Cross, Neil

Dowling, Joshua

Ferriera de Matos, Cristiana

Leyland, Rebecca

English

Sheffield Hallam University Research Archive

Investigation of TRAIL resistance in lung cancer cell linesDOWLING, Joshua, FERRIERA DE MATOS, Cristiana, LEYLAND, Rebecca and CROSS, Neil <http://orcid.org/0000-0003-2055-5815>Available from Sheffield Hallam University Research Archive (SHURA) at:http://shura.shu.ac.uk/23047/This document is the author deposited version.  You are advised to consult the publisher's version if you wish to cite from it.Published versionDOWLING, Joshua, FERRIERA DE MATOS, Cristiana, LEYLAND, Rebecca and CROSS, Neil (2018). Investigation of TRAIL resistance in lung cancer cell lines. In: British Society for Immunology: Yorkshire Immunology Group Symposium 2018, University of York, UK, 15th June 2018. (Unpublished) Copyright and re-use policySee http://shura.shu.ac.uk/information.htmlSheffield Hallam University Research Archivehttp://shura.shu.ac.ukInvestigation of TRAIL Resistance in Lung Cancer Cell Lines  Joshua Dowling, Cristiana de Matos, Rebecca Leyland, Neil Cross.  Biomedical Research centre, City Campus, Sheffield Hallam University, Howard Street, Sheffield, S1 1WB Introduction  TNF-related apoptosis-inducing ligand (TRAIL) is a important protein expressed by Natural Killer cells within the immune system.  It induces apoptosis preferentially in cancer cells and is a potential therapeutic target Many tumours are TRAIL-resistant,  and TRAIL-resistance emerges readily during therapy. TRAIL-sensitisors may overcome both existing, and emerging TRAIL insensitivity (see Fig 1) Non-proliferative quiescence cancer stem cell-like cells are TRAIL-resistant in some tumour models (Cross, NA. Unpublished observations).  Translational reprogramming agents such is EIF2 inhibitors may overcome the quiescent phenotype and sensitise cells to (Schewe & Aguirre-Ghiso, 2009).    Investigation of Translational reprogramming agent Salubrinal on TRAILR  Populations  The TRAIL sensitivity of 4 cell lines was determined (fig 4) and subsequently, TRAILR populations isolated after persistent TRAIL-treatment by culture of surviving cells after TRAIL treatment. TRAILR populations were confirmed to be less sensitive to TRAIL than parental populations (fig 5). TRAIL did not synergistically enhance TRAIL responses in TRAILR cells (Fig 6).        Figure 4. The percent apoptosis in lung cancer cell lines when exposed to TRAIL at 0150ng/ml for 24hours. Comparison of treated vs. untreated for each cell line is shown and assessed by Kruskal-Wallis multiple comparisons test (p<0.05=* P<0.01=** P<0.001).             Challenge of TRAILR cells with the translational reprogramming agent Salubrinal               Fig 6 Mean percent apoptosis in cell lines A549 (A) and A549 EML4-ALK (B) (ALK translocation) with combination treatments of Salubrinal (25µg/ml) and TRAIL (50ng/ml).  Conclusions and future work. The cell lines A549, and NCI-H2170 do not contain a quiescent population. However more recent work has successfully isolated quiescent cells from other lung cancer cell lines (Fig 3). TRAIL-sensitive cell lines can readily be made TRAILR by persistent treatment with sub-toxic doses of TRAIL, mirroring clinical findings. TRAILR cells could not be sensitised to TRAIL by Salubrinal Ongoing work is aimed at assessing gene expression changes in TRAILR cells and assessing TRAIL resistance in quiescent cells in the new TWIT-Q cell line. Aims and Hypothesis The hypothesis of this study was that quiescent cells isolated from lung cancer could be translationally reprogrammed to bring them out of a resistant state. Aims: 1) Isolate a quiescent phenotype within lung cancer cell lines. 2) Induce translational reprogramming using drug Salubrinal and re-sensitize the population. Methods Non-proliferating cells from lung cancer cells were distinguished using lipophilic membrane PKH67 dye which is lost on cell proliferation and flow cytometry to identify PKH67Hi non-proliferating cells. Apoptosis was assessed in response to TRAIL at 24 and 72 hours using Propidium Iodide and Hoechst 33342 staining.  Cells were re-challenged with to establish a TRAIL resistant (TRAILR) population vs. the parent line •TRAILR cells were treated with Salubrinal (10/25µm) in combination with TRAIL cells to assess the effects of translational reprogramming on acquired TRAILR   Results A549 does not contain a quiescent population, and all PKH67Hi cells were associated with spontaneous cell death (Fig 2)          Figure 2. (A) A549 cells stained with Hoechst 33342 and stimulated with 25ng/mL of TRAIL at 24 hours imaged using fluorescent microscopy to show apoptosis. (B) Images show PKH67Hi cells undergoing apoptosis when exposed to 25ng/mL of TRAIL at 72 hours in cell line A549 at 20x magnification.   Identification of quiescent cells Flow cytometry was used to identify a population of cells which retained PKH67 over a 12 day period. In the A549 cell lines, there was no high presence of PKH67 high cells.                                  Figure 3.  Flow cytometry and fluorescent microscopy data of NCI-H2170 (TOP), NCI-H838 (MIDDLE), and TWIT-Q (BOTTOM) cells. Column (A) represents PKH67 stain populations compared to the parent strain taken at day 1 and column (B) represents the same cells taken at day 12.  Microscopy images taken using the FITC camera of the cells at the same time as flow cytometry are displayed underneath the corresponding flow cytometry data.  A B Pkh67 Fluorescence Pkh67 Fluorescence Pkh67 Fluorescence Pkh67 Fluorescence Pkh67 Fluorescence Pkh67 Fluorescence B A 01020304050607080901000 5 25 50Percentage Dead Cells (%) TRAIL Concentration (ng/mL) Effects of TRAIL on Lung Cancer Cell lines. H2170HCC827A549 EMLY-ALKA549** * *** * ** ** *** * *** ** Figure 5. Mean percentage apoptosis in the parental lung cancer cell lines H2170 (A), A549 (B) and A549 EML4-ALK (C) compared to the TRAILR isolated populations. Data is shown as Mean +/- SD and significance determined by Kruskal-Wallis test (p<0.05=* P<0.01=**). * * * 0204060801000 25 50Percentage Dead cells (%) Trail concentration (ng/mL) ParentstrainTRAILTreatedstrainB 0204060801000 25 50Percentage Dead cells (%) Trail concentration (ng/mL) ParentstrainTRAILtreatedstrainC References Schewe, D& Aguirre-Ghiso (2009). Inhibition of eIF2  Dephosphorylation Maximizes Bortezomib Efficiency and Eliminates Quiescent Multiple Myeloma Cells Surviving Proteasome Inhibitor Therapy. Cancer Research, 69(4), 1545-1552. - Figure 1. Known TRAIL-sensitisers and mechanisms of action (Cross and Sayers, 2014) 0510152025Control 1% DSMO SAL 25ug/ml TRAIL 50ng/ml Sal25+TRAIL50Percentage Dead Cells (%) 0510152025Control 1% DSMO SAL 25ug/ml TRAIL 50ng/ml Sal25+TRAIL50Percentage Dead Cells (%) B A 

Investigation of TRAIL resistance in lung cancer cell lines

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Investigation of TRAIL resistance in lung cancer cell lines

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