427. 3D Modeling of Lung Adenocarcinoma With a Hydrogel Scaffold
Authors: Francesca del Bufalo, Valentina Hoyos, Shigeki Yagyu, Ignazio Caruana, Malcolm Brenner
Journal: Molecular Therapy (2015)

The contribution of the microenvironment to tumorigenesis is now widely recognized and encompasses both the 3D architectural structure and the interaction between malignant and normal stromal cells. This interactivity cannot be reproduced by conventional 2D cell culture systems. We have developed a fibrin-based hydrogel 3D lung cancer model that allows in vitro and in vivo modeling of tumor-stromal interaction under reproducible and tunable conditions. We engineered the model by culturing tumor cells (A549 or H1299), alone or in combination with fibroblasts (MRC5) and/or endothelial cells (HUVECs), within a PEG-fibrin hydrogel which is readily produced, biocompatible and stable (for up to 40 days). The hydrogel 3D culture can be monitored through microscopy and enables cells to acquire a more physiological 'tumor-like' morphology compared to 2D cultures. An enzymatic system (Nattokinase) enables efficient cell retrieval of almost 100% of the cells, which are >95% viable and retain their surface markers. Using this model we showed that A549 lung adenocarcinoma tumor cells organized into glandular clusters whose H&E structures and phenotype was modified by the accessory cells present. In the presence of the stromal cells, a more aggressive phenotype developed: more clusters of bigger size were revealed (cluster/10 hpf 6, 4 and 12, with sizes of 0.03-0.2 mm, 0.1-0.3 mm and 0.1-1.0 mm respectively in the A549, A549+MRC5 and A549+MRC5+HUVECs). Ki-67 staining confirmed a higher proliferation in the presence of both MRC5 and HUVECs (MIB1=15%), compared to MRC5 (MIB1=5%) or A549 only (MIB1=0%). We monitored A549 growth using the bioluminescence from transgenic Ff-luciferase and found significantly increased signal (log10) in the presence of MRC5, as early as D3 of culture (8.96±0.03 vs 8.66±0.08 of A549 only; p<0.0001). Having shown the feasibility of 3D-modeling a lung adenocarcinoma, we compared the response of these 3D tumors to the oncolytic adenovirus (OncAd) ICOVIR15 with the effects on 2D culture and compared both with the activity of the OncAd in vivo in a SCID-beige mice xenograft model by monitoring tumor bioluminescence. The 3D model more closely replicated the kinetics of tumor growth control in vivo than did the 2D model. Control required 14 days in vivo and in the3D model whereas in the 2D culture complete eradication was obtained by day 7 (fold increase in signal in untreated and treated groups respectively at day 14: in vivo, 14.56±7.81 and 6.50±5.24 vs 3D, 7.41±4.02 and 2.75±0.72 vs 2D, 328.3±72.71 and 0.01±0.01). To test the system in vivo, we injected the hydrogels (A549, A549 + MRC5 or A549+MRC5+HUVECs) i.p. into SCID-beige mice and let them grow for 2 weeks before retrieving and analyzing them. The tumor grew as islands and H&E confirmed a more aggressive phenotype, with larger tumor size and higher vascularization in the presence of MRC5 and HUVECs. In conclusion, we developed a 3D lung adenocarcinoma model with morphological and biological features of in vivo tumors. This model offers the opportunity to study, in vitro and in vivo, the role of human stroma in cancer progression and to more realistically predict tumor response to biological and other therapies including Onc Ad.