Journal: Science Advances

Article Title: TET2 regulates early and late transitions in exhausted CD8 + T cell differentiation and limits CAR T cell function

doi: 10.1126/sciadv.adp9371

Figure Lengend Snippet: ( A ) Left: Schematic of TRAC (top) and TET2 (bottom) loci alongside rAAV6 KI vectors. Right: Sanger sequencing electropherogram confirming integration of TRAC and TET2 KI constructs, underlined with dashed line. ( B ) Example plots of TET2 and TRAC-CAR19 single KI or dual TET2-TRAC-CAR19 KI T cells. ( C ) Example plots of CD3 loss detected by flow in TRAC-CAR19-KI T cells. ( D and E ) Schematic of in vitro ADCC assay (D) to deplete CRISPR-edited TET2-KI T cells. Example plots and data (E) of EGFR expression on TET2-KI T cells alone or in an NK cell coculture ± cetuximab incubation, gated on CD56 − populations, n = 4. ( F ) Cumulative fold expansion of TRAC-CAR19 and TET2-TRAC-CAR19 T cells during restimulation and at day 25, arrows represent addition of irradiated K562-CD19 + target cells, n = 5. ( G ) Proportions of CD4 + versus CD8 + T cells in TRAC-CAR19 and TET2-TRAC-CAR19 T cells after stimulation, n = 7. ( H ) Example plots showing distribution of central (CCR7 + CD45RO + ) and effector (CCR7 − CD45RO + ) memory-associated markers in CD8 + CAR T cell populations after restimulation, with summary after five stimulations, n = 5. ( I ) SPICE plot showing distribution of IR coexpression in CD8 + TRAC-CAR19 and TET2-TRAC-CAR19 T cells after 1 (acute) and 5 (chronic) stimulations, n = 6. ( J ) Data shown as means ± SEM [(F) and (G)] or individual values [(E) and (H)] from independent donors. ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001 by paired t test. Schematics [(A and (D)] created with BioRender.com .

Article Snippet: The following day, TET2 KI T cells were incubated with a cetuximab biosimilar (R&D Systems, no. MAB9577) at a concentration of 2000 ng/ml for 20 min. T cells were then cocultured at a 1:10 ratio with NK cells, with T cell numbers normalized to EGFR + expression.

Techniques: Sequencing, Construct, In Vitro, ADCC Assay, CRISPR, Expressing, Incubation, Irradiation

Journal: PLoS ONE

Article Title: Loss of p120ctn causes EGFR-targeted therapy resistance and failure

doi: 10.1371/journal.pone.0241299

Figure Lengend Snippet: (A) Cell viability assays in EPC1-C, -P, -E, and -PE cells show that EPC1-PE cells are resistant to EGFR therapy-induced death, (n = 8). (B) Cell viability assays show that EPC2-PE cells are resistant to cell death, (n = 4). Vehicle control was DMSO. * denotes p<0.05 for gefitinib treatment comparisons between EPC-E vs. EPC-C and EPC-PE vs. EPC-E. # denotes p<0.05 for erlotinib treatment comparisons between EPC-E vs. EPC-C and EPC-PE vs. EPC-E. ^ denotes p<0.05 for cetuximab treatment comparisons between EPC-E vs. EPC-C and EPC-PE vs. EPC-E.

Article Snippet: For viability testing, cells were treated with 5 μM gefitinib (#076091; Matrix Scientific, Columbia, SC), 10 μM erlotinib (#10483; Cayman Chemical; Ann Arbor, MI), or 10 nM cetuximab (#NBP2-75903; Novus Biologicals; Centennial, CO), with or without BAY 11–7085 (#B3033; ApexBio Technology; Houston, TX) at 2 μM or 3.3 μM for 48 hours.

Techniques: Control

Journal: PLoS ONE

Article Title: Loss of p120ctn causes EGFR-targeted therapy resistance and failure

doi: 10.1371/journal.pone.0241299

Figure Lengend Snippet: (A) A dose response curve for BAY 11–7085 using EPC1 cells demonstrates no significant cell death in any cell line at 2 uM. (B) Western blot analysis demonstrates a decrease in pNFkB expression when cells are treated with 2 uM BAY 11–7085. (C) Cell viability assays demonstrate that treatment of EPC1 cells with 2 uM BAY 11–7085 in combination with either gefitinib, erlotinib, or cetuximab results in a partial reduction in cell viability in EPC1-PE cells (n = 3). Vehicle refers to cells treated with 2 uM BAY 11–7085 and DMSO (as the EGFR diluent). * denotes p<0.05 for gefitinib treatment comparisons between EPC-E vs. EPC-C and EPC-PE vs. EPC-E. # denotes p<0.05 for erlotinib treatment comparisons between EPC-E vs. EPC-C and EPC-PE vs. EPC-E. ^ denotes p<0.05 for cetuximab treatment comparisons between EPC-E vs. EPC-C and EPC-PE vs. EPC-E.

Article Snippet: For viability testing, cells were treated with 5 μM gefitinib (#076091; Matrix Scientific, Columbia, SC), 10 μM erlotinib (#10483; Cayman Chemical; Ann Arbor, MI), or 10 nM cetuximab (#NBP2-75903; Novus Biologicals; Centennial, CO), with or without BAY 11–7085 (#B3033; ApexBio Technology; Houston, TX) at 2 μM or 3.3 μM for 48 hours.

Techniques: Western Blot, Expressing

Journal: PLOS One

Article Title: Microfluidic isolation and release of live disseminated breast tumor cells in bone marrow

doi: 10.1371/journal.pone.0319392

Figure Lengend Snippet: (A) Genes were all highly expressed in two cancer cell lines and barely expressed in BM samples. Specifically, there were no detectable levels of EGFR and EpCAM in both BM samples. MDA: MDA-MB-231; MCF7: MCF-7; BM1, BM2: bone marrow cells from healthy donors #1 and #2; BM1+1, +5, +50: 1, 5, 50 MDA-MB-231 cells were spiked into 1 million BM cells from healthy donor #1. (B) EGFR was highly expressed in MDA-MB-231 cells, an observation that aligned with the literature. Nucleated human BM cells were used as control. Streptavidin-PE was used to label biotinylated antibodies.

Article Snippet: Biotinylated antibody against epithelial cell adhesion molecule (anti-EpCAM) (eBioscience, Carlsbad, CA, USA) and biotinylated antibody against epidermal growth factor receptor (anti-EGFR, Research Grade Cetuximab Biosimilar) (R&D Systems, Minneapolis, MN, USA) were used as the tumor cell capture agents immobilized on the surface of the microchannels.

Techniques: Control