Expansion of airway basal epithelial cells from primary human non-small cell lung cancer tumors

Robert E Hynds, Assma Ben Aissa, Kate H C Gowers, Thomas B K Watkins, Leticia Bosshard-Carter, Andrew J Rowan, Selvaraju Veeriah, Gareth A Wilson, Sergio A Quezada, Charles Swanton, TRACERx Consortium, Sam M Janes, Robert E Hynds, Assma Ben Aissa, Kate H C Gowers, Thomas B K Watkins, Leticia Bosshard-Carter, Andrew J Rowan, Selvaraju Veeriah, Gareth A Wilson, Sergio A Quezada, Charles Swanton, TRACERx Consortium, Sam M Janes

Abstract

Pre-clinical non-small cell lung cancer (NSCLC) models are poorly representative of the considerable inter- and intra-tumor heterogeneity of the disease in patients. Primary cell-based in vitro models of NSCLC are therefore desirable for novel therapy development and personalized cancer medicine. Methods have been described to generate rapidly proliferating epithelial cell cultures from multiple human epithelia using 3T3-J2 feeder cell culture in the presence of Y-27632, a RHO-associated protein kinase (ROCK) inhibitor, in what are known as "conditional reprograming conditions" (CRC) or 3T3 + Y. In some cancer studies, variations of this methodology have allowed primary tumor cell expansion across a number of cancer types but other studies have demonstrated the preferential expansion of normal epithelial cells from tumors in such conditions. Here, we report our experience regarding the derivation of primary NSCLC cell cultures from 12 lung adenocarcinoma patients enrolled in the Tracking Cancer Evolution through Therapy (TRACERx) clinical study and discuss these in the context of improving the success rate for in vitro cultivation of cells from NSCLC tumors.

Trial registration: ClinicalTrials.gov NCT01888601.

Keywords: basal cells; cell culture; epithelial cells; lung cancer; stem/progenitor cells.

© 2018 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

Figures

Figure 1
Figure 1
Frequent expansion of normal human airway epithelial cells from non‐small cell lung cancer (NSCLC) tumors in 3T3‐J2 co‐culture in the presence of Y‐27632 (3T3 + Y). (a) Immunofluorescence staining for pan‐keratin, keratin 5, p63 and TTF‐1 in a representative culture (n = 3 patients; scale bar = 100 μm). (b) In vitro tracheosphere assay. Hematoxylin and eosin (H&E) staining (top panel, scale bar = 1 mm; bottom left panel, scale bar = 50 μm) demonstrated airway differentiation capacity of cell cultures expanded from NSCLC tumors (<passage 5; representative images, n = 4 patients). Immunofluorescence confirmed the presence of ACT+ ciliated cells and MUC5B+ mucosecretory cells (bottom right panel, scale bar = 50 μm). (c) Subcutaneous injection tumorigenesis model. Injection of cultured cells (<passage 5) into NSG mice did not lead to tumor formation but H&E analysis showed the presence of epithelium resembling that found in human airways after 3 months (3/3 patients; top panel scale bar = 500 μm; middle panel scale bar = 50 μm). Immunofluorescence confirmed the presence of ACT+ ciliated cells and MUC5B+ mucosecretory cells (bottom panel, scale bar = 50 μm). (d) Next‐generation sequencing using the Illumina MiSeq platform did not detect mutations found in the tumor of origin in 9/10 NSCLC cell cultures. “Multiple” indicates that more than one variant results from the specific mutation detected, i.e., it affects multiple isoforms at different positions. (e) H&E analysis showed bronchiolar epithelium within a human lung adenocarcinoma tumor (left panel, scale bar = 1 mm; top right panel, scale bar = 100 μm). Immunofluorescence confirmed the presence of p63+ basal epithelial cells within these bronchioles (bottom right panel, scale bar = 50 μm). [Color figure can be viewed at http://wileyonlinelibrary.com]
Figure 2
Figure 2
Expansion of primary human tumor cells from a KRAS‐mutant lung adenocarcinoma in 3T3‐J2 co‐culture in the presence of Y‐27632 (3T3 + Y). Cancer mutation‐bearing cells were detected in 1 of our 10 early passage patient cultures (CRUK0557) by next‐generation sequencing. Sanger sequencing of the same cell culture at later passage (P4; scale bar = 100 μm) revealed that the KRAS mutation was no longer detected (left panel). Injection of the early passage (P2) cell culture into an immune‐compromised (NSG) mouse generated a tumor with mutant KRAS (center panel). A hematoxylin and eosin (H&E)‐stained section is shown (scale bar = 500 μm). A magnified view of the black dotted box is shown below (scale bar = 100 μm). Re‐culture of cells from the cell culture‐derived xenograft in 3T3+Y was possible (right panel; scale bar = 100 μm) and mutant KRAS was again detected by Sanger sequencing. [Color figure can be viewed at http://wileyonlinelibrary.com]

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