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: International Journal of Molecular Sciences

Article Title: The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor–Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells

doi: 10.3390/ijms25094844

Figure Lengend Snippet: IC50 values for gefitinib in parental and gefitinib-resistant (GR) cell lines.

Article Snippet: EGFR-mutant (p.E746_A750del) NSCLC cell lines, HCC827 and PC9, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK), respectively.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor–Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells

doi: 10.3390/ijms25094844

Figure Lengend Snippet: Aberrant activation of EGFR signaling in GR cells. ( a , b ) Western blot analysis of EGFR expression and activation levels in HCC827 ( a ) and PC9 ( b ) parental and GR cells, in the absence or presence of gefitinib at the indicated concentrations. An anti-α-tubulin antibody was used for normalization. ( c , d ) Western blot analysis of expression and activation levels of ERK and AKT in HCC827 ( c ) and PC9 ( d ) parental and GR cells, in the absence or presence of gefitinib at the indicated concentrations. An anti-GAPDH antibody was used for normalization. Densitometric value ratios were calculated for phospho-ERK/total ERK (pERK/ERK) and phospho-AKT/total AKT (pAKT/AKT).

Article Snippet: EGFR-mutant (p.E746_A750del) NSCLC cell lines, HCC827 and PC9, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK), respectively.

Techniques: Activation Assay, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor–Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells

doi: 10.3390/ijms25094844

Figure Lengend Snippet: Evaluation of TGF-β1 expression in GR cells and its role in resistance and cell migration. ( a ) Immunofluorescence analysis of TGF-β1 expression (signaled in red) in HCC827 and PC9 parental and GR cell lines. Nuclei were stained with DAPI (signaled in blue). In the images, 20× magnification was used. ( b ) Immunoassay analysis of TGF-β1 levels in conditioned media from parental and GR cell lines. TGF-β1 levels were normalized for the cell number determined at the harvesting time and folds were calculated versus their respective parental cells. The values reported are the means from two independent experiments, each performed in duplicate. * p < 0.05 for comparison between parental versus GR cells (two-tailed Student’s t -test). ( c ) Cell proliferation assay on HCC827 parental and GR cells treated for 72 h with gefitinib (100 nM) and LY2109761 (LY, 5 µM), alone and in combination. *** p < 0.0005 for comparison between cells treated with gefitinib alone versus those treated with the drug combination (two-tailed Student’s t -test). ( d ) Analysis of wound-healing assays for HCC827 parental and GR-Low cells untreated and treated with LY (5 µM). *** p < 0.0005 for comparison between the treated versus untreated cells (two-tailed Student’s t -test).

Article Snippet: EGFR-mutant (p.E746_A750del) NSCLC cell lines, HCC827 and PC9, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK), respectively.

Techniques: Expressing, Migration, Immunofluorescence, Staining, Comparison, Two Tailed Test, Proliferation Assay