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: Genes & development

Article Title: Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer.

doi: 10.1101/gad.351037.123

Figure Lengend Snippet: Figure 1. Cancer-associated fibroblasts promote resistance to KRAS∗extinction in a PDAC model. (A) Crystal violet staining of iKPC1 cell colony formation assay, with Kras∗on (on dox), MRTX1133, or Kras∗off (off dox). The presence of 50% conditional medium from pCAF1, pCAF2, or CAF1 significantly increased the capacity of cell growth 4 d after treatment. (B) Measurement of cell viability using Celltiter-Glo in the same settings as in A 4 d after treatment. (C) Measurement of colony diameter of iKPC1 in Matrigel culturing assay in the same settings as in A 7 d after treatment. (D) Luciferase image of mice injected with 1 × 106 iKPC1-luc cells alone, iKPC2-luc cells alone, or iKPC1-luc or iKPC2-luc cells together with 1 × 106 pCAF1 or CAF1 cells 7 d after injection. Coinjection of iKPC and CAFs sig- nificantly increased KRAS∗-independent growth. (E) Quantification of luciferase signal from D. (F) Histology (HE, trichome, GFP, and KRASG12D staining) of tissue mass from iKPC + CAF1 injection from D. (G) Measurement of colony diameter of iKPC1 in Matrigel cul- turing assay 7 d after treatment with the indicated condition and treatment. (DMSO) Dox on with DMSO, (MRTX) dox on with 100 nM MRTX1133, (Kras∗off) dox off with DMSO, (CM) the presence of 50% CAF-CM in culture, (CM-HI) the presence of 50% heat-inactivated CAF-CM in culture. (H) Summary of the strategy for isolating CAF-secreted protein (CAF-pro). (I) Experimental settings and Western blot analysis of iKPC1 cells at the indicated time points after adding CAF-pro. (J) RTK array analysis of two iKPC cell lines after 5 min of CAF- pro or heat-inactivated CAF-pro (HI-CAF-pro) treatment. (K) Quantification of J. (L) Confirming ERBB2, ERBB3, and AKT phosphorylation in four different iKPC cell lines. Data are represented as mean ± SD for B, C, and G, and mean only for E. For B, C, E, and G, Student’s t-test was performed to calculate statistical values.

Article Snippet: Plasmid construction and gene knockdown, knockout, and overexpression LentiORFs were purchased from Origene for mouse Erbb2 (NM_001003817, MR227307), mouse Erbb3 (NM_010153, MR227559), mouse Nrg1 (NM_178591, MR226911L3), and human NRG1 (NM_013956, RC220134).

Techniques: Staining, Colony Assay, Luciferase, Injection, Western Blot, Phospho-proteomics

Journal: Genes & development

Article Title: Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer.

doi: 10.1101/gad.351037.123

Figure Lengend Snippet: Figure 2. Paracrine Nrg1–Erbb2/3 signaling activation confers KRAS∗resistance. (A) Western blot of NRG1 protein levels in various cell lines. (B) Multiplexed RNAscope immunofluorescence staining of iKPC tumor with NRG1-specific RNA probe (white), GFP (green), PDPN (red), and DAPI (blue). (C) qPCR results of Nrg1 on flow cytometry-sorted GFP+ PDAC cells or PDPN+ CAFs from iKPC GEM tu- mors. Two independent tumors were pooled together for dissociation. (D) Measurement of colony diameter of iKPC1 in 3D Matrigel cul- turing assay with the indicated treatment for 7 d. (on) Kras∗on with dox, (off) Kras∗off without dox, (MRTX) 100 nM MRTX1133 treatment, (CAF) the presence of CAF1-pro in culture, (NRG1) the presence of 10 ng/mL rmNRG1 in culture. Data are represented as mean ± SEM; Student’s t-test. (E) Western blot of the indicated iKPC1 knockout clones Kras∗off for 48 h and serum-starved for 2 h prior to 5-min stimulation of the indicated treatments (CAF1-pro or 10 ng/mL rmNRG1). (F) Western blot of the indicated iKPC1 ERBB3 knock- out or rescued clones K Kras∗off for 48 h and serum-starved for 2 h prior to 5-min stimulation of the indicated treatments (CAF1-pro or 10 ng/mL rmNRG1). (G) Western blot of iKPC1 cells with 48 h of K Kras∗off and 2 h of serum starvation prior to 5-min stimulation of the indicated treatments (CAF1-pro cells were preincubated with 100 μg/mL mouse IgG or the indicated concentration of NRG1 neutraliza- tion antibody YW538.24.71 for 1 h at 37°C). (H) Western blot of AsPC1, DMSO, or 100 nM MRTX1133 treatment for 24 h and serum star- vation for 2 h with the presence of 10 μg/mL control human IgG or 10 μg/mL pertuzumab prior to 5-min stimulation of the indicated treatments (CAF-pro, 10 ng/mL rmNRG1, or CAF-sgNrg1-pro). (I) Cell viability assay with AsPC1 with the indicated treatment for 4 d. (MRTX) Treated with 100 nM MRTX1133 (with pCAF1-pro, CAF1-pro, or 10 ng/mL rmNRG1), (mIgG + hIgG) treated with 10 μg/ mL control mouse IgG and 10 μg/mL control human IgG, (anti-NRG1) the presence of 10 μg/mL YW538.24.71, (pertuzumab) the presence of 10 μg/mL pertuzumab. Data are represented as mean ± SD; two-way ANOVA. (J) Luciferase assay of iKPC1-luc cells coinjected with pCAF3 or CAF1. One day after cell implantation, luciferase signal was measured to acquire the initial value of luciferase activity, and IgG or YW538.24.71 was given at a dose of 25 mg/kg. Luciferase signal was acquired again at 7 d after implantation. (K) Quantification of luciferase signal from J. Paired multiple t-test at day 7.

Article Snippet: Plasmid construction and gene knockdown, knockout, and overexpression LentiORFs were purchased from Origene for mouse Erbb2 (NM_001003817, MR227307), mouse Erbb3 (NM_010153, MR227559), mouse Nrg1 (NM_178591, MR226911L3), and human NRG1 (NM_013956, RC220134).

Techniques: Activation Assay, Western Blot, RNAscope, Immunofluorescence, Staining, Flow Cytometry, Knock-Out, Clone Assay, Concentration Assay, Control, Viability Assay, Luciferase, Activity Assay