Engineering cisplatin-resistant lung adenocarcinoma tissues
Non-small cell lung cancer (NSCLC) constitutes 85% of total lung cancer cases and standard therapy regimens employ the use of cisplatin – a platinum-based DNA damaging drug. However, treatment with cisplatin can often lead to relapse of aggressive tumors with high metastatic potential. A critical characteristic during tumor progression is drug resistance and can be contributed to, in part, the epithelial-to-mesenchymal transition (EMT). Several changes occur during the EMT which serve as a metric for drug resistance. But recapitulating drug resistance in vitro is arduous due to inevitable loss of resistance and increased culture maintenance. Here we show cisplatin-resistant A549s which maintain physiological relevance and allow for high-throughput application. A549 cells were exposed to cisplatin over 96 hour in two 48 hour intervals. Cell survival was verified using flow cytometry to determine optimal cisplatin concentration leading to resistance. A549 parent and 25uM cisplatin-treated spheroids were seeded at 1,000 cells/spheroid in non-adherent round bottom plates. DAPI, Phalloidin, and Ki67 expression was characterized in A549 parent and cisplatin-treated spheroids via immunohistochemistry. A549 parent and cisplatin-treated spheroids were co-cultured with cancer-associated fibroblasts (CAFs) and injected in collagen type I gels. Similarly, DAPI and Ki67 expression as well as α-SMA expression was characterized to elucidate CAF interactions among spheroids. We found that cisplatin- resistant spheroids formed a dysmorphic morphology relative to that of parent spheroids. Additionally, CAF interactions with cisplatin-treated A549 spheroids within collagen type I exhibit increased α-SMA expression along the periphery of the spheroid which could indicate spheroid crosstalk. Additionally, CAFs show an increased alignment among cisplatin-treated spheroid populations relative to untreated spheroid population.