OA 18.07 3D Low-Attachment Culture: A Putative Model for STAS And “Floating” Cancer Cells

Cancer cells “floating” in stroma, air space, or lymphovascular channels are often found in aggressive lung adenocarcinomas. Recently, the concept of STAS (spread through alveolar space) has been proposed, and studies have shown that STAS predicts early recurrence and poor patient prognosis. However, the biologic basis for the aggressive phenotype of the tumor with these histologic features remains elusive. In this study, we tested whether 3D (three-dimensional) low-attachment culture could be an in vitro model for STAS and “floating” cancer cells. Lung adenocarcinoma cell lines harboring diverse driver mutations (EGFR, KRAS, BRAF, HER2, RET) were used. Cells were subjected to detached condition by using low-attachment culture dishes to generate “floating” cancer cells in vitro. Cell growth assay, immunohistochemistry and Western blotting were used to characterize the biologic properties of “floating” cancer cells. Cancer cells were inoculated in pleural cavity or left ventricle of the heart of NOD/SCID mice to test their metastatic potential in vivo. Upon detachment, cancer cells formed solid, micropapillary, or hollow ring-like structures, admixed with single cells, recapitulating a histologic spectrum of STAS in primary tumors. Outlining with MUC1, a feature of micropapillary lung adenocarcinoma, was also demonstrated in the in-vitro-generated micropapillary clusters as well. Detached cells initially showed retarded cell growth, but some cell lines regained growth potential with time in culture. Expression analysis of various biomarkers revealed that detached cells showed features of cell stress and altered metabolism, as indicated by increased expression of phospho-p38 (a stress-activated MAP kinase) and GLUT-1 (glucose transporter-1), respectively, and these findings were confirmed by analysis of primary tumors. Finally, cancer cells that adapted to detached conditions exhibited increased metastasis in vivo. 3D low-attachment culture may be a convenient model to study the biology of aggressive lung cancer with STAS and “floating” cancer cells.