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: Scientific Data

Article Title: A single cell RNAseq benchmark experiment embedding “controlled” cancer heterogeneity

doi: 10.1038/s41597-024-03002-y

Figure Lengend Snippet: Cellranger statistics.

Article Snippet: PC9 (CSC-C4619J) human lung cancer cell line was purchased from Creative Bioarray; DV90 (ACC 307) and HCC78 (ACC 563) human lung cancer cell lines were provided from DSMZ Leibniz Institute.

Techniques:

Journal: Scientific Data

Article Title: A single cell RNAseq benchmark experiment embedding “controlled” cancer heterogeneity

doi: 10.1038/s41597-024-03002-y

Figure Lengend Snippet: Quality control of the cells. Low quality cells are characterised by having a low number of genes depicted as present, associated with low ribosomal content and high mitochondrial content. ( A ) A549, ( B ) CCL-185-IG, ( C ) CRL5868, ( D ) DV90, ( E ) HCC78, ( F ) HTB178, ( G ) PC9.

Article Snippet: PC9 (CSC-C4619J) human lung cancer cell line was purchased from Creative Bioarray; DV90 (ACC 307) and HCC78 (ACC 563) human lung cancer cell lines were provided from DSMZ Leibniz Institute.

Techniques: Control