Journal: Science Advances

Article Title: Immunosuppressive macrophages determine the effect of cellular senescence on tumor progression

doi: 10.1126/sciadv.adx2988

Figure Lengend Snippet: ( A ) Schematic representation of the construct used to generate SuSe mice. The bicistronic construct consists of a fusion protein FRB–caspase 8 (Casp8), followed by an internal ribosome entry site (IRES) and the mCherry autofluorescent protein, all under the control of the 1200–base pair (bp)–long mouse p16 promoter. Similarly, the bicistronic construct, FKBP–caspase 8, IRES, and GFP, is expressed under the control of the 1400-bp-long IL-6 promoter. The presence of mCherry and GFP allows for the identification of cells expressing high levels of p16 and IL-6, respectively. The addition of AP21967 induces the heterodimerization of FRB and FKBP, resulting in caspase 8–mediated apoptosis. ( B ) SuSe mice were crossed with MMTV-PyMT mice to characterize the effect of senescence during breast cancer progression. ( C ) SuSe/PyMT mice were euthanized at different time points corresponding to the indicated tumor stages. Samples from mammary glands were processed and stained for SA-β-Gal or analyzed by immunofluorescence to visualize the expression of the reporters under the control of p16 and/or IL-6 promoters, mCherry, and GFP, respectively. Results are expressed as averages ± SDs of three mice. ns, not significant. ( D ) The same mice were treated with vehicle or AP21967 every 3 days, starting at 4 weeks. At 6 weeks, mice were euthanized and analyzed as in (C).

Article Snippet: PyMT/SuSe mice were injected intraperitoneally (ip) every 3 days with 0.2 μg/g of body weight of AP21967 (#635055, Takara) from the fourth or ninth week of life until week 13.

Techniques: Construct, Control, Expressing, Staining, Immunofluorescence

Journal: Science Advances

Article Title: Immunosuppressive macrophages determine the effect of cellular senescence on tumor progression

doi: 10.1126/sciadv.adx2988

Figure Lengend Snippet: ( A ) SuSe/PyMT mice were treated with vehicle or AP21967 either at 4 weeks (early senolysis) or at 9 weeks (late senolysis), and tumor volumes were monitored at the specified time points. Typically, each mouse developed four or five tumors, and we calculated the average tumor volume per mouse. The results shown are the means of these average tumor volumes ± SD. Two-way analysis of variance (ANOVA) was used to determine statistical significance. ( B ) At the end of the experiment, mice were euthanized, and lungs were fixed, embedded in paraffin, and sectioned as indicated. ( C ) Representative examples of staining with anti–pan-keratin to visualize metastases. ( D ) The numbers of metastases (Mets) were determined in every slide and are represented as averages ± SD. t test was used to determine statistical significance. ( E ) The areas of all metastases are represented. Average number of metastases is represented in the y axes, and average metastasis areas are represented as circle areas. t test was used to determine statistical significance.

Article Snippet: PyMT/SuSe mice were injected intraperitoneally (ip) every 3 days with 0.2 μg/g of body weight of AP21967 (#635055, Takara) from the fourth or ninth week of life until week 13.

Techniques: Staining

Journal: Science Advances

Article Title: Immunosuppressive macrophages determine the effect of cellular senescence on tumor progression

doi: 10.1126/sciadv.adx2988

Figure Lengend Snippet: ( A ) Uniform Manifold Approximation and Projection (UMAP) of single cells from tumors treated as shown in , showing distinct clusters predominantly determined by cell type and status. T reg , regulatory T cells. ( B ) UMAP plots at 4 and 9 weeks for vehicle- and AP21967-treated mice, illustrating the distribution of different cell populations. ( C ) Total number of cells in each population that increased (Up) or decreased (Down) between 4 and 9 weeks and with or without AP21967 treatment. ( D ) Quantification of the log 2 fold change (FC) in the number of each cell type at 9 weeks after AP21967 treatment compared to vehicle treatment. ( E ) Immunohistochemical analyses of the infiltration of endothelial cells (CD31 + ) and lymphocytes (CD8 + ) in tumors from control mice and tumors from mice treated with AP21967, with accompanying bar graphs showing the percentage of CD31 + and CD8 + cells. t test was used to determine statistical significance.

Article Snippet: PyMT/SuSe mice were injected intraperitoneally (ip) every 3 days with 0.2 μg/g of body weight of AP21967 (#635055, Takara) from the fourth or ninth week of life until week 13.

Techniques: Immunohistochemical staining, Control

Journal: Science Advances

Article Title: Immunosuppressive macrophages determine the effect of cellular senescence on tumor progression

doi: 10.1126/sciadv.adx2988

Figure Lengend Snippet: ( A ) Tumor samples from mice treated with vehicle or AP21967 at 9 weeks were homogenized, and the levels of the indicated cytokines were analyzed using a cytokine array. The log 2 FC (AP21967/vehicle) for each cytokine is presented. ENA-78, epithelial-derived neutrophil-activating peptide 78; GROα, growth-regulated oncogene-alpha. ( B ) Levels of CCL2 and CXCL1 were quantified by ELISA from the same tumor homogenates as in (A). ( C ) UMAP plots showing the expression of the most up-regulated cytokines (CCL2, CCL3, and CCL7) and their receptors (CCR1, CCR2, and CCR5) in different cell populations. Relevant cell clusters are indicated. N, neutrophils; MØ, macrophages; E, endothelial cells; F, fibroblasts. ( D ) Flow cytometry analysis of myeloid cells purified from tumors obtained from SuSe mice treated with vehicle or AP21967. We identified TAMs and cells positive for the myeloid marker CD11B and for the macrophage marker F4/80. ( E ) The same cells as in (D) were cultured for 24 hours—in the presence of vehicle or anti-CCL2—and the presence of CCL2 in the conditioned medium was determined by ELISA. ( F ) Schematic representation of the specificities of receptors for CCL2, CCL3, and CCL7. ( G ) Flow cytometry analysis of CCR2 in the same cells as in (D). FSH-H, forward scatter height.

Article Snippet: PyMT/SuSe mice were injected intraperitoneally (ip) every 3 days with 0.2 μg/g of body weight of AP21967 (#635055, Takara) from the fourth or ninth week of life until week 13.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Purification, Marker, Cell Culture