Formation of Tumorspheres with Increased Stemness without External Mitogens in a Lung Cancer Model

Abstract

Like with most solid tumors, the presence of a subpopulation of cancer stem cells (CSCs) or cancer stem-like cells (CS-LCs) has been associated with chemoresistance and tumor relapse in lung cancer cells. In the absence of serum, CSCs/CS-LCs have the ability to grow as lung tumorspheres (LTSs), and this system is routinely used for isolation and characterization of putative CSCs/CS-LCs. Methods to isolate LTSs are usually performed in serum-free media supplemented with specific additives such as epidermal growth factor and basic fibroblast growth factor. In this study, we report the generation of LTSs without the addition of any external mitogenic stimulation. LTSs generated in this manner demonstrated several traits usually associated with increased stemness such as elevated expression of the stemness-associated marker Sox2 and increased chemoresistance to conventional anticancer drugs. In addition, we report that the FDA-approved drug Digitoxin, at concentration close to its therapeutic level, decreased the viability of LTSs and downregulated Sox2 independent of the PI3K/AKT pathway. The potential use of LTSs generated without the addition of any external mitogenic stimulation to study the role of specific factor(s) associated with stemness properties is also discussed.

Figure 1

Morphology of H460 cells grown as spheres without addition of external mitogens. Bars are 1000 μm (4x) and 100 μm (40x). The arrow indicates the same sphere at two different magnifications.

Figure 2

Expression of Sox2, Bcl2, Vimentin, β-actin, and GAPDH in H460 parental cells (P) and lung tumorspheres (LTSs) grown in SFM without external mitogens for 14 and 35 days.

Figure 3

Chemosensitivity of lung tumorspheres compared to the parental H460 cell line. (a) Representative images of lung tumorspheres treated with DMSO (control), Paclitaxel (PX), or Digitoxin (DIG). In these images, the dark appearance of the tumorspheres is because pictures have been taken after incubation with MTT solution to show that the cells are metabolically active and able to reduce the MTT. Bars are 1000 μm (4x). (b) Quantification of the effects of different anticancer agents on parental H460 cells (black columns) and LTSs (white columns). In all cases, H460 parental cells and lung tumorspheres were incubated with the indicated concentration of drugs for 72 h. Cell viability was assessed by the MTT assay for H460 parental cells and by the CCK assay for lung tumorspheres. Data (mean ± ES) are representative of three independent experiments performed in sextuplicate. ^∗ P < 0.05 compared to control value. ^∗∗ P < 0.05 compared between parental and spheres.

Figure 4

DIG downregulates Sox2 expression likely independent of the PI3K/AKT pathway. Expression of Sox2, pAKT, AKT, and GAPDH in untreated and DIG-treated H460 parental cells (P) and lung tumorspheres (LTSs) grown in SFM without external mitogens for 14 days.