It was proposed that the hybrid-E/M state is controlled by a regulatory network consisting of mir-200/Zeb and Snail/miR34 . Methods NSCLC A549 and H460 cells were treated with pemetrexed for 72?h. In addition, 24?h of cisplatin treatment was initiated at day 1, 2 AZ32 or 3 3 resulting in either simultaneous pemetrexed application or pemetrexed pretreatment for 24 or 48?h, respectively. Cell growth and colony formation as well as senescence induction were quantified after treatment. Cell cycle distribution and phosphorylation of histone variant H2AX as a surrogate marker for DNA damage was quantified by flow cytometry. Relative changes in gene expression were determined by quantitative real time PCR. Results Prolonged pemetrexed pretreatment for 48?h prior to cisplatin treatment maximally delayed long-term cell growth and significantly reduced the number of recovering clones. Moreover, apoptosis and senescence were augmented and recovery from treatment-induced DNA damage was delayed. Interestingly, a cell population was identified that displayed an epithelial-to-mesenchymal transition (EMT) and which had a stem cell phenotype. This population was highly resistant to concomitant pemetrexed-cisplatin treatment but was sensitized by pemetrexed pretreatment. Conclusions Adaptation of the standard treatment schedule to include pretreatment with pemetrexed optimizes the anticancer efficiency of pemetrexed-cisplatin combination therapy, which correlates with a persistence of treatment-induced DNA damage. Therefore, this study warrants further investigations to elucidate whether such an adaptation could enhance the effectiveness of the standard clinical LAIR2 treatment regimen. In addition, a subpopulation of therapy resistant cells with EMT and cancer stem cell features was identified that was resistant to the standard treatment regimen but sensitive to pemetrexed pretreatment combined with cisplatin. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2117-4) contains supplementary material, which is available to authorized users.  and we have recently shown that blocking EMT abrogates resistance to MTA in NSCLC . Mesenchymal cells are characterized by a loss of cell-to-cell contact and a spindle-shaped morphology (reviewed in ). Expression of NANOG, Sox2, CD44 is associated with stemness in various tissues and has allowed AZ32 the identification of normal stem cells and subsequently also of cancer stem cells (CSCs; reviewed in . For lung cancer, CSCs were identified by means of numerous markers, e.g. drug-resistant side-population, CD133+, ALDHhigh and EpCAM+ cells (for references, see ). However, similar to the latest discoveries concerning the EMT status, more recent findings indicate that increased plasticity might also be present within cancer populations, enabling bidirectional interconvertibility between CSCs and non-CSCs (reviewed in ). In this study, we aimed to optimize the MTA-cisplatin anticancer modality and subsequently performed an in-depth molecular and cellular analysis to elucidate the molecular mechanisms underlying the observed benefit of sequential combination therapy. We demonstrated that prolonged MTA pretreatment improved the combination therapys efficiency. This effect correlated with the induction of persistent DNA damage, increased apoptosis and senescence initiation. The occurrence of resistant clones was thereby diminished, however those that did remain featured an epithelial-to-mesenchymal phenotype and were enriched for stem cell traits. Methods Cell culture and reagents The NSCLC cell lines A549 (CCL-185) and H460 (HTB-177) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbeccos modified Eagles AZ32 medium nutrient mixture F-12 Ham (Cat. #D6421, Sigma-Aldrich, St. Louis, MO, USA), supplemented with 10?% fetal bovine serum (Cat. #10270-106; Life Technologies, Grand Island, NY, USA), 1?% Penicillin/Streptomycin solution (Cat. #P0781, Sigma-Aldrich) and 1?%?L-Glutamine (Cat. #25030-024, Sigma-Aldrich) at 37?C in a humidified 5?% CO2 incubator. Cell lines were previously DNA fingerprinted (Microsynth, Bern, Switzerland). Medium was changed every 3?days. Pemetrexed/MTA (commercial name ALIMTA; Cat #VL7640) was purchased from Eli Lilly (Suisse) S.A. (Vernier/Geneva, Switzerland). Cisplatin (commercial name Cisplatin Ebewe) was purchased from Sandoz Pharmaceuticals AG (Steinhausen/Cham, Switzerland). Drug response and senescence associated -galactosidase assay To determine cell growth during the treatment and the AZ32 initial recovery phase, 1106 cells were seeded into 150?mm 20?mm tissue culture treated plates (Cat. #20151, SPL Life Sciences Co., Ltd, Korea). Parallel experiments were performed in triplicate and samples were subsequently processed for flow cytometry as described below..