Journal: iScience

Article Title: From byproduct to biotherapeutic: Comparative study of buffy coats and leukoreduction system chambers for NK cell-based immunotherapies

doi: 10.1016/j.isci.2026.114907

Figure Lengend Snippet: Functional characterization of NK cells (A) Degranulation capacity of NK cells following a 2 h co-culture with K562 leukemia cells, measured by surface CD107a expression ( n = 5). (B and C) Europium-based cytotoxicity assay assessing the specific lysis of K562 target cells at E:T ratios of 10:1, 5:1, 3:1, 1:1, and 0.5:1 on day 7 (B) and day 14 (C) of culture to monitor functional decline over time ( n = 6 for both time points). Here, all comparisons were not significant and are summarized as such (n.s.: p > 0.05). (D) IFN-γ release following 16 h of co-culture of NK cells with K562 cells, quantified via Luminex assay ( n = 5). (E) Kinetic live-cell killing assay using the Incucyte monitoring of Nuclight Red signal loss in K562 cells over 72 h at an E:T ratio of 1:1 ( n = 6). (F) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis thresholds during Incucyte co-culture with K562 cells. (G) CD107a degranulation assay of NK cells with or without pre-incubation with trastuzumab (anti-HER2 antibody) during a 2 h co-culture with MDA-MB-453 cells ( n = 5). (H) Incucyte-based cytotoxicity assay shows the real-time lysis of MDA-MB-453 cells by trastuzumab-loaded NK cells at an E:T ratio of 1:1 over 72 h ( n = 6). (I) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis during the ADCC assay. (n varies as not all samples reached the benchmark values within the observation period). All ADCC experiments have been performed after 14 days of in vitro NK cell expansion. N/A indicates that statistical analysis was not possible for this group. 9J) CD107a degranulation of unmodified NK cells and ErbB2-CAR-NK cells after 2 h co-culture with MDA-MB-453 cells ( n = 5). (K) Incucyte-based cytotoxicity assay showing killing dynamics of ErbB2-CAR-NK cells against MDA-MB-453 cells at an E:T ratio of 1:1 over 72 h ( n = 6). (L) Proportion of donors reaching 25%, 50%, and 75% cumulative lysis during the CAR-mediated cytotoxicity assay. (n varies as not all samples reached the benchmark values within the observation period). All CAR-killing experiments have been performed after 14 days of in vitro NK cell expansion. Unless otherwise stated, n refers to biologically independent donor samples and is indicated per group. Data are shown as mean ± SEM. p-values were determined by Student’s t test with Welsh correction (A, D, F, G, I, J, and L) or by one-way ANOVA (B and C) with Tukey post-test (K and L).

Article Snippet: Mouse Monoclonal anti-CD340 (Her2) , Miltenyi Biotec , Cat# 130-124-474.

Techniques: Functional Assay, Co-Culture Assay, Expressing, Cytotoxicity Assay, Lysis, Luminex, Degranulation Assay, Incubation, ADCC Assay, In Vitro

Journal: Clinical Cancer Research

Article Title: PI3Kα Inhibitor and Degrader Inavolisib Can Co-opt FGFR2 to Enhance Responses in Patients with PIK3CA -Mutated Solid Tumors and in Preclinical Models

doi: 10.1158/1078-0432.CCR-25-1459

Figure Lengend Snippet: Inavolisib sensitivity depends on high FGFR2 expression. A, Legend related to panels in B–I . B, Cell lines were treated with 0.03 μmol/L of the FGFR2i or 2-μmol/L lapatinib for 1 hour followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). C, Cell lines were treated with inavolisib or the FGFR2i at various concentrations for 1 hour. Cell lysates were immunoprecipitated with an antibody against p85β, followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). IB, immunoblotting. D, Following RAS-GTP pulldown, cell lines were treated with the FGFR2i or lapatinib for different durations and immunoblotted with the antibodies indicated at left. Representative results from experiments ( n = 2). E, Cell lysates from cells treated with inavolisib alone or in combination with the FGFR2i or lapatinib for 4 hours were immunoprecipitated with RAS antibody and blotted with p110α antibody. F, Mechanistic model of the effects of FGFR2 and HER2 inhibition on HER3 and RAS activity. FGFR2-high–expressing cell lines induced PI3K signaling through both HER3 and WT RAS activity (top) compared with HER2-induced PI3K signaling through HER3 but not RAS activity (bottom). G, FGFR2-high–expressing cell lines, MFM223 and SUM52PE, were treated with inavolisib, FGFR2i, or lapatinib for 1 hour. Membrane fractions were analyzed by reciprocal co-IP with one another using HER3 or FGFR2 antibody and Western blotting with FGFR2, HER3, RAS, and p85β antibody. Representative results from experiments ( n = 2). H, SUM52PE, MFM223, and MFE280 cells were treated with inavolisib single-agent or in combination with the FGFR2i or lapatinib for 6 hours. Ubiquitinated proteins were pulled down from the membrane fraction with TUBE1 reagent and blotted with p110α antibody. Representative results from experiments ( n = 2). I, Western blots of the inhibitor response in PI3K signaling (pHER3 and pAKT) in PIK3CA mutant MFM223 and PIK3CA WT SUM52PE; cell lines were treated with 0.5-μmol/L inavolisib or 1-μmol/L alpelisib for different durations. Representative results from experiments ( n = 2). J, Ratio of inavolisib and alpelisib GR 50 values in FGFR2-high ( n = 12) vs. FGFR2-low ( n = 9) expressing cell lines harboring PIK3CA mutations, as assessed in a 5-day viability assay. Data are represented as median (center line) ± IQR (25th to 75th percentile, box) and ± full range (minimum to maximum, whiskers). P value was calculated using Wilcoxon rank-sum test. Representative results from experiments ( n = 2). WB, Western blotting.

Article Snippet: For HER3 and FGFR2 IP, lysates were incubated overnight with a biotin-conjugated HER3 or FGFR2 antibody, respectively (Invitrogen, cat. No. MA5-13037, RRID: AB_10983790; and OriGene, cat. No. TA502917AM), which was followed by the addition of 50 μL of streptavidin agarose beads to each sample and an additional 2 hours of incubation.

Techniques: Expressing, Western Blot, Immunoprecipitation, Inhibition, Activity Assay, Membrane, Co-Immunoprecipitation Assay, Mutagenesis, Viability Assay