Journal: Pharmacogenomics and Personalized Medicine

Article Title: Toward precision medicine with next-generation EGFR inhibitors in non-small-cell lung cancer

doi: 10.2147/PGPM.S55339

Figure Lengend Snippet: Summary of clinical trials of commercially available EGFR tyrosine kinase inhibitors versus chemotherapy as first-line therapy in non-small-cell lung carcinoma with activating EGFR mutations

Article Snippet: A different approach has been the discovery and development of the second-generation pan-human epidermal growth factor receptor (HER) kinase inhibitors afatinib and dacomitinib (PF-00299804; Pfizer; and ).

Techniques: Clinical Proteomics

Journal: Pharmacogenomics and Personalized Medicine

Article Title: Toward precision medicine with next-generation EGFR inhibitors in non-small-cell lung cancer

doi: 10.2147/PGPM.S55339

Figure Lengend Snippet: Summary of clinical trials of EGFR tyrosine kinase inhibitors in development in NSCLC with EGFR mutations

Article Snippet: A different approach has been the discovery and development of the second-generation pan-human epidermal growth factor receptor (HER) kinase inhibitors afatinib and dacomitinib (PF-00299804; Pfizer; and ).

Techniques: Clinical Proteomics, Mutagenesis

Journal: bioRxiv

Article Title: HB-EGF and zinc activate EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures

doi: 10.1101/2022.11.02.514820

Figure Lengend Snippet: Increased expression of EGFR in the hippocampal neurogenic niche and activation of the EGFR signaling pathway are early events in MTLE-HS. A, Schematic time-course of hippocampus dissection after intrahippocampal injection of KA (1nmol). B, Determination of EGFR mRNA expression by RT-qPCR showing an early increase after KA injection. One-way ANOVA *p < 0.05, **p < 0.01. Bars show mean ± SEM. n=3. Dots show individual data. C, Determination of changes in normal and phosphorylated protein levels involved in EGFR signaling by WB of hippocampal samples. p-EGFR, p-STAT3, p-AKT and p-ERK are increased. Data are representative of 3 biological replicates, with representative blots cropped and marked with solid lines. D, Quantification of the ratio of phosphorylated versus non-phosphorylated forms of EGFR. E, Quantification of the ratio of phosphorylated versus non-phosphorylated forms of STAT3. F, Quantification of the ratio of phosphorylated versus non-phosphorylated forms of AKT. G, Quantification of the ratio of phosphorylated versus non-phosphorylated forms of ERK after WB. Phosphorylation changes are compared respect to control after WB. One-way ANOVA in (D, G) and Kruskal-Wallis in (E, F) *p < 0.05, **p < 0.01, ***p < 0.001. Bars show mean ± SEM. Dots show individual data. n=5. (H) Confocal microscopy images after immunostaining for EGFR in the SGZ of the hippocampus of control (upper panel) and MTLE-HS (lower panel) Nestin-GFP showing increased expression in NSCs and ANPs early after intrahippocampal KA injection. Scale bar 20 μm. I, Quantification of EGFR immunofluorescent signal in the GCL, hilar region and NSCs. Mann-Whitney for GCL and Student’s t-test for hilar region and NSCs *p < 0.05, **p < 0.01. Bars show mean ± SEM. Dots show individual data. n=3.

Article Snippet: The epidermal growth factor receptor (EGFR) irreversible inhibitor afatinib dimaleate (Ref. S7810, Selleck Chemicals LLC, Houston, TX, USA) was diluted with saline to a concentration of 2 mg/Kg and injected intrahippocampally immediately prior to KA delivery following the same steps as for the KA injection.

Techniques: Expressing, Activation Assay, Dissection, Injection, Quantitative RT-PCR, Phospho-proteomics, Control, Confocal Microscopy, Immunostaining, MANN-WHITNEY

Journal: bioRxiv

Article Title: HB-EGF and zinc activate EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures

doi: 10.1101/2022.11.02.514820

Figure Lengend Snippet: (A) RT-qPCR determination of changes of FGFR1 and FGFR2 mRNA expression during the initial 3d after intrahippocampal injection of KA (1 nmol). One-way ANOVA for FGFR1 and Kruskal Wallis for FGFR2. Bars show mean ± SEM. Dots show individual data. n=3. (B) WB of hippocampi dissected at different time points after KA (1 nmol) showing no changes in FGFR1 expression. (C) Confocal microscopy images after immunostaining for FGFR1 in the SGZ of Nestin-GFP mice 24 h after KA. Scale bar is 10 μm. (D) Immunofluorescence images of cultured hippocampal NSPCs stimulated with EGF and FGF showing EGFR staining. Scale bar is 10 μm.

Article Snippet: The epidermal growth factor receptor (EGFR) irreversible inhibitor afatinib dimaleate (Ref. S7810, Selleck Chemicals LLC, Houston, TX, USA) was diluted with saline to a concentration of 2 mg/Kg and injected intrahippocampally immediately prior to KA delivery following the same steps as for the KA injection.

Techniques: Quantitative RT-PCR, Expressing, Injection, Confocal Microscopy, Immunostaining, Immunofluorescence, Cell Culture, Staining

Journal: bioRxiv

Article Title: HB-EGF and zinc activate EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures

doi: 10.1101/2022.11.02.514820

Figure Lengend Snippet: (A) Immunofluorescence images showing the increase in EGFR expression in cultured hippocampal NSPCs during mitosis. Arrows mark cell localization. Scale bar 10 μm. (B) Quantification of the cell area and pixel intensity (EGFR staining) of cultured NSPCs undergoing mitosis or in interphase. Student’s t-test for cell area and Mann Whitney for EGFR pixel intensity ***p < 0.001. Bars show mean ± SEM. Dots show individual data. (C) Schematic of the culture strategy to evaluate the effect of the irreversible EGFR inhibitor afatinib on cell proliferation. NSPCs were cultured in presence of EGF or afatinib pretreatment plus EGF during 48h. A pulse of BrdU 10 μM 1 h before fixation was applied to label mitotic cells. (D) Representative immunofluorescence images of cultured NSPCs from Nestin-GFP mice showing the reduction of nuclear BrdU incorporation 24 h post afatinib treatment. Scale bar is 20 μm. (E) Quantification of BrdU+ cells expressed as percentage of total number of cells. Mann Whitney *p < 0.05. Bars show mean ± SEM. Dots show individual data. (F) Representative immunofluorescence images of cultured NSPCs 48 h post-afatinib treatment, illustrating the cell loss. Scale bar is 20 μm. (G) Quantification of BrdU+ cells showing the drastic reduction due to afatinib treatment. Mann Whitney ***p < 0.001. Bars show mean ± SEM. Dots show individual data. (H) Schematic of the in vivo strategy to assess the effect of afatinib on the neurogenic niche. Afatinib or vehicle was administered together with KA and mice were sacrificed 3 d later. (I) Confocal microscopy image showing the massive cell death provoked in the hippocampus by afatinib administration in a Nestin-GFP mouse, supporting the in vitro results.

Article Snippet: The epidermal growth factor receptor (EGFR) irreversible inhibitor afatinib dimaleate (Ref. S7810, Selleck Chemicals LLC, Houston, TX, USA) was diluted with saline to a concentration of 2 mg/Kg and injected intrahippocampally immediately prior to KA delivery following the same steps as for the KA injection.

Techniques: Immunofluorescence, Expressing, Cell Culture, Staining, MANN-WHITNEY, BrdU Incorporation Assay, In Vivo, Confocal Microscopy, In Vitro

Journal: bioRxiv

Article Title: HB-EGF and zinc activate EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures

doi: 10.1101/2022.11.02.514820

Figure Lengend Snippet: Inhibition of EGFR signaling with gefitinib reduces proliferation of NSPCs. A, EGFR of cultured hippocampal NSPCs stimulated with EGF was phosphorylated at Tyr845 and Tyr1068 residues as analyzed by WB. Its downstream effector ERK was also phosphorylated. Pretreatment with the EGFR inhibitor gefitinib blocked phosphorylation of the receptor, as well as of ERK. B, Quantification of the ratio of phosphorylated versus non-phosphorylated forms for EGFR Tyr845 and Tyr1068 and ERK (four independent replicates) showed the consistent inhibitory effect of gefitinib *p < 0.05, Mann Whitney for Tyr845 and Student’s t-test for Tyr1068 and ERK. Bars show mean ± SEM. Dots show individual data. n=4. C, Paradigm of the strategy to evaluate the effect on cultured cell proliferation. Cells were cultured in presence of EGF or gefitinib pretreatment plus EGF during 48h. A 1h-pulse of BrdU 10 μM was given before fixation to label mitotic cells. D, Representative immunofluorescence images of cultured NSPCs from Nestin-GFP mice showing the reduction of BrdU+ cells in gefitinib-treated cells. Scale bar 10 μm. E, Quantification of the number of BrdU+ cells expressed as percentage respect to total BrdU+ cells in the control condition showing the strong effect of gefitinib. Mann Whitney ***p < 0.001. Bars show mean ± SEM. Dots show random fields of two pooled independent experiments.

Article Snippet: The epidermal growth factor receptor (EGFR) irreversible inhibitor afatinib dimaleate (Ref. S7810, Selleck Chemicals LLC, Houston, TX, USA) was diluted with saline to a concentration of 2 mg/Kg and injected intrahippocampally immediately prior to KA delivery following the same steps as for the KA injection.

Techniques: Inhibition, Cell Culture, Phospho-proteomics, MANN-WHITNEY, Immunofluorescence, Control

Journal: bioRxiv

Article Title: HB-EGF and zinc activate EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures

doi: 10.1101/2022.11.02.514820

Figure Lengend Snippet: Inhibition of EGFR signaling with gefitinib in vivo reduces the induction of React-NSC induction and partially restores neurogenesis in MTLE-HS. A, Confocal microscopy images of MTLE-HS mice treated with intranasal gefitinib (10 mg/Kg) or vehicle, 3 days after intrahippocampal KA injection (1nmol) after staining for nestin, GFAP and Ki67. Scale bar, 50 μm. B, Quantification of proliferating (Ki67+) NSCs, ANPs and overall proliferation in the SGZ expressed as percentage respect to control. Student’s t-test **p < 0.01. Bars show mean ± SEM. Dots show individual data. n=4. C, Confocal microscopy images of DG sections after immunostaining for GFP, DAPI was used to label all cell nuclei. Scale bar, 20 μm. D, Quantification of changes in the length of the processes. E, Quantification of changes in cell volume. F, Quantification of changes in the number of branch points. Changes were quantified after 3D-sholl analysis and expressed as percentage respect to the control condition (sal+DMSO). One-way ANOVA ***p < 0.001. Dots show individual cells (D-F). G, Quantification of the volume of the GCL in saline+DMSO, saline+gefitinib, KA+DMSO and KA+gefitinib treated animals 14dpKA. Kruskal-Wallis *p < 0.05, **p < 0.01, ***p < 0.001. Bars show mean ± SEM. Dots show individual data. n=3.

Article Snippet: The epidermal growth factor receptor (EGFR) irreversible inhibitor afatinib dimaleate (Ref. S7810, Selleck Chemicals LLC, Houston, TX, USA) was diluted with saline to a concentration of 2 mg/Kg and injected intrahippocampally immediately prior to KA delivery following the same steps as for the KA injection.

Techniques: Inhibition, In Vivo, Confocal Microscopy, Injection, Staining, Control, Immunostaining, Saline

Journal: bioRxiv

Article Title: HB-EGF and zinc activate EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures

doi: 10.1101/2022.11.02.514820

Figure Lengend Snippet: (A) Confocal microscopy images showing an increase of the EGFR ligand HB-EGF in the hilus and the molecular layer of Nestin-GFP mice 3dpKA. Scale bar 20 μm. (B) Quantification of HB-EGF by ELISA at different time points during the first 3dpKA. Values are represented as pg of HB-EGF in 100 μg of tissue. One-way ANOVA *p < 0.05, ***p < 0.001. Bars show mean ± SEM. n=3. Dots show individual data. (C) Immunofluorescence images of NSPCs in vitro showing HB-EGF expression. Scale bar is 10 μm. (D) Quantification by ELISA of HB-EGF released in vitro ,expressed as pg of ligand per mL. Kruskal Wallis *p < 0.05. Bars show mean ± SEM. Dots show individual data.

Article Snippet: The epidermal growth factor receptor (EGFR) irreversible inhibitor afatinib dimaleate (Ref. S7810, Selleck Chemicals LLC, Houston, TX, USA) was diluted with saline to a concentration of 2 mg/Kg and injected intrahippocampally immediately prior to KA delivery following the same steps as for the KA injection.

Techniques: Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Immunofluorescence, In Vitro, Expressing

Journal: bioRxiv

Article Title: HB-EGF and zinc activate EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures

doi: 10.1101/2022.11.02.514820

Figure Lengend Snippet: (A) WB for phosphorylated-ERK of cultured NSPCs after 15 min, 2 h and 6 h of growth factors starvation. (B) WB for EGFR and phosphorylated-1068-EGFR of cultured NSPCs stimulated either with EGF (20ng/mL) or starved and stimulated with zinc (200μM), or zinc with gefitinib (2μM) pretreatment. (C) Quantification of the ratio of phosphorylated-EGFR to EGFR. Kruskal-Wallis, *p < 0.05. (D) Confocal microscopy images of the neurogenic niche from Nestin-GFP mice after immunostaining for EGFR 24h post intrahippocampal zinc injection. Scale bar 20 μm. (E) Quantification of EGFR expression (in pixels) in NSCs represented as the percentage respect to control. Student’s t-test. Bars show mean ± SEM. Dots show individual data. n=3.

Article Snippet: The epidermal growth factor receptor (EGFR) irreversible inhibitor afatinib dimaleate (Ref. S7810, Selleck Chemicals LLC, Houston, TX, USA) was diluted with saline to a concentration of 2 mg/Kg and injected intrahippocampally immediately prior to KA delivery following the same steps as for the KA injection.

Techniques: Cell Culture, Confocal Microscopy, Immunostaining, Injection, Expressing, Control

Journal: bioRxiv

Article Title: HB-EGF and zinc activate EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures

doi: 10.1101/2022.11.02.514820

Figure Lengend Snippet: Gefitinib blocks the zinc-induced activation of the EGFR signaling pathway in NSPCs. A, Pretreatment with gefinitib (2μM) prevents the phosphorylation of EGFR induced by EGF and by zinc (200μM) as shown by WB. B, WB of in vitro cultured NSPCs showing that activation of p-ERK downstream signaling in presence of zinc (200μM) is blocked by gefitinib (2μM). Note that β-Actin shows no variation on loading inputs. C, Ratio of phosphorylated to non-phosphorylated EGFR Tyr845 after Kruskal-Wallis analysis. D, Ratio of phosphorylated to non-phosphorylated ERK after one-way ANOVA. *p < 0.05. Bars show mean ± SEM. Dots show individual data (C, D). E, the presence of HB-EGF and zinc stimulates EGFR phosphorylation at Tyr845 and Tyr1068 sites and shows a tendency to stimulate p-ERK signaling. Gefitinib reduces both phosphorylation of EGFR and p-ERK. F, Quantification of the phosphorylated to the non-phosphorylated ratio EGFR Tyr845. G, Quantifications of the phosphorylated to the non-phosphorylated ratio EGFR Tyr1068 followed by One-way ANOVA analysis (F, G). (H) Quantifications of the phosphorylated to the non-phosphorylated ratio for ERK followed by Kruskal-Wallis analysis. *p < 0.05, **p < 0.01. Bars show mean ± SEM. Dots show individual data (F-H). I, Schematic of intranasal gefitinib treatment after intrahippocampal zinc injection. BrdU was given 24h prior to sacrifice to identify mitotic cells. J, Confocal microscopy images of saline, zinc and zinc+gefitinib (10mg/Kg) 3d after zinc showing the increase of BrdU+ cells in the SGZ of zinc-injected animals, that is reversed by the administration of gefitinib. Scale bar 50 μm. K, Quantification of total BrdU+ cells in the SGZ. There is a significant increase of BrdU+ cells in the SGZ after zinc administration, that is however contained administering gefitinib. One-way ANOVA *p < 0.05. Bars show mean ± SEM. Dots show individual data. n=3.

Article Snippet: The epidermal growth factor receptor (EGFR) irreversible inhibitor afatinib dimaleate (Ref. S7810, Selleck Chemicals LLC, Houston, TX, USA) was diluted with saline to a concentration of 2 mg/Kg and injected intrahippocampally immediately prior to KA delivery following the same steps as for the KA injection.

Techniques: Activation Assay, Phospho-proteomics, In Vitro, Cell Culture, Injection, Confocal Microscopy, Saline

Journal: Life

Article Title: Galvanotactic Migration of Glioblastoma and Brain Metastases Cells

doi: 10.3390/life12040580

Figure Lengend Snippet: Effects of the EGF receptor inhibitor afatinib on cell proliferation and apoptosis. ( A ) Glioblastoma cells (HROG02, HROG05, HROG15, HROG17, and HROG24) and brain metastasis cell (HROBMC01 and HROBML01) growing in 96-well half-area microplates were treated with afatinib (striated bars) or solvent (black bars) for 48 h, before DNA synthesis was assessed with the BrdU incorporation assay. One hundred percent BrdU incorporation corresponds to cells cultured with solvent only. Data are presented as mean ± SEM ( n ≥ 18 separate cultures); * p < 0.05 versus control cultures (Kruskal–Wallis test with post hoc Dunn’s test). ( B ) Subconfluent-growing glioblastoma cells were challenged with afatinib (striated bars) or solvent control (black bars) for 6 h followed by caspase 3/7 enzyme activity quantification. Data are presented as mean ± SEM ( n ≥ 9 separate cultures for caspase activity assay); * p < 0.05 versus control cultures (Kruskal–Wallis test with post hoc Dunn’s test).

Article Snippet: To gauge the effects of the EGF receptor antagonist afatinib and pan-AKT inhibitor capivasertib (both from Selleck Chemicals, Houston, TX, USA) on DNA synthesis, a 5-bromo-2′-deoxyuridine (BrdU) incorporation assay kit (Roche Diagnostics GmbH, Mannheim, Germany) was employed.

Techniques: Solvent, DNA Synthesis, BrdU Incorporation Assay, Cell Culture, Control, Activity Assay, Caspase Activity Assay

Journal: Life

Article Title: Galvanotactic Migration of Glioblastoma and Brain Metastases Cells

doi: 10.3390/life12040580

Figure Lengend Snippet: Effects of AKT inhibitors and afatinib on galvanotactic migration of ( A ) HROG02, ( B ) HROG15, and ( C ) HROG17. Glioblastoma cells growing on coverslips were treated with afatinib, capivasertib, MK-2206, or solvent (DMSO) at the indicated doses. Median is shown as a black colored line and the mean as a red line. Data are based on 5–12 independent biological replicates (in each replicate migration of up to 40 cells was analyzed), * p < 0.05 versus control cultures w/o DC (Mann–Whitney U test).

Article Snippet: To gauge the effects of the EGF receptor antagonist afatinib and pan-AKT inhibitor capivasertib (both from Selleck Chemicals, Houston, TX, USA) on DNA synthesis, a 5-bromo-2′-deoxyuridine (BrdU) incorporation assay kit (Roche Diagnostics GmbH, Mannheim, Germany) was employed.

Techniques: Migration, Solvent, Control, MANN-WHITNEY