Tumor Initiation Capacity and Therapy Resistance Are Differential Features of EMT-Related Subpopulations in the NSCLC Cell Line A549

Abstract

Cell lines are essential tools to standardize and compare experimental findings in basic and translational cancer research. The current dogma states that cancer stem cells feature an increased tumor initiation capacity and are also chemoresistant. Here, we identified and comprehensively characterized three morphologically distinct cellular subtypes in the non–small cell lung cancer cell line A549 and challenge the current cancer stem cell dogma. Subtype-specific cellular morphology is maintained during short-term culturing, resulting in the formation of holoclonal, meroclonal, and paraclonal colonies. A549 holoclone cells were characterized by an epithelial and stem-like phenotype, paraclone cells featured a mesenchymal phenotype, whereas meroclone cells were phenotypically intermediate. Cell-surface marker expression of subpopulations changed over time, indicating an active epithelial-to-mesenchymal transition (EMT), in vitro and in vivo. EMT has been associated with the overexpression of the immunomodulators PD-L1 and PD-L2, which were 37- and 235-fold overexpressed in para- versus holoclone cells, respectively. We found that DNA methylation is involved in epigenetic regulation of marker expression. Holoclone cells were extremely sensitive to cisplatin and radiotherapy in vitro, whereas paraclone cells were highly resistant. However, inhibition of the receptor tyrosine kinase AXL, whose expression is associated with an EMT, specifically targeted the otherwise highly resistant paraclone cells. Xenograft tumor formation capacity was 24- and 269-fold higher in holo- than mero- and paraclone cells, respectively. Our results show that A549 subpopulations might serve as a unique system to explore the network of stemness, cellular plasticity, tumor initiation capacity, invasive and metastatic potential, and chemo/radiotherapy resistance.

A Mechanism for Epithelial-Mesenchymal Heterogeneity in a Population of Cancer Cells

“…Hybrid E / M cells play a key role in the collective dissemination of tumor cells as clusters, an aggressive mechanism of cancer metastasis (Jolly et al, 2015). Further, EMP-associated phenotypes differ in their tumor-seeding abilities (Grosse-Wilde et al, 2018;Neelakantan et al, 2017) and in their sensitivity to drugs (Creighton et al, 2009;Tièche et al, 2018). Understanding the mechanisms driving EMP will thus be a critical step in the development of more effective anti-cancer therapies.…”

HSP90/AXL/eIF4E-regulated unfolded protein response as an acquired vulnerability in drug-resistant KRAS-mutant lung cancer

“…Our models recapitulate tumor progression upon cancer drugs, which might differ from that occurring under pathological circumstances such as de novo intratumor heterogeneity. A549 cells displayed marked heterogeneity 28–30 , evidenced by coexistence of epithelial (holoclone) and mesenchymal (paraclone) subpopulations under standard culture conditions (Supplementary Fig. 8A).…”

“…8A). Transcriptomic profiling of the A549 holoclone and paraclone 30 revealed overexpression of epithelial [ CDH1 (E-Cadherin), TJP3 (tight junction protein ZO-3), CLDN4/7 (epithelial tight junction proteins Claudin 4/7)], and mesenchymal ( SNAI2 , ZEB2 and VIM ) markers, respectively. Notably, numerous genes encoding key components of the resistance pathway, most prominently GAS6 [encoding growth arrest-specific protein 6 (GAS-6), a cognate ligand of AXL], AXL (AXL), MKNK1 (MNK1), HSP90AA1 , and HSP90AB1 that encode the cytosolic isoforms HSP90α and HSP90β, respectively, were expressed at significantly higher levels in A549 paraclones than the holoclone (Fig.…”

Distinct characteristics of dasatinib‑induced pyroptosis in gasdermin E‑expressing human lung cancer A549 cells and neuroblastoma SH‑SY5Y cells

“…In a recent report, pyroptosis was shown to be induced in A549 cells by the chemotherapy agent cisplatin, but not by paclitaxel (29). These differences may be due to the heterogeneity of A549 cell populations as three sub-types were discovered according to cell morphological and molecular features (30).…”