Journal: Frontiers in Immunology

Article Title: Senescent human fibroblasts have increased FasL expression and impair the tumor immune response

doi: 10.3389/fimmu.2025.1685269

Figure Lengend Snippet: Therapy-induced senescence in tumor spheroids impairs the viability of infiltrated immune cells. (A) Schematic representation of the 3D tumor spheroid model. In brief, A549 tumor cells alone or with an equal number of HDF (5000 cells for each) were mixed to form monospheroids or mixed-cell spheroids, respectively. Five days after, spheroids were treated with doxorubicin (0.1µM) or irradiated (15 Gy) to induce senescence. Then, four days later PBMCs (5 x 10 5 ) were added, and their infiltration into the spheroid was quantified 48 hours later by flow cytometry. (B) Representative images of spheroids stained for β-galactosidase activity six days after being treated with doxorubicin or irradiation. Unstained spheroids or spheroids not exposed to therapy were used as controls. The scale bar represents 100 μm. (C) Graph showing the average size of spheroids represented by the largest brightfield object area metric (µm 2 ) over time following treatments as detected by IncuCyte imaging. Shown is the mean ± SEM of three independent experiments. Statistical differences were identified by mixed-effects modeling with Tukey’s multiple comparisons. ****p < 0.0001. (D) Representative images of immunostained spheroid sections showing CD45 + immune cell infiltration (white), the presence of HDF (GFP, green), FasL expression (red), and cell nuclei (DAPI, blue). Scale bar = 100 μm. (E) Graph showing the proportion of live (Annexin V - /PI - ) CD45 + immune cells infiltrated in spheroids from the indicated groups. Each dot represents the average of infiltrated cells in n=6 spheroids collected from four independent experiments. Shown is the mean ± SEM. Statistical analysis between groups was performed by a one-way ANOVA with Tukey’s multiple comparisons. *p < 0.05. (F) Schematic of the in vivo experimental design. NSG-SGM3 mice were injected intravenously (i.v) with 4X10 5 non-senescent HDF. After 29 days mice received a single dose of whole thorax radiation (12Gy). Seven days after, mice were sacrificed, and lungs were collected for analysis. (G) Representative immunofluorescence staining of HDF (GFP in green), FasL (in red), and cell nuclei (DAPI in blue) from irradiated and non-irradiated lung tissues. The scale bar represents 100 μm. (H) Confocal images shown at higher magnification of a tissue section as described in (G) .

Article Snippet: Sections were mounted on gelatin-coated slides and subjected to immunofluorescence staining using a GFP polyclonal antibody (A-11122; Thermo Fisher Scientific, USA), a FasL antibody (NOK-1, sc-33716; Santa Cruz Biotechnology), and DAPI for nuclear counterstaining.

Techniques: Irradiation, Flow Cytometry, Staining, Activity Assay, Imaging, Expressing, In Vivo, Injection, Immunofluorescence