Journal: Clinical Cancer Research

Article Title: Anti-EGFR Antibody–Drug Conjugate Carrying an Inhibitor Targeting CDK Restricts Triple-Negative Breast Cancer Growth

doi: 10.1158/1078-0432.CCR-23-3110

Figure Lengend Snippet: Stochastic conjugation of cetuximab to CDK inhibitor and ADC internalization in live breast cancer cells. A, Flow cytometric evaluation of surface EGFR expression (TNBC: MDA-MB-468, HCC1143, HCC1806, MDA-MB-231, HCC1937, SUM149, and CAL51; HER2+: SKBR3; ER+: MCF7, T47D; nontumorigenic epithelial cell model: MCF10A; immune cell model: human B lymphocytes RPMI8866, RPMI8226, and monocytic cell line U937; human primary melanocyte: melanocyte). B, EGFR mRNA expression from the Cancer Cell Line Encyclopedia database showed a positive correlation with surface EGFR measured by flow cytometry in A (Spearman’s rank coefficient, r = 0.723). A high level of correlation was found between EGFR and cyclin E ( r = 0.738), but not with cyclin A or CDK2. Nonsignificant P values are marked as NS. C, Top, Schematic diagram of stochastic ADC conjugation by antibody reduction with TCEP and then conjugation to SNS-032 via MC-Val–Ala-PAB. Middle, HIC analysis confirmed an average DAR of 4.4. Bottom, SEC trace indicates negligible ADC aggregation and minimal free linker–payload (less than 0.8%). D, Surface plasmon resonance analysis demonstrated similar binding affinity ( K D ) for cetuximab (0.73 nmol/L) and ADC (1.28 nmol/L). Isotype IgG1 and isotype ADC showed no measurable binding. E, Monitoring internalization of Fabfluor-pH-labeled cetuximab, ADC, or isotype control (10 nmol/L) by Incucyte live-cell imaging. Phase and red fluorescence time-course images were captured for 24 hours. Images of internalized antibody display in cytosolic, low pH lysosomal vesicle-associated red fluorescence in cells. Scale bar, 0.2 mm. F, Cells were seeded in Matrigel for 5 days, allowing the formation of spheroids. Fabfluor-pH-labeled antibodies or ADC (10 nmol/L) were introduced in the Matrigel and showed rapid internalization in EGFR-high MDA-MB-468 and MDA-MB-231, whereas EGFR-low CAL51 displayed little red fluorescence signals. A low level of internalization was observed for isotype or isotype-ADC controls. Scale bar, 0.5 mm. P values determined by two-tailed unpaired t test of three independent experiments compared with isotype control.

Article Snippet: Human primary epidermal melanocytes were cultured in Melanocyte Growth Medium (Cell Applications Inc.).

Techniques: Conjugation Assay, Expressing, Flow Cytometry, SPR Assay, Binding Assay, Labeling, Control, Live Cell Imaging, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Overcoming efflux of fluorescent probes for actin imaging in living cells

doi: 10.1101/2020.02.17.951525

Figure Lengend Snippet: Confocal and nanoscopy images of living cells and tissues stained with the new actin probes. (a) Comparison of confocal and STED images of human fibroblasts stained with 6-610CP-JAS. (b) Human melanocytes co-stained with 6-580CP-JAS and 5-SiR-Hoechst. (c) live-cell imaging of body wall muscle of dissected D. melanogaster larva costained with 6-580CP-JAS and 6-SiR-CTX. (d) Max intensity projection of a frog erythrocyte stained with 6-610CP-JAS. (e) Rat primary neuron culture co-stained with 6-610CP-JAS and neurofascin (AlexaFluor 488). Scale bars 10 μm (a-d), 5 μm (e).

Article Snippet: Normal neonatal human melanocytes (MatTek Corporation, Cat. NHM-CRY-NEO) were cultured in Normal human melanocyte cells growth medium (MatTek Corporation, Cat. NHM-GM) in a humidified 5% CO2 incubator at 37 °C.

Techniques: Staining, Comparison, Live Cell Imaging