Journal: Cancer Science

Article Title: EGFR inhibition in EGFR ‐mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment

doi: 10.1111/cas.15701

Figure Lengend Snippet: CD24 expression is induced in EGFR‐mutant NSCLC cells upon EGFR‐TKI treatment. (A) Differential gene expression between EGFR‐TKI‐treated versus ‐untreated EGFR‐mutant NSCLC cells in GSE75308 (left) and GSE57156 (right) were analyzed. (B) EGFR‐mutant PC9 and H1975 cells, and EGFR‐wild‐type RERF‐LC‐Ad1 and H522 cells were treated with osimertinib for 72 h in vitro. CD24 expression was analyzed by flow cytometry. Gray: isotype control; dotted line: untreated; line: treated. Data are representative of four independent experiments. (C) Numbers of CD24 molecules expressed on cells with or without EGFR‐TKI treatment in vitro. Data are presented as mean ± SEM from four independent experiments. Numbers of the receptors were calculated using the BD QuantiBrite kit as described in Materials and Methods. (D) CD24 gene expression in tumor cells with or without EGFR‐TKI treatment in vitro were analyzed by qRT‐PCR. Data are presented as mean ± SEM of technical replicates from at least two independent experiments. (E) EGFR‐mutant PC9 and EGFR‐wild‐type H522 cells were treated either with osimertinib, gefitinib, or afatinib for 72 h in vitro. CD24 expression was analyzed by flow cytometry. Data are representative of two independent experiments. * q < 0.01.

Article Snippet: The lung cancer cell line, PC9, was obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Expressing, Mutagenesis, Gene Expression, In Vitro, Flow Cytometry, Control, Quantitative RT-PCR

Journal: Cancer Science

Article Title: EGFR inhibition in EGFR ‐mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment

doi: 10.1111/cas.15701

Figure Lengend Snippet: CD24 induction in EGFR‐mutant NSCLC cells is a target of antibody‐dependent cellular phagocytosis. (A) The fraction of immune cell types in the tumor microenvironment of NSCLC from the TCGA‐LUAD dataset (B) Arbitrary gene expression levels of SIGLEC‐10 and MRC1 across macrophages in the tumor microenvironment of NSCLC (C) Siglec‐10 expression was analyzed by flow cytometry. Gray: isotype control, line: Siglec‐10. Human monocyte‐derived macrophages were differentiated according to the protocols shown in Supplementary Table . Data are a representative of at least two independent experiments from three healthy donors. (D) M‐CSF differentiated macrophages and EGFR‐TKI‐treated PC9 cells were incubated for 9 h with anti‐CD24 antibodies or isotype control antibodies. Cytochalasin D was used to inhibit macrophage phagocytosis as described in Materials and Methods. Data are presented as the mean ± SEM of technical replicates from at least two independent experiments. *** p < 0.001.

Article Snippet: The lung cancer cell line, PC9, was obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Mutagenesis, Gene Expression, Expressing, Flow Cytometry, Control, Derivative Assay, Incubation

Journal: Cancer Science

Article Title: EGFR inhibition in EGFR ‐mutant lung cancer cells perturbs innate immune signaling pathways in the tumor microenvironment

doi: 10.1111/cas.15701

Figure Lengend Snippet: EGFR‐TKIs accelerate the release of the cell‐free DNA activating type I IFN response in a STING‐dependent manner. (A) EGFR‐mutant tumor cells were treated with either osimertinib (EGFR‐TKI) or paclitaxel. Data are presented as the mean ± SEM of technical triplicates from at least two independent experiments. (B) cfDNA concentrations in the supernatant were measured as described in Materials and Methods. Data are presented as the mean ± SEM of technical triplicates from at least two independent experiments. (C) THP‐1‐Dual and THP‐1‐Dual STING‐knockout (KO) cells were cultured either with LPS (500 ng/ml), IFNa2a (10,000 U/ml), tumor‐derived cfDNA (PC9 and H1975 cfDNA)/lipofectamine, or lipofectamine alone. IRF (left) and NF‐κB (right) activities were analyzed as described in Materials and Methods. (D) PMA‐differentiated THP‐1 monocytes were pretreated either with lipofectamine, poly(dA:dT) (100 ng/ml), tumor‐derived cfDNA (PC9) (100 ng/ml)/lipofectamine, or IFNa2a (20,000 U/ml) followed by IFN‐γ stimulation (100 ng/ml). (E) Expression levels of CXCL9 and CXCL10 were analyzed using flow cytometry. * p < 0.05, ** p < 0.01, **** p < 0.0001, * q < 0.01. ns, not significant.

Article Snippet: The lung cancer cell line, PC9, was obtained from the European Collection of Authenticated Cell Cultures.

Techniques: Mutagenesis, Knock-Out, Cell Culture, Derivative Assay, Expressing, Flow Cytometry

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: IC 50 values (nM) for the mutant EGFR-expressing Ba/F3 cells, PC9 cells or MGH121 cells.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Mutagenesis, Expressing

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a ) The results of screening the growth-inhibitory activity of 30 drugs in Ba/F3 cells expressing four types of EGFR-del19 with or without T790M or C797S mutations are shown in a heat map. Ba/F3 cells expressing each EGFR mutant were treated with 100 nM of the indicated inhibitors. After 72 h of drug treatment, the cell viability was measured using the CellTiter-Glo assay. Relative cell viability was calculated from each value divided by the DMSO control. Among the inhibitors, only brigatinib and ponatinib were sufficiently efficacious against the triple-mutant EGFR. AZD3463 acted as a weak inhibitor to the triple mutation. ( b ) Growth inhibition assessed by the CellTiter-Glo assay of EGFR-C797S/T790M/del19 (triple-del19)-mutated Ba/F3 cells treated with gefitinib, osimertinib and brigatinib.; N =3. Results are expressed as mean±s.d. IC 50 values were calculated using growth inhibition assay. ( c ) Phosphorylation of EGFR and downstream signals were significantly inhibited by brigatinib in Ba/F3 cells expressing triple-del19 even though afatinib and osimertinib did not suppress at all the EGFR signalling of triple-del19.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Activity Assay, Expressing, Mutagenesis, Glo Assay, Control, Inhibition, Growth Inhibition Assay, Phospho-proteomics

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a ) Chemical structures of six ALK–TKIs were very similar. ( b , c ) IC 50 values in Ba/F3 cells expressing four mutation types of EGFR-del19 were obtained by treatment with brigatinib, AP26113-analog, AZD3463, TAE684, ceritinib and ASP3026 for 72 h. Those of C797S/T790M/del19 were shown by bar graph ( b ) and those of all mutation types were demonstrated by a table ( c ). The CellTiter-Glo assay was used to measure cell viability. ( d , e ) Ba/F3 cells expressing T790M/del19 ( d ) or C797S/T790M/del19 ( e ) were treated with the indicated concentrations of brigatinib, AP26113 analog, TAE684, ceritinib or ASP3026 for 6 h. Phosphorylation of EGFR and its downstream signals were evaluated by western blotting with the indicated antibodies.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Expressing, Mutagenesis, Glo Assay, Phospho-proteomics, Western Blot

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a – e ) PC9 (del19) ( a ), PC9-T790M (T790M/del19) ( b ), PC9-triple mutant (C797S/T790M/del19) ( c ), MGH121 parent (T790M/del19) ( d ) and MGH121 resistant-2 (C797S/T790M/del19) ( e ) cells were treated with serially diluted gefitinib, osimertinib and brigatinib for 72 h. Cell viability was measured using the CellTiter-Glo assay.; N =3. Results are expressed as mean±s.d. ( f ) Western blotting of PC9 triple mutant (C797S/T790M/del19) cells indicated that brigatinib and AP26113 analog, but not afatinib or osimertinib, suppressed phosphorylation of EGFR and its downstream signalling. ( g ) Similar results were obtained in MGH121 resistant-2.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Mutagenesis, Glo Assay, Western Blot, Phospho-proteomics

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a ) The cell growth inhibition of Ba/F3 cells expressing EGFR-C797S/T790M/del19 (EGFR-triple-del19) treated with brigatinib, AP26113-analog, AZD3463 and osimertinib at indicated concentrations combined with or without cetuximab (10 μg ml −1 ) for 72 h assessed by CellTiter-Glo assay. ( b ) Inhibition of EGFR signal pathway in BaF3 EGFR-triple-del19 cells treated with brigatinib+cetuximab (10 μg ml −1 ) for 6 h was evaluated using western blotting. ( c , d ) The cell growth inhibition of PC9 triple-mutant cells ( c ) and MGH121-res2 cells ( d ) treated with brigatinib and osimertinib at indicated concentrations combined with or without cetuximab (10 μg ml −1 ) for 72 h assessed by CellTiter-Glo assay. ( e , f ) Inhibition of EGFR signal pathway in PC9 triple-mutant cells ( e ) and MGH121-res2 cells ( f ) treated with brigatinib+cetuximab (10 μg ml −1 ) for 6 h was evaluated using western blotting.; Results in a , c , e are expressed as mean±s.d. ( N =3). The significance of difference between indicated groups are calculated by Student's t -test (NS; not significant, * P <0.05, ** P <0.01).

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Inhibition, Expressing, Glo Assay, Western Blot, Mutagenesis

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a , b ) PC9 cells expressing EGFR-C797S/T790M/del19 were subcutaneously implanted into Balb-c nu/nu mice. When the average tumour volume reached ∼200 mm 3 , the mice were randomized into vehicle control or treatment groups (50 mg kg −1 of osimertinib, 75 mg kg −1 of brigatinib, 1 mg per mouse of cetuximab three times a week or 75 mg kg −1 of brigatinib combined with cetuximab administered as previously described) and treated once daily by oral gavage for the indicated period. Tumour volume ( V ) was calculated as 0.5 × length × width 2 , and body weights (B.W.) of mice were measured twice weekly.; N =6. Results are expressed as mean±s.d. The significance of difference between the mean tumour volume of control and of brigatinib on day 7, between brigatinib and brigatinib+cetuximab on day 23, respectively, are calculated by Mann–Whitney U test (** P <0.01). ( c ) Survival periods of mice in each treatment arm were demonstrated using the Kaplan–Meier curve. ( d ) Phosphorylation of EGFR and its downstream signalling in two tumour samples obtained from each group were evaluated using western blotting. ( e , f ) In vivo experiment of PC9 triple-mutant cells following a similar protocol as in , using panitumumab 0.5 mg per mouse two times a week administered peritoneally instead of cetuximab.; N =6. Results are expressed as mean±s.d. The significance of difference between the mean tumour volume of control and of brigatinib on day 16, between brigatinib and brigatinib+panitumumab on day 23, respectively, are calculated by Mann–Whitney U test (** P <0.01). ( g ) A Kaplan–Meier curve of the survival of the mice in each treatment arm. ( h ) Phosphorylation of EGFR and its downstream signalling in two tumour samples obtained from xenografts of PC9-triple mutant cells treated for 8 days with the indicated drugs (brigatinib: 75 mg kg −1 daily, administered orally; panitumumab: 0.5 mg per mouse two times a week, administered peritoneally) were assessed by western blotting with the indicated antibodies.

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Expressing, Control, MANN-WHITNEY, Phospho-proteomics, Western Blot, In Vivo, Mutagenesis

Journal: Nature Communications

Article Title: Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

doi: 10.1038/ncomms14768

Figure Lengend Snippet: ( a , b ) MGH121-res2 expressing EGFR-C797S/T790M/del19 were subcutaneously implanted into SCID-beige mice. When the average tumour volume reached ∼200 mm 3 , the mice were randomized into vehicle control and treatment groups (50 mg kg −1 of osimertinib (po), 75 mg kg −1 of brigatinib (po), 1 mg per mouse of cetuximab two times a week and 75 mg kg −1 of brigatinib combined with cetuximab administered as previously described, respectively) and treated for the indicated period. Tumour volume ( V ) was calculated as 0.5 × length × width 2 , and the body weights (B.W.) of the mice were measured twice weekly. N =6. Results are expressed as mean±s.d. The significance in difference between the mean tumour volume of control and of osimertinib, brigatinib and cetuximab, between cetuximab and brigatinib+cetuximab, respectively, on day 42 are calculated by Mann–Whitney U test (NS: not significant, * P <0.05, ** P <0.01).

Article Snippet: PC9-EGFR- C797S/T790M/del19 (PC9-triple-mutant) cells (6 × 10 6 ) or PC9-EGFR-T790M/del19 (PC9-T790M) cells (6 × 10 6 ) were suspended in 100 μl of 1:2 Matrigel and subcutaneously implanted into Balb-c nu/nu mice (Charles River).

Techniques: Expressing, Control, MANN-WHITNEY

Journal: Translational Cancer Research

Article Title: Chronic nicotine exposure affects programmed death-ligand 1 expression and sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer

doi: 10.21037/tcr.2019.05.02

Figure Lengend Snippet: Morphology of cells after treatment with gefitinib in PC9 and PC9/N cells. Cells were seeded in six-well culture plates, and after 24 hrs, the cells were treated with gefitinib (0, 0.1, or 1 µM) for 48 hrs. Cells were photographed under a phase contrast inverted microscope. Cells were stained with Wright and Giemsa (original magnification 200×). These results are representative of technical triplicate.

Article Snippet: Human NSCLC cell line PC9 (EGFR exon 19 deletion) was purchased from Korean Cell Line Bank (Seoul, Korea).

Techniques: Inverted Microscopy, Staining

Journal: Translational Cancer Research

Article Title: Chronic nicotine exposure affects programmed death-ligand 1 expression and sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer

doi: 10.21037/tcr.2019.05.02

Figure Lengend Snippet: Cell viability assay for PC9 and PC9/N cells cultured in the presence of gefitinib. Cell growth inhibition in response to gefitinib was evaluated by using the MTT assay. Cells were treated with the indicated concentrations of gefitinib and cell viability was determined 48 hrs later. There are mean of independent triplicate experiments. The data are presented as means ± SEM from technical triplicate. PC9/N: nicotine (1 µM) expose for 3 months in PC9 cells. *, P<0.05; **, P<0.01 and ***, P<0.001 compared with PC9 + gefitinib 0 µM. ## , P<0.01 and ### , P<0.001 compared with PC9/N + gefitinib 0 µM. $ , P<0.05 between two cells with gefitinib 0.01 µM. Data are presented as mean ± standard error of the mean (SEM). SEM, standard error.

Article Snippet: Human NSCLC cell line PC9 (EGFR exon 19 deletion) was purchased from Korean Cell Line Bank (Seoul, Korea).

Techniques: Viability Assay, Cell Culture, Inhibition, MTT Assay

Journal: Translational Cancer Research

Article Title: Chronic nicotine exposure affects programmed death-ligand 1 expression and sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer

doi: 10.21037/tcr.2019.05.02

Figure Lengend Snippet: The mRNA expression of cells after treatment with gefitinib in PC9 and PC9/N cells. Cells were treated with gefitinib (0, 0.1, or 1 µM) for 48 hrs, and the mRNA expressions of α1-nAchR, and PD-L1 were examined by quantitative reverse transcription (qRT)-PCR (A) and RT-PCR (B). These results are representative of technical triplicate. *, P<0.05 and ***, P<0.001 compared with PC9 cells. ### , P<0.001 compared with PC9 cells + gefitinib 0 µM and $$$ , P<0.001 compared with PC9/N + gefitinib 0 µM. nAchR, nicotinic acetylcholine receptors; PD-L1, programmed death ligand 1.

Article Snippet: Human NSCLC cell line PC9 (EGFR exon 19 deletion) was purchased from Korean Cell Line Bank (Seoul, Korea).

Techniques: Expressing, Reverse Transcription, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction

Journal: Translational Cancer Research

Article Title: Chronic nicotine exposure affects programmed death-ligand 1 expression and sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer

doi: 10.21037/tcr.2019.05.02

Figure Lengend Snippet: The protein expression levels of cells after treatment with gefitinib in PC9 and PC9/N cells. The cells were cultured with gefitinib (0, 0.1, or 1 µM) for 48 hrs, and EGFR, mTOR, AKT, PD-L1 and α1-nAchR was detected by Western blot. β-actin was used as an internal control. Western blot was quantified by densitometry and ImageJ. These results are representative of technical triplicate. EGFR, epidermal growth factor receptor; PD-L1, programmed death ligand 1; nAchR, nicotinic acetylcholine receptors.

Article Snippet: Human NSCLC cell line PC9 (EGFR exon 19 deletion) was purchased from Korean Cell Line Bank (Seoul, Korea).

Techniques: Expressing, Cell Culture, Western Blot, Control

Journal: Translational Cancer Research

Article Title: Chronic nicotine exposure affects programmed death-ligand 1 expression and sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer

doi: 10.21037/tcr.2019.05.02

Figure Lengend Snippet: Immunofluorescence staining of PD-L1 and phosphorylation of EGFR in PC9 and PC9/N cells treated with 0.1 µM gefitinib. The localizations of PD-L1 and p-EGFR (green signal; Alexa488) in PC9 and PC9/N cells were shown by immunofluorescence counterstained with DAPI (blue signal) and analyzed by confocal microscopy (original magnification 400×). These results are representative of technical triplicate. DAPI, 4',6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor.

Article Snippet: Human NSCLC cell line PC9 (EGFR exon 19 deletion) was purchased from Korean Cell Line Bank (Seoul, Korea).

Techniques: Immunofluorescence, Staining, Phospho-proteomics, Confocal Microscopy

Journal: Translational Cancer Research

Article Title: Chronic nicotine exposure affects programmed death-ligand 1 expression and sensitivity to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer

doi: 10.21037/tcr.2019.05.02

Figure Lengend Snippet: Overall proportion of PD-L1 TPS expression according to pack-year in NSCLC patients harboring activating EGFR mutation. Heavy smokers ( ≥ 30 PY) tended to have higher expression ( ≥ 50% PD-L1 TPS) than never and light smokers, however, Fisher exact test showed no statistical significance (P value =0.628). PD-L1, programmed death ligand 1; TPS, tumor proportion score; NSCLC, non-small cell lung cancer; EGFR, epidermal growth factor receptor; PY, pack-year.

Article Snippet: Human NSCLC cell line PC9 (EGFR exon 19 deletion) was purchased from Korean Cell Line Bank (Seoul, Korea).

Techniques: Expressing, Mutagenesis

Journal: Antioxidants

Article Title: H 2 S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine

doi: 10.3390/antiox13010051

Figure Lengend Snippet: NSCLC cell lines present cysteine reliance on H 2 S production. ( A ) NSCLC cell lines showed similar basal levels of H 2 S production. To verify if H 2 S production was directly dependent on cysteine degradation, cells were exposed to AOAA/PAG treatment. ( B ) NSCLC cells cultured in the presence of the H 2 S donor NaHS alone and in combination with cysteine showed that H 2 S levels decreased when exposed to AOAA and PAG. Upon cysteine treatment, PC-9 showed a tendency to decrease the average levels of H 2 S in control conditions and compensated production of H 2 S upon exposure to inhibitors. ( C ) Upon cysteine supplementation, NSCLC cell lines tended to decrease H 2 S production at T = 0 h but compensated the CBS and CSE inhibition, maintaining (H292 and PC-9) or increasing (A549) H 2 S levels upon AOAA and PAG exposure. Analysis of H 2 S at T = 0 h indicated that A549 and H292 cell lines presented decreased levels of H 2 S in the presence of cysteine with and without glycolysis inhibition with BPA, while the PC-9 cell line presented no significant changes in all conditions. ( D ) Analysis of H 2 S levels showed that all cell lines presented a similar basal ability to produce H 2 S. Maximum score ( E ) or the average levels ( F ) of ATP production indicated that A549 cells increased the production of ATP upon cysteine supplementation independently of the presence of inhibitors, while H292 and PC-9 were able to maintain ATP levels in all experimental conditions. Maximum ATP score and average levels indicated that A549 cells increased the levels of ATP upon BPA and/or cysteine supplementation, and H292 and PC-9 cells maintained the ATP levels in all culture conditions independently of glycolysis inhibition. All data were normalized to the control condition and are represented as mean ± SD. * p < 0.5, ** p < 0.01, **** p < 0.0001.

Article Snippet: Human adenocarcinoma cell line A549 (CCL-185™), mucoepidermoid carcinoma cell line H292 (CRL-1848™), tubular renal cell line HK2 (CRL-2190™), and keratinocyte cell line HaCaT (PCS-200-011™) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and adenocarcinoma cell line PC-9 (90071810) was obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK).

Techniques: Cell Culture, Inhibition

Journal: Antioxidants

Article Title: H 2 S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine

doi: 10.3390/antiox13010051

Figure Lengend Snippet: NSCLC cells express distinct expression patterns of enzymes and transporters involved in cysteine metabolism. ( A ) mRNA expression level analysis showed that H292 cells expressed noteworthy levels of CBS and SLC7A11 compared to A549 cells, while PC-9 cells showed low overall expression of CTH , MPST , SLC1A1, and SLC7A11 . ( B ) Immunofluorescence analysis showed that protein expression followed mRNA expression patterns, reporting similar differences. ( C ) mRNA expression level analysis further showed that H292 cells highly expressed GOT2 , while PC-9 showed high expression levels of GOT2 and CDO1 compared to A549 cells. All mRNA expression level data are relative to HPRT1 and represented as mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human adenocarcinoma cell line A549 (CCL-185™), mucoepidermoid carcinoma cell line H292 (CRL-1848™), tubular renal cell line HK2 (CRL-2190™), and keratinocyte cell line HaCaT (PCS-200-011™) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and adenocarcinoma cell line PC-9 (90071810) was obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK).

Techniques: Expressing, Immunofluorescence

Journal: Antioxidants

Article Title: H 2 S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine

doi: 10.3390/antiox13010051

Figure Lengend Snippet: Distinct molecular backgrounds induce individual metabolic patterns in NSCLC cells. ( A – C ) 1 H-Nuclear magnetic resonance (NMR) of the NSCLC panel studied indicated alterations between A549, H292, and PC-9 regarding levels of intracellular amino acids and peptides, sugars and organic acids, and other metabolites. ( D – F ) NMR of the NSCLC panel studied further indicated alterations between A549, H292, and PC-9 regarding levels of extracellular amino acids and peptides and sugars and organic acids. Data are represented as mean ± SD. * p < 0.5, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ( G , H ) PCA showed no differences in the endometabolome ( G ), but the exometabolome ( H ) indicated that A549 and PC-9 cells present distinct metabolic profiles, and H292 cells had common metabolic patterns with A549 and PC-9 cells. ( I ) PLS-DA analysis of the exometabolome allowed the discrimination of the three cell lines. From the PLS-DA analysis of the metabolites present in the cell media, it was possible to discriminate PC-9 from the other cell lines in the first component, while A549 could be discriminated in the second component (upper panel), Q 2 = 0.873. ( J ) The loading plot (lower panel) showing the metabolites important for the discrimination, colored by VIP in the first component. 2-Oxoisocaproate increased in A549, while aspartate, lactate, phosphocholine, and glycerophosphocholine increased in PC-9, making them important for discrimination between these cell lines. ( K ) From the PLS-DA analysis of the metabolites present in the cell media, it was possible to discriminate between A549 and PC-9 cell lines (upper panel), Q 2 = 0.975. ( L ) The loading plot (lower panel) showing the metabolites important for the discrimination, colored by VIP in the first component. ( M ) Metabolites significantly different between PC-9 and A549. Volcano plot (fold change >2 and p value < 0.05) of the intracellular metabolites between A549 and PC-9. Arginine, proline, and nicotinurate were significantly increased in A549 and guanosine in PC-9. ( N ) Nicotinurate was only present in A549 cells. Box plot of nicotinurate intracellular concentrations in the three cell lines (ANOVA analysis p value = 0.00028097 × 10 4 .

Article Snippet: Human adenocarcinoma cell line A549 (CCL-185™), mucoepidermoid carcinoma cell line H292 (CRL-1848™), tubular renal cell line HK2 (CRL-2190™), and keratinocyte cell line HaCaT (PCS-200-011™) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and adenocarcinoma cell line PC-9 (90071810) was obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK).

Techniques: Nuclear Magnetic Resonance

Journal: Antioxidants

Article Title: H 2 S-Synthesizing Enzymes Are Putative Determinants in Lung Cancer Management toward Personalized Medicine

doi: 10.3390/antiox13010051

Figure Lengend Snippet: NSCLC cell lines are chemoresistant, and SeChry@PURE G4 -FA induces decreased cell viability in NSCLC cells, with specificity toward tumor cells rather than nontumoral cells. ( A ) NSCLC cell lines exposed to the most commonly used therapy regimens showed overall resistance to these drugs, except for cisplatin. ( B ) EC 50 curves and values for SeChry, Sechry@PURE G4- FA, and PURE G4- FA in NSCLC indicated a higher sensitivity of A549 and H292 to the treatment than PC-9. Empty nanoparticles (PURE G4. FA) did not induce relevant toxicity on cell viability. ( C ) EC 50 curves and values for SeChry, Sechry@PURE G4- FA, and PURE G4- FA in noncancer cell lines (HaCaT and HK2) indicated no effect on cell viability. Empty nanoparticles (PURE G4. FA) did not induce relevant toxicity on cell viability. ( D ) Detection of FR-α by immunofluorescence indicated high protein levels in NSCLC but not in nontumoral cell lines. FR- α is labeled in green, and nuclei were counterstained with DAPI (blue). Magnification 400×, scale 20 µm. *** p < 0.001, **** p < 0.0001.

Article Snippet: Human adenocarcinoma cell line A549 (CCL-185™), mucoepidermoid carcinoma cell line H292 (CRL-1848™), tubular renal cell line HK2 (CRL-2190™), and keratinocyte cell line HaCaT (PCS-200-011™) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), and adenocarcinoma cell line PC-9 (90071810) was obtained from European Collection of Authenticated Cell Cultures (ECACC, Porton Down, Salisbury, UK).

Techniques: Immunofluorescence, Labeling

Journal: Technology in Cancer Research & Treatment

Article Title: KRT6A Promotes EMT and Cancer Stem Cell Transformation in Lung Adenocarcinoma

doi: 10.1177/1533033820921248

Figure Lengend Snippet: Keratin 6A knockdown undermine the proliferation and migration ability of lung cancer cells. A and B, KRT6A was knocked down by siRNA, siRNA significantly knocked down KRT6A mRNA expression and protein level in A549 and PC-9 cell lines (the Western blots were quantified by Image J). C and D, CCK8 assay shown si-KRT6A could significantly undermine the proliferation of A549 and PC-9 cells. E, Would healing assay shown that KRT6A knockdown significantly undermined the migration ability in lung cancer cells. F, By measuring the supernatant LDH level, the cell death rate was determined, no significant difference was found between si-KRT6A and NC groups, which indicate KRT6A knockdown did not influence the cell death rate between NC and KRT6A knockdown group. CCK8 indicates Cell Counting Kit-8; KRT6A, keratin 6A; mRNA, messenger RNA; NC, negative control; siRNA, small interfering RNA.

Article Snippet: The lung cancer cell line, A549 was obtained from the ATCC (American Type Culture Collection) and PC-9 was obtained from NanJing KeyGen Biotech Co, Ltd. A549 and PC-9 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, Inc, Waltham, MA; cat. no. 12491-015) supplemented with 10% fetal bovine serum (FBS; cat. no. 10500064; Gibco; Thermo Fisher Scientific, Inc) and penicillin/streptomycin cultured in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Knockdown, Migration, Expressing, Western Blot, CCK-8 Assay, Cell Counting, Negative Control, Small Interfering RNA

Journal: Technology in Cancer Research & Treatment

Article Title: KRT6A Promotes EMT and Cancer Stem Cell Transformation in Lung Adenocarcinoma

doi: 10.1177/1533033820921248

Figure Lengend Snippet: Keratin 6A is involved in CSCs transformation. A and B, CXCR4 high /CD133 high cells were identified as CSCs, compared with si-KRT6A groups, NC groups have significantly expanded CSCs population (Q2). C and D, The colony formation assay shown that compared with si-KRT6A A549 and PC-9 cells the normal control cancer cells (NC groups) have greater biological advantage in forming colonies. CSC indicates cancer stem cell; KRT6A, keratin 6A; NC, negative control.

Article Snippet: The lung cancer cell line, A549 was obtained from the ATCC (American Type Culture Collection) and PC-9 was obtained from NanJing KeyGen Biotech Co, Ltd. A549 and PC-9 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific, Inc, Waltham, MA; cat. no. 12491-015) supplemented with 10% fetal bovine serum (FBS; cat. no. 10500064; Gibco; Thermo Fisher Scientific, Inc) and penicillin/streptomycin cultured in a humidified incubator at 37°C and 5% CO 2 .

Techniques: Transformation Assay, Colony Assay, Control, Negative Control