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: Oncology Letters

Article Title: Assessment of ALK gene fusions in lung cancer using the differential expression and exon integrity methods

doi: 10.3892/ol.2016.4157

Figure Lengend Snippet: Detection of anaplastic lymphoma kinase fusion in urine from non-small cell lung cancer patients using the differential expression method.

Article Snippet: NSCLC cells and clinical samples The NCI-H3122 human NSCLC cell line (ALK fusion-positive) was provided by AstraZeneca Innovation Center (Shanghai, China).

Techniques: Quantitative Proteomics, Fluorescence In Situ Hybridization, Control

Journal: CytoJournal

Article Title: Oncogene 5’-3’ exoribonuclease 2 enhances epidermal growth factor receptor signaling pathway to promote epithelial–mesenchymal transition and metastasis in non-small-cell lung cancer

doi: 10.25259/Cytojournal_49_2024

Figure Lengend Snippet: XRN2 was upregulated in NSCLC. (a) The messenger RNA levels of XRN2 in the H460 NSCLC cell line and BEAS-2B human bronchial epithelial cells. (b and c) Protein expression levels of XRN2 in H460 and BEAS-2B cells. n = 6. (*** P < 0.001). (XRN2: 5’-3’ exoribonuclease 2, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, H460 cells: NSCLC cell line; BEAS-2B: Human bronchial epithelial cells, NSCLC: Non-small-cell lung cancer.)

Article Snippet: Human NSCLC cell line H460 (iCell-h160) and normal human bronchial epithelial cells BEAS-2B (iCell-h023) were obtained from iCell Biological Science Company (Shanghai, China).

Techniques: Expressing

Journal: CytoJournal

Article Title: Oncogene 5’-3’ exoribonuclease 2 enhances epidermal growth factor receptor signaling pathway to promote epithelial–mesenchymal transition and metastasis in non-small-cell lung cancer

doi: 10.25259/Cytojournal_49_2024

Figure Lengend Snippet: XRN2 promoted migration and EMT progression in NSCLC cells. (a-c) Validation of XRN2 overexpression and knockdown efficiency in H460 cells. (d-g) Migration and invasion assay of H460 cells after XRN2 overexpression and knockdown. (h-k) Protein levels of E-cadherin, N-cadherin, and vimentin in H460 cells after XRN2 overexpression and knockdown. n = 6. (** P < 0.01 and *** P < 0.001). (Ov-NC: Overexpress negative control, XRN2: 5’-3’ exoribonuclease 2, Sh-NC: ShRNA negative control, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, H460 cells: NSCLC cell line, BEAS-2B: Human bronchial epithelial cells, NSCLC: Non-small-cell lung cancer.)

Article Snippet: Human NSCLC cell line H460 (iCell-h160) and normal human bronchial epithelial cells BEAS-2B (iCell-h023) were obtained from iCell Biological Science Company (Shanghai, China).

Techniques: Migration, Over Expression, Knockdown, Invasion Assay, Negative Control, shRNA

Journal: CytoJournal

Article Title: Oncogene 5’-3’ exoribonuclease 2 enhances epidermal growth factor receptor signaling pathway to promote epithelial–mesenchymal transition and metastasis in non-small-cell lung cancer

doi: 10.25259/Cytojournal_49_2024

Figure Lengend Snippet: XRN2 promoted angiogenesis in NSCLC lung metastasis. (a and b) HE stained images of lung metastases. (c and d) IHC analysis of CD31 + cells in lung metastatic lesions. (e and f) Images of tube formation by HUVECs co-cultured with H460-Ov-XRN2 or H460-Sh-XRN2 cells. (g-i) mRNA and protein expression levels of VEGFA in HUVECs under co-culture conditions. n = 6. (** P < 0.01; *** P < 0.001). (Ov-NC: Overexpress negative control, XRN2: 5’-3’ exoribonuclease 2, Sh-NC: ShRNA negative control, CD31: Cluster of differentiation 31, FOV: Field of view, VEGFA: Vascular endothelial growth factor A, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, H460 cells: NSCLC cell line, BEAS-2B: Human bronchial epithelial cells, NSCLC: Non-small-cell lung cancer, Ov-XRN2: XRN2 overexpression, HUVECs: Human umbilical vein endothelial cells .)

Article Snippet: Human NSCLC cell line H460 (iCell-h160) and normal human bronchial epithelial cells BEAS-2B (iCell-h023) were obtained from iCell Biological Science Company (Shanghai, China).

Techniques: Staining, Cell Culture, Expressing, Co-Culture Assay, Negative Control, shRNA, Over Expression

Journal: CytoJournal

Article Title: Oncogene 5’-3’ exoribonuclease 2 enhances epidermal growth factor receptor signaling pathway to promote epithelial–mesenchymal transition and metastasis in non-small-cell lung cancer

doi: 10.25259/Cytojournal_49_2024

Figure Lengend Snippet: XRN2 overexpression promoted the phosphorylation of EGFR in NSCLC cells. (a) The protein bands of p-EGFR and EGFR. (b) Relative expression levels of the p-EGFR/EGFR protein ratio. (c) The EGFR mRNA levels in H460 cells. n = 6. (*** P < 0.001). (Ov-NC: Overexpress negative control, XRN2: 5’-3’ exoribonuclease 2, Sh-NC: ShRNA negative control, EGFR: Epidermal growth factor receptor, p-EGFR: Phosphorylation epidermal growth factor receptor, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, NSCLC: Non-small-cell lung cancer.)

Article Snippet: Human NSCLC cell line H460 (iCell-h160) and normal human bronchial epithelial cells BEAS-2B (iCell-h023) were obtained from iCell Biological Science Company (Shanghai, China).

Techniques: Over Expression, Expressing, Negative Control, shRNA

Journal: CytoJournal

Article Title: Oncogene 5’-3’ exoribonuclease 2 enhances epidermal growth factor receptor signaling pathway to promote epithelial–mesenchymal transition and metastasis in non-small-cell lung cancer

doi: 10.25259/Cytojournal_49_2024

Figure Lengend Snippet: EGFR mediated the biological functions of XRN2 in NSCLC metastasis. (a-d) Transwell assays of H460 cells after transfection with Sh-XRN2 and Ov-EGFR. (e-h) The protein expression levels of E-cadherin, N-cadherin, and vimentin in H460 cells post-transfected with Sh-XRN2 and Ov-EGFR. (i and j) Tube formation by HUVECs co-cultured with H460-Sh-XRN2 or H460-Sh-XRN2+Ov-EGFR. n = 6. (* P < 0.05, ** P < 0.01 and *** P < 0.001). (XRN2: 5’-3’ exoribonuclease 2, Sh-NC: ShRNA negative control, Ov-NC: overexpression negative control, EGFR: Epidermal growth factor receptor, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, NSCLC: Non-small cell lung cancer, Ov-EGFR: EGFR overexpression, HUVECs: Human umbilical vein endothelial cells.)

Article Snippet: Human NSCLC cell line H460 (iCell-h160) and normal human bronchial epithelial cells BEAS-2B (iCell-h023) were obtained from iCell Biological Science Company (Shanghai, China).

Techniques: Transfection, Expressing, Cell Culture, shRNA, Negative Control, Over Expression

Journal: American Journal of Cancer Research

Article Title: Cathepsin C regulates tumor progression via the Yes-associated protein signaling pathway in non-small cell lung cancer

doi:

Figure Lengend Snippet: CTSC affects tumorigenesis in NSCLC in vivo. A. H1975-mock and H1975-CTSC cells are subcutaneously injected into both flanks of BALB/c nude mice (n = 5). Representative images showing the tumor-bearing mice and sizes of tumors injected with H1975-mock and H1975-CTSC cells at 18 days. Representative images of the removed tumors. B, C. Tumor growth is significantly increased in the CTSC overexpression group. D. Representative images of H&E, Ki-67, and CTSC staining. Magnification: × 400. E. H1975-shCtrl and H1975-shCTSC cells are subcutaneously injected into both flanks of BALB/c nude mice (n = 5). Representative images showing the tumor-bearing mice and sizes of tumors injected with H1975-shCtrl and H1975-shCTSC cells at 20 days. Representative images of the removed tumors. F, G. Tumor weight and volume are evaluated after dissecting tumors from the mice in each group. Tumor growth is significantly impaired in the CTSC knockdown group. H. Representative images of H&E, Ki-67, and CTSC staining. Magnification: × 400. Scale bar, 50 μm. *P < 0.1.

Article Snippet: The human NSCLC cell lines H520, H1975, and H1299 were purchased from the Korean Cell Line Bank (Seoul, Korea).

Techniques: In Vivo, Injection, Over Expression, Staining, Knockdown

Journal: BMC Medicine

Article Title: Targeting the epidermal growth factor receptor in non-small cell lung cancer cells: the effect of combining RNA interference with tyrosine kinase inhibitors or cetuximab

doi: 10.1186/1741-7015-10-28

Figure Lengend Snippet: Relevant genotypic changes in the NSCLC cell lines studied

Article Snippet: The human NSCLC cell lines H292 (CRL-1848TM, mucoepidermoid pulmonary carcinoma) was kindly provided by Prof Dr Filip Lardon (Universiteit Antwerpen-CDE Geneeskunde-Oncologie).

Techniques: Mutagenesis

Journal: BMC Medicine

Article Title: Targeting the epidermal growth factor receptor in non-small cell lung cancer cells: the effect of combining RNA interference with tyrosine kinase inhibitors or cetuximab

doi: 10.1186/1741-7015-10-28

Figure Lengend Snippet: Effect of TKI treatment on cell growth inhibition and apoptosis induction . Effect of TKIs gefitinib, erlotinib, and afatinib and cetuximab on the growth (A) and caspase3/7 activity (B) of HCC827, H292, H358, H1650, and H1975 cells after 72-h incubation. Independent experiments were repeated six times and one representative result is shown. Points, mean value of six identical wells of a single representative experiments; bars, SD (n = 6).

Article Snippet: The human NSCLC cell lines H292 (CRL-1848TM, mucoepidermoid pulmonary carcinoma) was kindly provided by Prof Dr Filip Lardon (Universiteit Antwerpen-CDE Geneeskunde-Oncologie).

Techniques: Inhibition, Activity Assay, Incubation