulation of in vivo models. Treatment of PCLS with the PPARγ agonizing chemoprevention agent iloprost produced similar effects on gene expression and downstream signaling as in vivo models. This occurred in both wildtype tissue and in Frizzled 9 knockout tissue, a transmembrane receptor required for iloprost’s preventive activity. We explored new areas of iloprost mechanisms by measuring immune and inflammation markers in PCLS tissue and media, and immune cell presence with immunofluorescence. To demonstrate potential for drug screening, we treated PCLS with additional lung cancer chemoprevention agents and confirmed activity markers in culture. PCLS offers an intermediate step for chemoprevention research between in vitro and in vivo models that can facilitate drug screening prior to in vivo studies and support mechanistic studies with more relevant tissue environments and functions than in vitro models.