Journal: EMBO Molecular Medicine

Article Title: Humanized avian embryo models replicate an immune tumor environment for rapid immunotherapy studies

doi: 10.1038/s44321-026-00398-5

Figure Lengend Snippet: ( A ) Representative FACS profiles of CD69 and CD25 activation markers or TIM-3 and PD-1 exhaustion marker expressions within the CD3+ CD4+ population of hu-PBMCs infiltrated in MDA-MB-231 tumor site or in equivalent control tissues. Data are presented for 37 pooled embryos intra-venously injected with hu-PBMCs from donor 5. ( B ) Histograms showing the quantification of tumor volumes in embryos grafted with CFSE+ MDA-MB-231 cell line, and intra-venously injected or not with hu-PBMCs from donor 4. N = 1 experiment ( n = 5 embryos in control group and n = 8 embryos in I.V injected group). Dots represent volumes normalized to each embryo’s BSA. Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Mann–Whitney test, exact P -values indicated on graph. ns: not significant. ( C ) Histogram showing the fraction of Caspase 3 positive cells within the avian somitic tissue, in presence and absence of hu-PBMCs-MDA-MB-231 cells (hu-PBMCs from donor 6). For each experimental group, n = 7 sections from 3 embryos. Mann–Whitney test, exact P -values indicated on the graph. ns: not significant. ( D ) Microphotographs of immunofluorescent labeling of cryosections of chick embryos at 48 h post co-grafting of CFSE+ MDA-MB-231 cells (green) and hu-PBMCs from donor 6. Immune cells were detected with anti-human CD45 antibody (red), and apoptotic cells with anti-cleaved Caspase 3 antibody (white). Nuclei of avian and human cells were stained with Hoechst (blue). The images show somitic tissues free or populated by grafted human cells. White arrow indicates avian Caspase 3+ cells. ( E ) Histogram showing the global distribution pattern of hu-PBMCs from donor 10 when grafted alone ( n = 9 embryos) or in combination with MDA-MB-231 cells at a ratio 1:1 ( n = 11 embryos), N = 1. Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Each dot represents the average distance of hu-PBMCs from the site of injection for individual embryos. Mann–Whitney test, exact P -value indicated on graph. ** P < 0.01. ( F , G ) Histograms showing the differences of hu-PBMC subpopulation fractions for two donors, between conditions of pre-grafting, grafting alone or grafting in combination with MDA-MB-231 or HCT 116 cells. Embryos were harvested 48 h post grafting. N = 3 for pre- and post-graft groups. For the histogram in ( F ), N = 3 for co-grafted experimental group ( n = 8 to 11 pooled embryos); For the histogram in ( G ), N = 2 for co-engrafted experimental group ( n = 7 to 9). The histogram of post-graft is also presented in Fig. . Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Chi-square test, P -values are indicated. **** P < 0.0001, *** P < 0.001 ( P = 0.0003), * P < 0.05 ( P = 0.0373). ( H , I ) Histograms showing the percentage of CD69 positive cells ( H ) and TIM-3 positive cells ( I ) within the CD3+ CD8+ and CD3+ CD4+ populations, for 3 donors in two experimental groups of hu-PBMCs grafted alone or co-grafted with MDA-MB-231 cells. Donors are color-coded (red: donor 1, orange: donor 6 and blue: donor 8). Each dot represents the result of pooled embryos, see details Fig. . Mann–Whitney test, P -values are indicated on the graph. * P < 0.05, ns: not significant.

Article Snippet: The MDA-MB-436 (ATCC-HTB-130) and MDA-MB-231 (ATCC-HTB-26) TNBC cell lines were obtained from American Type Culture Collection and HCT 116 from Cytion (116:300195) and were not re-authenticated.

Techniques: Activation Assay, Marker, Control, Injection, MANN-WHITNEY, Labeling, Staining

Journal: EMBO Molecular Medicine

Article Title: Humanized avian embryo models replicate an immune tumor environment for rapid immunotherapy studies

doi: 10.1038/s44321-026-00398-5

Figure Lengend Snippet: ( A ) Schematic drawing of the humanization process: CFSE-labeled MDA-MB-231 cells were mixed with hu-PBMCs at defined ratio. In some experiments, PBMCs were labeled with Orange Cell Tracker. Mix of cells was micro-injected in the developing somites of chick embryos at E2 (HH14). Engrafted embryos were harvested at E4 (HH25) for a panel of analyses. ( B ) Histogram depicting the position of the 10 Orange Cell Tracker-labeled hu-PBMCs that were the most distal to the grafting site, per grafted embryo. Hu-PBMCs from donor 10 were co-engrafted with MDA-MB-231 in avian embryo at a ratio 1:1 ( n = 11 analyzed embryos) or engrafted alone ( n = 9), N = 1. Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Mann–Whitney test, exact P -value indicated on graph. **** P < 0.0001. ( C ) Representative light sheet microscopy photographs illustrating the quantitative analysis of distribution pattern. Blue circles represent the grafting site, pink cercles represent the area covered by hu-PBMCs. Scale bar: 200 µm. ( D ) Light sheet microscopy photographs of whole embryos illustrating the co-engraftment process, 48 h post-grafting. The green signal depicts MDA-MB-231 tumor cells, the red signal the hu-PBMCs and the white signal the co-localized fraction. Volumes were calculated using Imaris software (V). Scale bars: 100 µm. ( E ) Analysis of the respective location of cancer cells and hu-PBMCs. Diameters of 20 Orange Cell Tracker PBMCs (donor 10) and 20 CFSE+ MDA-MB-231 tumor cells were measured per embryo ( n = 10) with Imaris software to determine an average cell size of each cell types. Schematic showing the calculation of distances between cells. A measure of 8.5 µm, representing the average distance between the centers of mass of two adjacent immune-cancer cells, was used as the threshold for qualifying contact. The histogram presents the fractions of adjacent and non-adjacent immune-cancer cells calculated with Imaris software for 10 engrafted embryos, N = 1. Chi-square test, Exact P -value indicated on the graph, ns: not significant. ( F ) Microphotographs of immunolabeled cryosections of E4 chick embryo grafted at E2. MDA-MB-231 cells were detected with CFSE (green) and hu-PBMCs from donor 6 with anti-human CD45 antibody (red). Chick and human cell nuclei are labeled with Hoechst (blue). The images show CD45+ cells in contact with cancer cells, the magnification showing immune cell processes surrounding cancer cells. Scale bars: 20 µm. ( G ) Histograms showing the hu-PBMC subpopulation fractions for donor 8, between conditions of pre-grafting, grafting alone or grafting in combination with MDA-MB-231 or HCT 116 cells. Embryos were harvested 48 h post grafting. N = 3 per experimental group; post graft ( n = 9 to 17 pooled embryos); co-engrafted with MDA-MB-231 ( n = 9 to 11); co-engrafted with HCT 116 ( n = 12 to 18). The histogram of post-graft is also presented in Fig. . Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Chi-square test. **** P < 0.0001, *** P < 0.001 (exact P = 0.0002), ** P < 0.01 (exact P = 0.001). ( H , I ) Histograms showing the fraction of CD69 ( H ) and TIM-3 ( I ) positives cells within both CD3+ CD8+ and CD3+ CD4+ populations, for 3 different donors (1, 6 and 8) and 3 experimental groups representing conditions of pre-grafting, grafting alone and combined with MDA-MB-231 cells. Box-plots represent the median, interquartile range (25th–75th percentiles), with whiskers indicating the minimum and maximum values. Each dot represents pooled embryos that were engrafted with the same donor. N = 3 for all donors in pre-graft and post-graft. In post-graft group, N = 1 for donor 1 ( n = 6 to 7 pooled embryos); N = 4 for donor 6 ( N = 8 to 13); N = 3 for donor 8 ( n = 9 to 16). For post co-graft group, N = 1 for donor 1 ( n = 51); N = 2 donor 6 ( n = 17 to 33); N = 3 donor 8 ( n = 24 to 27). Experiments with fewer than 20 CD3+ CD8+ or 20 CD3+ CD4+ cells were excluded from analysis. Unpaired T test was performed between all pre-graft and post-graft groups except for CD69 in CD3+ CD4+ population. Mann–Whitney test was performed in all other comparisons, exact P -values indicated on graphs. **** P < 0.0001*** P < 0.001, ** P < 0.01, * P < 0.05, ns: not significant. ( J ) Histograms showing the quantification of the tumor volumes of MDA-MB-231 engrafted alone or co-engrafted with hu-PBMCs from donor 6 or 8. For each embryo, the volume was normalized to the BSA. Dots represent individual embryos. N = 1 experiment per group ( n = 11 embryos in engrafted group; n = 17 in co-engraftment with donor 6, and n = 13 in co-engraftment with donor 8). Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Unpaired T-test (donor 6) or Mann–Whitney test (donor 8). Exact P -values indicated on graph. ns: not significant. .

Article Snippet: The MDA-MB-436 (ATCC-HTB-130) and MDA-MB-231 (ATCC-HTB-26) TNBC cell lines were obtained from American Type Culture Collection and HCT 116 from Cytion (116:300195) and were not re-authenticated.

Techniques: Labeling, Injection, MANN-WHITNEY, Microscopy, Software, Immunolabeling

Journal: EMBO Molecular Medicine

Article Title: Humanized avian embryo models replicate an immune tumor environment for rapid immunotherapy studies

doi: 10.1038/s44321-026-00398-5

Figure Lengend Snippet: ( A ) Representative FACS profiles of PD-L1 expression in indicated cell lines (MDA-MB-231, MDA-MB-436, HCT 116). ( B ) Table presenting the morphological analysis of embryos treated with different pembrolizumab doses. ( C ) Histogram showing the fraction of PD-1 positive cells in CD3+ populations in embryos grafted with a mix of hu-PBMCs from donor 1 and MDA-MB-231 cells, treated either with anti-PD-1 (pembrolizumab) at 2 doses (178 in red or 890 mg/kg in blue) or control (NaCl 0.9%). Percentages represent the PD-1 expression diminution in pembrolizumab condition compared to control. N = 1, for 178 mg/kg dose ( n = 51 pooled embryos in control group; n = 54 pooled embryos in pembrolizumab group) also shown in Fig. ; for 890 mg/kg ( n = 29 and n = 36). No statistical test was performed. ( D ) Representative FACs profiles of CD69 and CD25 expression in the CD3+ CD8+ population of hu-PBMCs in embryos grafted with hu-PBMCs from donors 1 or 6, combined with MDA-MB-231 cells and treated either with control (NaCl 0.9%) or anti-PD-1 (pembrolizumab). Donor 1 ( n = 52 pooled embryos per experimental group); donor 6 ( n = 20 in control group; n = 26 in pembrolizumab group). ( E ) Histogram showing the fraction of CD69 positive cells in CD3+ CD8+ and CD3+ CD4+ populations in embryos grafted with a mix of hu-PBMCs and MDA-MB-231 cells and treated either with anti-PD-1 (pembrolizumab) or control (NaCl 0.9%). Each dot represents pooled embryos in experiments conducted with 3 donors (red: donors 1, orange: donor 6; blue: donor 8), see details Fig. . Wilcoxon test, exact P -values indicated on the graph. * P < 0.05, ns: not significant. ( F ) Histograms showing the fraction of CD25 positive cells in the CD3+ CD8+ and CD3+ CD4+ populations in hu-PBMCs co-grafted with MDA-MB-231 cells in embryos treated either with anti-PD-1 (pembrolizumab) or with control (NaCl 0.9%). Each dot represents pooled embryos in experiments conducted with 3 donors (donors 1, 6 and 8). N = 1 for donor 1 ( n = 51 pooled embryos in control group; n = 54 pooled embryos in pembrolizumab group); N = 2 donor 6 ( n = 17 to 33; n = 27 to 36); N = 3 donor 8 ( n = 24 to 27; n = 19 to 23). Experiments with fewer than 20 CD3+ CD8+ or 20 CD3+ CD4+ cells were excluded from analysis. Box-plots represent the median, interquartile range (25th–75th percentiles), with whiskers indicating the minimum and maximum values. Wilcoxon test, exact P -values indicated on the graph, ns: not significant. ( G ) Representative FACS profiles of TIM-3 expression in the CD3+ CD69+ population in embryos grafted with hu-PBMCs from donor 1 or 8, combined with MDA-MB-231 cells and treated either with control (NaCl 0.9%) or anti-PD-1 (pembrolizumab). Donor 1 ( n = 52 pooled embryos per experimental group); donor 8 ( n = 24 in control group; n = 19 in pembrolizumab group). ( H ) Histogram showing the fraction of TIM-3 positive cells in CD3+ CD69+ population in embryos grafted with a mix of hu-PBMCs and MDA-MB-231 cells and treated either with anti-PD-1 (pembrolizumab) or control (NaCl 0.9%). Each dot represents pooled embryos in experiments conducted with 3 donors (red: donors 1, orange: donor 6; blue: donor 8), see details Fig. . Wilcoxon test, exact P -value indicated on the graph. * P < 0.05.

Article Snippet: The MDA-MB-436 (ATCC-HTB-130) and MDA-MB-231 (ATCC-HTB-26) TNBC cell lines were obtained from American Type Culture Collection and HCT 116 from Cytion (116:300195) and were not re-authenticated.

Techniques: Expressing, Control

Journal: EMBO Molecular Medicine

Article Title: Humanized avian embryo models replicate an immune tumor environment for rapid immunotherapy studies

doi: 10.1038/s44321-026-00398-5

Figure Lengend Snippet: ( A ) Analysis of survival rate (left axis) and mean body surface area (BSA, right axis) of chick embryos injected with increasing doses of anti-PD-1 pembrolizumab. Each doses and control excipient (Ctl, NaCl 0.9%) were injected to 15 embryos, in N = 1. Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SD. Unpaired T-test compared to excipient, exact P -values indicated on graphs, ns: non-significant. ( B ) Schematic drawing of the humanization process with treatment injection. CFSE-labeled MDA-MB-231 or HCT 116 cells were mixed at effector:target ratio cells of 4:10 with hu-PBMCs. The mix was micro-injected in the developing somites at E2 (HH14). Pembrolizumab at 178 mg/kg or NaCl 0.9% was injected intra-venously 24 h post-grafting. Grafted embryos were harvested at E4 stage (HH25) for a panel of analyses. ( C , D ) Histograms depicting the fraction of PD-1 positive cells at the surface of CD3+ T cells, 24 h post-treatment with anti-PD-1 (pembrolizumab) or control (NaCl) of embryos grafted either with hu-PBMCs ( C ) or the mix tumor cells/hu-PBMCs ( D ). The experiments were conducted with 6 color-coded donors. For graph ( C ), data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM or for individual donors (dark red: donor 1, red: donor 2, light blue: 4, orange: donor 6, blue: donor 8, dark blue: donor 9). N = 1 experiment per donor, donor 1 ( n = 18 pooled embryos per experimental group); donor 2 ( n = 18); donor 4 ( n = 9); donor 6 ( n = 17); donor 8 ( n = 3 for control and n = 9 for pembrolizumab); donor 9 ( n = 8). Wilcoxon test, exact P -value indicated on graph. * P < 0.05. For graph ( D ), data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM or for individual donors (dark red: donor 1, orange: donor 6 and blue: donor 8). N = 1 for donor 1 ( n = 51 pooled embryos in control group; n = 54 pooled embryos in pembrolizumab group); N = 2 for donor 6 ( n = 14 to 17; n = 16 to 27); N = 2 for donor 8 ( n = 24 to 27 n = 19 to 23). Paired T test, exact P -value indicated on graph. * P < 0.05. ( E ) Histogram showing the fraction of CD69 positive cells in CD3+ CD8+ or CD3+ CD4+ populations in embryos grafted with MDA-MB-231 cells and hu-PBMCs, treated with anti-PD-1 (pembrolizumab) or control (NaCl 0.9%). Box-plots represent the median, interquartile range (25th–75th percentiles), with whiskers indicating the minimum and maximum values. Each dot represents an independent experiment performed with 3 donors (donors 1, 6 and 8). N = 1 for donor 1 ( n = 51 pooled embryos in control group; n = 54 pooled embryos in pembrolizumab group); N = 2 for donor 6 ( n = 17 to 32 and n = 27 to 36); N = 3 for donor 8 ( n = 24 to 27 and n = 19 to 23). Experiments with fewer than 20 CD3+ CD8+ or 20 CD3+ CD4+ cells were excluded from analysis. Wilcoxon test, exact P -values indicated on the graphs. * P < 0.05, ns: not significant. ( F ) Representative FACS profiles of CD69 and CD25 expressions within the CD3+ CD8+ population of hu-PBMCs co-grafted with MDA-MB-231 cells in embryos treated either with control (NaCl 0.9%) or with anti-PD-1 (pembrolizumab). Data are presented for pooled embryos engrafted with donor 8 ( n = 24 pooled embryos for control; n = 19 pooled embryos for pembrolizumab). ( G ) Histogram showing the fraction of TIM-3 positive cells within the CD3+ CD69+ population of hu-PBMCs co-grafted with MDA-MB-231 cells in embryos treated either with anti-PD-1 (pembrolizumab) or control (NaCl 0.9%). Box-plots represent the median, interquartile range (25th–75th percentiles), with whiskers indicating the minimum and maximum values. Each dot represents pooled embryos for 3 donors (donors 1, 6 and 8). N = 1 for donor 1 ( n = 51 pooled embryos in control group; n = 54 pooled embryos in pembrolizumab group); N = 3 for donor 6 ( n = 17 to 32 and n = 27 to 36); N = 3 for donor 8 ( n = 24 to 27 and n = 19 to 23). Experiments with fewer than 20 CD3+ CD69+ cells were excluded from analysis. Wilcoxon test, exact P -value indicated on the graph. * P < 0.05. ( H ) Representative FACS profiles of TIM-3 and CTLA-4 expressions within the CD3+ CD69+ population of hu-PBMCs grafted with MDA-MB-231 cells in embryos treated either with control (NaCl 0.9%) or with anti-PD-1 (pembrolizumab). Data are presented for pooled embryos engrafted with donor 6 ( n = 20 pooled embryos for control; n = 25 pooled embryos for pembrolizumab). .

Article Snippet: The MDA-MB-436 (ATCC-HTB-130) and MDA-MB-231 (ATCC-HTB-26) TNBC cell lines were obtained from American Type Culture Collection and HCT 116 from Cytion (116:300195) and were not re-authenticated.

Techniques: Injection, Control, Labeling

Journal: EMBO Molecular Medicine

Article Title: Humanized avian embryo models replicate an immune tumor environment for rapid immunotherapy studies

doi: 10.1038/s44321-026-00398-5

Figure Lengend Snippet: ( A ) Histograms showing the quantification of the tumor volumes of MDA-MB-231 cells co-engrafted with hu-PBMCs from donor 8. Embryos were treated either with control (NaCl 0.9%) or with 3 increasing doses of pembrolizumab (35.6, 178, 890 mg/kg). Dots represent volumes normalized to each embryo’s BSA. N = 1 ( n = 25 embryos in control group, n = 7 in 35.6 mg/kg dose, n = 25 in 178 mg/kg dose and n = 12 in 890 mg/kg dose). Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. The percentage represents the average volume reduction per experimental group. Unpaired T test for the two highest doses and Mann–Whitney test for 35.6 mg/kg dose. Exact P -values indicated on graph. ** P < 0.01, ns: not significant. ( B ) Representative light sheet microscopy 3D images of tumors in E4 chick embryo co-engrafted with MDA-MB-231 cells and hu-PBMCs from donor 8, in pembrolizumab (178 mg/kg) and control conditions. The right panels illustrate the extraction of the fluorescent signal for measuring tumor volume (V) with Imaris software (in green). Scale bars: 150 µm. ( C ) Histograms showing the quantification of the tumor volumes of MDA-MB-231 cells co-engrafted with hu-PBMCs from donor 8, pre-incubated or not with anti-MHC I antibodies at 20 µg/mL. For each group, embryos were treated either with control (NaCl 0.9%) or with anti-PD-1 (pembrolizumab) at 178 mg/kg. Dots represent volumes normalized to each embryo’s BSA. N = 1 experiment ( n = 23 embryos in control group, n = 28 in anti-PD-1 group, n = 27 in control group with anti-MHC I and n = 29 in anti-PD-1 group with anti-MHC I). Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. The percentage represents the average volume reduction between treated and control for each group. Statistical analyses were conducted with Mann–Whitney when comparing to control group with anti-MHC I, and Unpaired T test when comparing to control group or anti-PD-1 groups. Exact P -values indicated on graph. * P < 0.05, ** P < 0.01, ns: not significant. ( D ) Histograms showing the quantification of the tumor volumes of MDA-MB-436 and HCT 116 cells co-engrafted with hu-PBMCs from donor 8. Embryos were treated either with control (NaCl 0.9%) or anti-PD-1 (pembrolizumab). Dots represent volumes normalized to each embryo’s BSA. N = 1 experiment per cell line for MDA-MB-436 ( n = 15 in control group and n = 19 in pembrolizumab group); for HCT 116 ( n = 17 and n = 18). Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. The percentage represents the average volume reduction. Statistical analyses were conducted with Unpaired T-test for MDA-MB-436, Mann–Whitney for HCT 116. Exact P -values indicated on graphs. * P < 0.05, ns: not significant. ( E ) Graph showing the variation of average volume reduction of MDA-MB-231 tumors co-grafted with hu-PBMCs from different donors in embryos treated with anti-PD-1 (pembrolizumab) or control (NaCl 0.9%). N = 1 for donor 1 ( n = 13 embryos in control group and n = 19 embryos in pembrolizumab group); N = 1 for donor 2 ( n = 9 and n = 9); N = 1 donor 6 ( n = 17 and n = 21); N = 1 for donor 8 ( n = 25 and n = 24); N = 1 for donor 9 ( n = 19 and n = 17). Donors are color-coded (orange round: donor 1, green diamond: donor 2, blue square: donor 6, red upward triangle: donor 8, black downward triangle: donor 9). Statistical analysis was done with Mann–Whitney (donor 6) or Unpaired T test (donor 1, 2, 8 and 9). ** P < 0.01 ( P = 0.0016 for donor 1 and P = 0.0057 for donor 8), **** P < 0.0001 (donor 9), ns: not significant ( P = 0.916 for donor 2 and P = 0.3631 for donor 6). ( F ) Histogram showing the fraction of CSFE+ Caspase 3+ cells in tumors of MDA-MB-231 cells co-grafted with hu-PBMCs from donor 6 in embryos treated with anti-PD-1 (pembrolizumab) and control. n = 35 sections for control from 3 embryos; n = 30 sections for pembrolizumab condition from 3 embryos. Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Mann–Whitney test, exact P -value indicated on the graph. * P < 0.05. ( G ) Microphotographs illustrating immunofluorescent labeling of chick embryo cryosections, 48 h after co-grafting of CFSE+ MDA-MB-231 cells and hu-PBMCs from donor 6 and treatment with anti-PD-1 (pembrolizumab) or control. Immune cells were labeled with anti-human CD45 antibody (red), apoptotic cells with anti-human cleaved Caspase 3 antibody (white). Avian and human nuclei were stained with Hoechst (blue). White arrow points CFSE+ Caspase 3+ cells. Scale bars: 20 µm. ( H , I ) Histograms showing the quantification of the tumor volumes of MDA-MB-231 cells co-grafted with hu-PBMCs from donor 8 and treated either with control, anti-PD-1 (pembrolizumab at 178 mg/kg), combination of 2 chemotherapies or combination of the three treatments (tritherapies). In ( H ), embryos were treated with gemcitabine-carboplatin (0.68 mg/kg and 92.3 mg/kg, respectively) or tritherapy (combination of pembrolizumab-gemcitabine-carboplatin). N = 1 ( n = 11 embryos in control group, n = 12 in anti-PD-1, n = 12 in gemcitabine-carboplatin and n = 8 in tritherapy). In ( I ), embryos were treated with docetaxel-carboplatin (1.4 mg/kg and 92.3 mg/kg, respectively) or the tritherapy (combination of pembrolizumab-docetaxel-carboplatin). N = 1 ( n = 9 embryos in control group, n = 14 in anti-PD-1, n = 11 in docetaxel-carboplatin and n = 9 in tritherapy). Dots represent volumes normalized to each embryo’s BSA. Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. The percentage represents the average volume reduction compared to control. Statistical analyses were conducted with Mann–Whitney (tritherapy group for histogram ( H ); anti-PD-1 group for ( I )) or Unpaired T test (anti-PD-1 and chemotherapies groups for ( H ); tritherapy and chemotherapies groups for ( I )). Exact P -values indicated on graphs. * P < 0.05, ** P < 0.01, ns: not significant. .

Article Snippet: The MDA-MB-436 (ATCC-HTB-130) and MDA-MB-231 (ATCC-HTB-26) TNBC cell lines were obtained from American Type Culture Collection and HCT 116 from Cytion (116:300195) and were not re-authenticated.

Techniques: Control, MANN-WHITNEY, Microscopy, Extraction, Software, Incubation, Labeling, Staining

Journal: EMBO Molecular Medicine

Article Title: Humanized avian embryo models replicate an immune tumor environment for rapid immunotherapy studies

doi: 10.1038/s44321-026-00398-5

Figure Lengend Snippet: ( A ) Histograms showing the quantification of the tumor volumes of MDA-MB-231, treated either with control (NaCl 0.9%) or anti-PD-1 (pembrolizumab). Dots represent volumes normalized to each embryo’s BSA. N = 1 ( n = 13 in control group and n = 15 in anti-PD-1 group). Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. The percentage represents the average volume reduction. Unpaired T test, exact P -value indicated on graph, ns: not significant. ( B ) Histograms showing the quantification of the tumor volumes of MDA-MB-436 cells, and HCT 116 cells co-engrafted with hu-PBMCs from donor 1 or 6, respectively. Embryos were treated either with control (NaCl 0.9%) or anti-PD-1 (pembrolizumab). Dots represent volumes normalized to each embryo’s BSA. N = 1 experiment per cell line, for MDA-MB-436 ( n = 8 in control group and n = 9 in anti-PD-1 group); for HCT 116 ( n = 26 and n = 32). Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. The percentage represents the average volume reduction. Statistical analyses were conducted with Mann–Whitney for MDA-MB-436 and T test with Welch’s correction for HCT 116. Exact P -values indicated on graphs. ** P < 0.01, ns: not significant. ( C ) Table showing the characterization of HLA-A2 haplotypes of the different hu-PBMC donors and cancer cell lines. ( D ) Analysis of survival rate (left axis) and mean body surface area (BSA, right axis) of chick embryos injected with increasing doses of docetaxel (0.28, 1.4, 7, 35 mg/kg). Each dose and control (NaCl 0.9%) were injected to 15 embryos, N = 1. The dose 1.4 mg/kg was chosen as MTD. Data are represented as mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SD. Unpaired T-test compared to excipient, exact P -values indicated on the graph, ns: non-significative.

Article Snippet: The MDA-MB-436 (ATCC-HTB-130) and MDA-MB-231 (ATCC-HTB-26) TNBC cell lines were obtained from American Type Culture Collection and HCT 116 from Cytion (116:300195) and were not re-authenticated.

Techniques: Control, MANN-WHITNEY, Injection

Journal: Molecular cell

Article Title: Dietary supplement chondroitin-4-sulfate exhibits oncogene-specific pro-tumor effects on BRAF V600E melanoma cells

doi: 10.1016/j.molcel.2018.02.010

Figure Lengend Snippet: Key Resources Table

Article Snippet: Human: NB4 , CLS , Cat# 300299/p638_NB-4; RRID: CVCL_0005.

Techniques: Virus, Recombinant, RNA Extraction, Transfection, SYBR Green Assay, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Malachite Green Assay, Activity Assay, shRNA, Sequencing, Mutagenesis, CRISPR, Plasmid Preparation, Software, Expressing

Journal: International Journal of Nanomedicine

Article Title: Magnetic thermoablation stimuli alter BCL2 and FGF-R1 but not HSP70 expression profiles in BT474 breast tumors

doi: 10.2147/IJN.S77372

Figure Lengend Snippet: Effect of magnetic thermoablation on the BCL2, HSP70, and FGF-R1 protein expression pattern in BT474 tumors. Notes: Representative pictures after IHC staining of BCL2, HSP70, and FGF-R1 were chosen. Blue: nuclei, red: protein-specific antibody, scale bar: 100 μm. Relative semi-quantitative expression of investigated proteins is depicted in diagrams. In diagrams, bars represent the relative number of slides with a specific protein expression normalized to the total number of all slides in the specific treatment group. Bar colors represent the amount of specifically stained area within the viable tumor region: unstained/negative (light gray), less than 50% of vital area stained (dark grey), more than 50% of vital area stained (black). Whereas a downregulation of BCL2 and FGF-R1 can be assumed, HSP70 expression remained unchanged. Abbreviations: BCL2, B-cell lymphoma 2; HSP70, heat shock protein; FGF-R1, fibroblast growth factor receptor 1; IHC, immunohistochemistry; MNP, magnetic nanoparticles; AMF, alternating magnetic field.

Article Snippet: For tumor implantation, 200 μL Matrigel (BD MatrigelTM Basement Membrane Matrix; Becton Dickinson GmbH, Germany) containing 1×10 7 BT474 cells (human mammary-adenocarcinoma, Cell Lines Service, Germany) were injected subcutaneously between the shoulder blades of 6–15 week old female severe combined immune deficiency (SCID)–Balb/c mice (Harlan Laboratories, the Netherlands).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Frontiers in Nutrition

Article Title: Active fractions of golden-flowered tea ( Camellia nitidissima Chi ) inhibit epidermal growth factor receptor mutated non-small cell lung cancer via multiple pathways and targets in vitro and in vivo

doi: 10.3389/fnut.2022.1014414

Figure Lengend Snippet: Inhibition of proliferation and clonogenic ability of NSCLC cell lines by four active fractions of CNC. (A) Inhibitory effects of different concentrations of CLP, CLS, CFP, and CFS (6.25, 12.5, 25, 50 μg⋅mL –1 ) on NCI-H1975, HCC827 and A549 cells, respectively. (B) IC50 of different concentrations of CLP, CLS, CFP, and CFS (6.25, 8, 10, 12.5, 15, 20, 25, and 50 μg⋅mL –1 ) on NCI-H1975, HCC827, and A549 cells, respectively. (C) Inhibitory effects of different concentrations of CLS, CLP, CFS, and CFP (1.5625, 3.125, 6.25 μg⋅mL –1 ) on colony formation of NCI-H1975 cells, respectively. *Indicates p < 0.05, **indicates p < 0.01 and *** indicates p < 0.001 relative to the control by ANOVA. The data are presented as the mean ± standard deviation ( n = 3).

Article Snippet: NCI-H1975 cells were incubated in different concentration of CLS for 24 h, the cells were cultured with 1 μM DCFH 2 -DA (Solarbio, D6470) for 30 min at 37°C.

Techniques: Inhibition, Control, Standard Deviation

Journal: Frontiers in Nutrition

Article Title: Active fractions of golden-flowered tea ( Camellia nitidissima Chi ) inhibit epidermal growth factor receptor mutated non-small cell lung cancer via multiple pathways and targets in vitro and in vivo

doi: 10.3389/fnut.2022.1014414

Figure Lengend Snippet: Inhibition of proliferation rate of NCI-H1975 cells by four active fractions of CNC. (A–D) Representative field of view of immunofluorescence assay with anti-EdU antibody is shown, where in NCI-H1975 cells, the signal of incorporating with EdU was green and the nucleus was stained blue by Hoechst. Histograms showed the mean values of cell proliferation rates after administration of different concentrations of CLS, CLP, CFS, and CFP (6.25, 12.5, 25 μg⋅mL –1 ). *Indicates p < 0.05, **indicates p < 0.01 and ***indicates p < 0.001 relative to the control by ANOVA. The data are presented as the mean ± standard deviation ( n = 3).

Article Snippet: NCI-H1975 cells were incubated in different concentration of CLS for 24 h, the cells were cultured with 1 μM DCFH 2 -DA (Solarbio, D6470) for 30 min at 37°C.

Techniques: Inhibition, Immunofluorescence, Staining, Control, Standard Deviation

Journal: Frontiers in Nutrition

Article Title: Active fractions of golden-flowered tea ( Camellia nitidissima Chi ) inhibit epidermal growth factor receptor mutated non-small cell lung cancer via multiple pathways and targets in vitro and in vivo

doi: 10.3389/fnut.2022.1014414

Figure Lengend Snippet: CLS induced programmed NSCLC death through pyroptosis. (A) TdT-mediated dUTP Nick-End Labeling (TUNEL) positive rate of NCI-H1975 cells after administration of different concentrations of CLS (6.25, 12.5, 25 μg⋅mL –1 ). (B) Reactive oxygen species (ROS) positive rate of NCI-H1975 cells after administration of different concentrations of CLS (6.25, 12.5, 25 μg⋅mL –1 ). (C) Annexin V-FITC/propidine iodide (PI) staining of NCI-H1975 cells after 48 h was performed to identify early/late apoptosis, and the data were analyzed via flow cytometry. (D) SEM was used to detect the morphological changes of NCI-H1975 cells. (E,F) Relative uperoxide dismutase (SOD) activity and relative lactate dehydrogenase (LDH) activity of NCI-H1975 cells after administration of different concentrations of CLS (6.25, 12.5, 25 μg⋅mL –1 ). (G) Relative IL-1β activity and relative LDH activity of NCI-H1975 cells after administration of different concentrations of CLS (6.25, 12.5, 25 μg⋅mL –1 ). *Indicates p < 0.05, **indicates p < 0.01 and ***indicates p < 0.001 relative to the control by ANOVA. The data are presented as the mean ± standard deviation ( n = 3).

Article Snippet: NCI-H1975 cells were incubated in different concentration of CLS for 24 h, the cells were cultured with 1 μM DCFH 2 -DA (Solarbio, D6470) for 30 min at 37°C.

Techniques: End Labeling, TUNEL Assay, Staining, Flow Cytometry, Activity Assay, Control, Standard Deviation

Journal: Frontiers in Nutrition

Article Title: Active fractions of golden-flowered tea ( Camellia nitidissima Chi ) inhibit epidermal growth factor receptor mutated non-small cell lung cancer via multiple pathways and targets in vitro and in vivo

doi: 10.3389/fnut.2022.1014414

Figure Lengend Snippet: Transcriptome analysis of NCI-H1975 cells between the control group and CLS-treated group. (A) Volcano plot with 1,077 DEGs up-regulated and 1,008 DEGs down-regulated. (B) GO enrichment analysis of up-regulated and down-regulated DEGs. (C) KEGG enrichment analysis of up-regulated and down-regulated DEGs. (D) The expression heatmap of 22 DEGs related to Cytokine-cytokine receptor interaction and Rheumatoid arthritis signaling pathway in each sequencing sample. (E) The expression heatmap of 23 DEGs related to PIK-Akt signaling pathway in each sequencing sample. (F) The expression heatmap of 16 DEGs related to MAPK signaling pathway in each sequencing sample. (H,I) Interaction relationships of DEGs on four signaling pathways, including TGF-β signaling pathway, TNF signaling pathway, PIK-Akt signaling pathway and MAPK signaling pathway.

Article Snippet: NCI-H1975 cells were incubated in different concentration of CLS for 24 h, the cells were cultured with 1 μM DCFH 2 -DA (Solarbio, D6470) for 30 min at 37°C.

Techniques: Control, Expressing, Sequencing, Protein-Protein interactions

Journal: Frontiers in Nutrition

Article Title: Active fractions of golden-flowered tea ( Camellia nitidissima Chi ) inhibit epidermal growth factor receptor mutated non-small cell lung cancer via multiple pathways and targets in vitro and in vivo

doi: 10.3389/fnut.2022.1014414

Figure Lengend Snippet: Comparative analysis of quantitative real-time PCR (RT-qPCR) data and Western Blot (WB) data. (A) Relative mRNA levels of nine genes including TGFB2, INHBB, TNFRSF10C, PIK3R3, ITGB8, EIF4E1B, NTRK, CACN1D, MEF2C. (B) Protein levels of INHBB, PIK3R3, TGFB2, ITGB8, TrkB, CACNA1D detected in CLS-treated NCI-H1975 cells were detected by Western blot with quantification of each protein, n = 3 biologically independent embryos. *Indicates p < 0.05, **indicates p < 0.01 and ***indicates p < 0.001 relative to the control by ANOVA. The data are presented as the mean ± standard deviation ( n = 3).

Article Snippet: NCI-H1975 cells were incubated in different concentration of CLS for 24 h, the cells were cultured with 1 μM DCFH 2 -DA (Solarbio, D6470) for 30 min at 37°C.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Control, Standard Deviation

Journal: Neural Regeneration Research

Article Title: Maintaining moderate levels of hypochlorous acid promotes neural stem cell proliferation and differentiation in the recovery phase of stroke

doi: 10.4103/1673-5374.392889

Figure Lengend Snippet: Microglia are a crucial source of hypochlorous acid in the stroke recovery phase. (A–C) Co-localization and statistical analysis of Iba-1 + microglia (Cy3, red) and hypochlorous acid (HKOCl-3, green) in ischemic SVZ of MCAO rats. Hypochlorous acid was visualized using a HKOCl-3 probe. Hypochlorous acid levels (normalized to day 1) increased on the 3 rd and 5 th days after MCAO. Fluorescence intensity of Iba-1 increased on the 5 th day, but decreased on the 7 th and 14 th days after MCAO. Scale bar: 100 μm. (D) Hypochlorous acid production in BV2 cells and neutrophils with or without O/R challenge. (E) BV2/C17.2 co-culture system to examine the effect of hypochlorous acid produced by BV2 cells, with or without O/R challenge. (F) Neutrophil/C17.2 co-culture system to examine the effect of hypochlorous acid produced by neutrophils on C17.2 cell viability under O/R conditions. Taurine (100 μM) and 4-ABAH (100 μM) were used to scavenge or prevent hypochlorous acid production, respectively, in O/R-challenged neutrophils. Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (B, C: one-way analysis of variance followed by Dunnett’s post hoc test; D–F: one-way analysis of variance followed by Tukey’s post hoc test). DAPI: 4,6-Diamidino-2-phenylindole; Iba1: ionized calcium binding adapter molecule 1; O/R: oxygen-glucose deprivation/reoxygenation; SVZ: subventricular zone.

Article Snippet: The mouse NSC line C17.2 (kindly provided by Professor Jiangang Shen, RRID: CVCL_4511), microglia cell line BV2 (Cat# 305156, CLS Cell Lines Service GmbH, Eppelheim, Germany, RRID: CVCL_0182), and hippocampal neuronal cell line HT22 (Cat# 305158, CLS Cell Lines Service GmbH, RRID: CVCL_0321) were cultured in complete high-glucose DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin.

Techniques: Fluorescence, Co-Culture Assay, Produced, Binding Assay