Journal: SLAS discovery : advancing life sciences R & D

Article Title: High throughput flow cytometry identifies small molecule inhibitors for drug repurposing in T-ALL

doi: 10.1177/2472555218774248

Figure Lengend Snippet: Drug response profiles of the T-ALL cell lines. (A) Heatmap indicating the responses of five T-ALL cell lines to 31 small molecule inhibitors after 72 hr incubation under normoxic conditions. The columns indicate the T-ALL cell lines and the rows indicate the tested drugs. The color bar reflects the log10 transformed EC50 values obtained for three independent experiments. The darkest red indicates the most sensitivity (lowest EC50 values) of the cell lines to the tested inhibitors. EC50 values are only reported for compounds that yielded a maximum response values of 20% or greater. For those samples which yielded maximal responses < 20% and/or had EC50 values greater than 100µM, we report those EC50 values as “>100 µM” (light grey). (B) Maximum responses of each T-ALL cell line to the small molecule inhibitors at the highest tested dose, which was 100 µM for each drug except for *alvocidib, *bortezomib and *carfilzomib, for which the highest tested dose was 1 µM. The heatmap shows the percentage of dead cells, where darkest red indicates 100% propidium iodide positive cells. (C) Representative high throughput dose response curves of T-ALL cell lines to tyrosine kinase inhibitors. The cells were treated with a series of drug concentrations (0.005 – 100 µM for afatinib, crizotinib and ponatinib; 0.05 – 1000 nM for alvocidib) and incubated for 72 hr, followed by cell viability analyses by flow cytometry using propidium iodide. The data represent the average of three separate experiments. Error bars denote SEM.

Article Snippet: Compounds We investigated several drug classes, including: TKIs (n = 26), cyclin dependent kinase inhibitors (n = 2; alvocidib and palbociclib), Hedgehog signaling inhibitor (n = 1; vismodegib) and proteasome inhibitors (n = 2; bortezomib and carfilzomib), all purchased from LC Laboratories (Woburn, MA) ( Suppl.

Techniques: Incubation, Transformation Assay, High Throughput Screening Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: Monolayer culture alters EGFR inhibitor response through abrogation of microRNA-mediated feedback regulation

doi: 10.1038/s41598-024-56920-7

Figure Lengend Snippet: The effects of culture method on cells’ response to first-line lung adenocarcinoma therapies. ( a ) HCC827 cells were cultured for four days in either monolayer (2D) or hanging drop spheroid (3D) culture and challenged with erlotinib or gefitinib for 72 h. ( b ) The IC50 for cells in 2D culture aligns with that of the GDSC dataset while cells in 3D culture show increased resistance. ( c ) HCC827 cells were cultured in 2D and 3D for six days; ATP concentration was measured every 24 h. Cells in 2D culture begin proliferating earlier and show greater overall proliferation than cells in 3D culture. ( d ) HCC827 cells in 3D culture show increased resistance to the taxane docetaxel, but no significant difference in response to the platinum drug cisplatin. ( b – d ) Viability and ATP concentration were assessed via CellTiter Glo assay (Promega). ( b , d ). T-test with Welch’s correction; data represents the mean of three experiments; each experiment had six replicates per dose; error bars represent standard deviation. The dotted line indicates the IC50 for the indicated drug in HCC827 cells as published in the Genomics of Drug Sensitivity in Cancer (GDSC2) dataset .

Article Snippet: Cells were cultured for 4 days, then challenged with EGFR TKIs erlotinib (Chemie-Tek #CT-EL-002) or gefitinib (MedChemExpress #HY-50895), or taxane docetaxel (MedChemExpress #HY-B0011) in eight concentrations from 1 nM to 10 μM, or platinum drug cisplatin (MedChemExpress #HY-17394) from 1 nM to 100 μM for 3 days.

Techniques: Cell Culture, Concentration Assay, Glo Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Monolayer culture alters EGFR inhibitor response through abrogation of microRNA-mediated feedback regulation

doi: 10.1038/s41598-024-56920-7

Figure Lengend Snippet: EGFR, but not MKI67, shows a discrepancy between relative transcript and relative protein expression. ( a ) HCC827 cells in 3D culture express lower levels of MKI67 as determined by RNA-seq (left); this is reflected at the protein level through immunostaining for Ki-67, which shows roughly 50% difference between the two conditions (middle, right). ( b ) EGFR transcript levels do not differ significantly between conditions (left), although immunostaining for EGFR shows significantly higher levels of EGFR protein in the 2D cells (middle, right). Student’s t-test, mean of three slides per condition. Error bars represent standard deviation.

Article Snippet: Cells were cultured for 4 days, then challenged with EGFR TKIs erlotinib (Chemie-Tek #CT-EL-002) or gefitinib (MedChemExpress #HY-50895), or taxane docetaxel (MedChemExpress #HY-B0011) in eight concentrations from 1 nM to 10 μM, or platinum drug cisplatin (MedChemExpress #HY-17394) from 1 nM to 100 μM for 3 days.

Techniques: Expressing, RNA Sequencing, Immunostaining, Standard Deviation

Journal: Scientific Reports

Article Title: Monolayer culture alters EGFR inhibitor response through abrogation of microRNA-mediated feedback regulation

doi: 10.1038/s41598-024-56920-7

Figure Lengend Snippet: miRNA targeting EGFR is upregulated in spheroids and affects the response to EGFR TKI. ( a ) Cells were cultured in 2D for four days; half of the cells were then plated in 3D and the other half were stored at -80C. After four additional days, half of the spheroids were stored at -80C and the other half were returned to 2D culture for an additional four days and then stored at -80C. RNA isolation was performed in parallel. ( b ) miR-146a-5p shows a significant increase of expression in 3D culture, and a significant return to 2D levels in 3D-2D. ( c ). Transfection of spheroids with a miR-146a-5p inhibitor reduced expression by more than 50% compared to non-transfected cells. Expression measured by RT-qPCR, relative to non-transfected control. Error bars represent standard deviation. ( d ) Spheroids transfected with miR-146a-5p inhibitor show increased sensitivity to 150 nM erlotinib compared to non-transfected cells. Survival normalized to untreated controls; plot shows mean ± standard deviation. Kruskal–Wallis test with Dunn’s correction for multiple comparisons; n = 6. ( e ) Three additional miRNA that target EGFR show significant upregulation in 3D compared to 2D. ( b , e ). miRNA expression measured by miRNA microarray. Two-way ANOVA with Tukey’s test for multiple comparisons; n = 3; error bars represent standard deviation.

Article Snippet: Cells were cultured for 4 days, then challenged with EGFR TKIs erlotinib (Chemie-Tek #CT-EL-002) or gefitinib (MedChemExpress #HY-50895), or taxane docetaxel (MedChemExpress #HY-B0011) in eight concentrations from 1 nM to 10 μM, or platinum drug cisplatin (MedChemExpress #HY-17394) from 1 nM to 100 μM for 3 days.

Techniques: Cell Culture, Isolation, Expressing, Transfection, Quantitative RT-PCR, Control, Standard Deviation, Microarray

Journal: Scientific Reports

Article Title: Monolayer culture alters EGFR inhibitor response through abrogation of microRNA-mediated feedback regulation

doi: 10.1038/s41598-024-56920-7

Figure Lengend Snippet: miR-146a-5p regulation and effect on drug response may be mutation-specific . ( a ) RNAseq shows that in both cases, EGFR transcripts are expressed at similar levels in both culture conditions and the tissue of origin. ( b ) Case A shows miR-146a-5p downregulation in 2D, reflecting the data collected from HCC827, while Case B shows no significant change. RT-qPCR; fold change relative to flash frozen control. Each point represents the mean of three technical replicates. Kruskal–Wallis test with Dunn’s correction for multiple comparisons. ** p = 0.0045. ( c ) When challenged with 75 nM erlotinib for 72 h, Case A shows significantly higher resistance in 3D culture, which aligns with that seen in the HCC827 cells. Case B shows a nonsignificant difference in response. Each point represents a single well (2D) or spheroid (3D), and all spheroids of consistent size and shape were used. Kolmogorov–Smirnov test, p = 0.0476. ( d ) RNAseq shows that Case A carries the same EGFR exon 19 deletion as HCC827, while Case B carries an exon 20 insertion.

Article Snippet: Cells were cultured for 4 days, then challenged with EGFR TKIs erlotinib (Chemie-Tek #CT-EL-002) or gefitinib (MedChemExpress #HY-50895), or taxane docetaxel (MedChemExpress #HY-B0011) in eight concentrations from 1 nM to 10 μM, or platinum drug cisplatin (MedChemExpress #HY-17394) from 1 nM to 100 μM for 3 days.

Techniques: Mutagenesis, Quantitative RT-PCR, Control