Journal: Nature Communications

Article Title: An ex vivo uterine system captures implantation, embryogenesis, and trophoblast invasion via maternal-embryonic signaling

doi: 10.1038/s41467-025-60610-x

Figure Lengend Snippet: A A representative 3D image of whole-mount immunofluorescence for FOXA2, a marker of uterine glands as well as embryonic PrE. See also Movie S . B Representative 3D images of whole-mount immunofluorescence for CD45 and F4/80, markers of leukocytes and macrophages, respectively. White arrows indicate the presence of leukocytes and macrophages. See also Movie S . C Slices from a representative 3D image of whole-mount immunofluorescence for a decidualization marker KLF5. White arrows indicate the induction of KLF5 in the embryo-attached subluminal stroma. Dotted line indicates the boundary between LE and stroma. D Representative 3D images of whole-mount immunofluorescence for FLK-1 and αSMA, markers of VE and VSM, respectively. White dotted areas indicate the avascular region in the vicinity of the embryo attachment site corresponding to in vivo implantation. See also Movie S . E A slice from representative 3D images of whole-mount immunofluorescence for a proliferation marker Ki67. The cultured ex vivo uterus sustains an appropriate proliferation-differentiation switching (PDS) pattern, an index of uterine receptivity to the embryos. Note the cessation of proliferation in LE essential for ensuing trophoblast invasion. See also Movie S . Dotted line indicates the boundary between LE and stroma. F Immunofluorescence of CK8 on histological sections at 36 h shows the onset of trophoblast invasion (arrowheads) into the stromal layer of the endometria. G A slice from a representative 3D image of whole-mount immunofluorescence for E-cadherin at 48 h shows the breach and elimination of uterine LE. tdTomato-negative uterine E-cadherin breaks at the boundary with the embryo indicated by white arrows. H H&E staining at 48 h exhibits enlarged nuclei in invading trophoblasts. White and orange dotted lines represent the surface of LE and the boundary between LE and stroma, respectively. PrE, primitive endoderm; Em, embryo; LE, luminal epithelium; GE, glandular epithelium; Str, stroma; Dec, decidua. DsRed2 or tdTomato indicates the mitochondria or cell membrane of all embryonic/extraembryonic cell lineages. Scale bars: 50 μm. Three independent experiments with different recipient animals were performed for all the data.

Article Snippet: For CD45, F4/80, KLF5, αSMA, FLK-1, Ki67, and E-cadherin, primary antibodies for CD45 (1:300, rat, #103101, BioLegend, USA), F4/80 (1:300, rat, MCA497, BIORAD), KLF5 (1:300, rabbit, #21017-1-AP, Proteintec, USA), αSMA (1:300, rabbit, #19245, Cell Signaling, USA), FLK-1 (1:300, rat, #550549, BD Biosciences, USA), Ki67 (1:300, rabbit, MA5-14520, Thermo Fisher Scientific, USA), and E-cadherin (1:300, rabbit, #3195, Cell Signaling, USA) were used with the appropriate secondary antibodies with the same protocol as pAKT/AKT staining.

Techniques: Immunofluorescence, Marker, In Vivo, Cell Culture, Ex Vivo, Staining, Membrane

Journal: Journal for immunotherapy of cancer

Article Title: Understanding the dynamics of TKI-induced changes in the tumor immune microenvironment for improved therapeutic effect.

doi: 10.1136/jitc-2024-009165

Figure Lengend Snippet: Figure 1 TKI therapy alters the tumor immune microenvironment. (A) Representative mIF images of pretreatment tumor and resected samples analyzed for immune-related biomarkers. (B) Densities (cells/mm2) of CD8+, CD8+GB+, CD8+PD-1+, CD163+, CD68+, and CD163+CD68+ by mIF quantification in paired pretreatment tumor samples and resected tumors. (C) Cell viability CCK-8 assay for cells treated with TKIs (osimertinib: 10 nM, lorlatinib: 10 nM), activated T cells (1:1 ratio to cancer cells), or the combination. (D) T cell-mediated cancer cell-killing assay. PC-9 and H3122 cells co-cultured with activated T cells for 48 hours with or without TKIs (osimertinib: 10 nM, lorlatinib: 10 nM) were subjected to crystal violet staining. Ratio of cancer cells to T cells: 1:1. (E) Ki67 incorporation assay on PC-9 and H3122 cells treated as indicated. Activated T cells (1:1 ratio to cancer cells) or TKIs (osimertinib: 10 nM, lorlatinib: 10 nM) were added to the culture medium for 48 hours. Cells were then counterstained with DAPI. (F) PC-9 cells were injected into mice (n=3 mice per group) on day 0, hu-PBMCs were injected into mice via the tail vein on day 7, and osimertinib was administered as indicated. (G) Macroscopic appearance of tumors after drug application for 4 weeks. (H) The tumor weight (g) for each mouse is shown. *p<0.05, **p<0.01. ns, no significance. (I) Immunofluorescence staining with an antibody against CD8 to detect T cells and antibodies against CD68 and CD206 to detect macrophages in TKI-resistant non-small cell lung cancer tissues (11 cases of EGFR-TKI resistance, 5 cases of ALK-TKI resistance). Scale bar: 50 µm. ALK, anaplastic lymphoma kinase; DAPI, 4′,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; ALKi,anaplastic lymphoma kinase inhibitor; EGFRi,epidermal growth factor receptor inhibitor; hu-PBMC,human-Peripheral blood mononuclear cell; mIF, multiplex immunofluorescence; PD-1, programmed cell death protein 1; TKI, tyrosine kinase inhibitor.

Article Snippet: Then, the cells were fixed and incubated overnight with Ki67 (#M00254- 8, Boster, Wuhan, China).

Techniques: CCK-8 Assay, Cell Culture, Staining, Injection, Immunofluorescence, Multiplex Assay

Journal: Journal for immunotherapy of cancer

Article Title: Understanding the dynamics of TKI-induced changes in the tumor immune microenvironment for improved therapeutic effect.

doi: 10.1136/jitc-2024-009165

Figure Lengend Snippet: Figure 6 Aspirin enhances the antitumor immunity response. (A) PC-9 cells were injected into mice (n=3 mice per group) on day 0, hu-PBMCs were injected into the tail veins of mice on day 7, and osimertinib/osimertinib plus aspirin was administered as indicated. (B) Macroscopic appearance of tumors after drug application for 4 weeks. The tumor weight (g) of each mouse is shown. *p<0.05, ***p<0.001. (C) Cell viability CCK-8 assay for cells treated with aspirin (200 µM), activated T cells (1:2 ratio to cancer cells), or the combination for 48 hours. Data are shown as the mean±SEM. *p<0.01. (D) T cell-mediated cancer cell-killing assay. PC-9OR and HCC827OR cells co-cultured in the indicated groups for 48 hours were subjected to crystal violet staining. Ratio of cancer cells to T cells: 2:1. (E) Ki67 incorporation assay on PC-9OR and HCC827OR cells treated as indicated. Activated T cells (1:2 ratio to cancer cells) or aspirin (200 µM) were added to the culture medium for 48 hours. Cells were then counterstained with DAPI. (F) Flow cytometry analysis of the exhaustion- and activation-related molecule Foxp3 in activated T cells co-cultured with the indicated cells (ratio of cancer cells to T cells: 1:1) for 48 hours with or without aspirin (200 µM). (G) PD-L1 levels in total protein extracts from indicated cells treated with aspirin for 48 hours, analyzed by western blotting. (H) LAMC2 levels in total protein extracts from the indicated cells treated with aspirin for 48 hours, analyzed by western blotting. (I) Immunofluorescence staining with an antibody against LAMC2 in PC-9 xenografts treated with osimertinib or osimertinib plus aspirin. (J) Schematic diagram of a T-cell chemotaxis assay directly regulated by the treatment of PC-9OR cells with aspirin (200 µM). Representative images of infiltrating T cells stained with CFSE dye. (K) Representative images of the immunofluorescence staining of CD68 (red), CD206 (green), and DAPI (blue) in mouse tumor tissue sections. (L) Immunofluorescence staining with antibodies against CD8 (red) and LAMC2 (green) in non-small cell lung cancer tissues treated with osimertinib or osimertinib plus aspirin. Scale bar: 50 µm.hu-PBMC,human-Peripheral blood mononuclear cell; S.C,Subcutaneous; CFSE, carboxyfluorescein diacetate succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; Foxp3, forkhead box P3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LAMC2, laminin subunit γ−2; PBS, phosphate-buffered saline; PD-L1, programmed death ligand 1.

Article Snippet: Then, the cells were fixed and incubated overnight with Ki67 (#M00254- 8, Boster, Wuhan, China).

Techniques: Injection, CCK-8 Assay, Cell Culture, Staining, Flow Cytometry, Activation Assay, Western Blot, Immunofluorescence, Chemotaxis Assay, Saline