Journal: Cancer Cell

Article Title: TIM3 + breast cancer cells license immune evasion during micrometastasis outbreak

doi: 10.1016/j.ccell.2025.06.015

Figure Lengend Snippet: Metastasis immune pressure positively selects TIM3 + metastatic cells (A) Experimental design using immunocompetent (IC) Balb/c mice and immunodeficient (ID) NOD scid gamma (NSG) mice for the assessment of metastatic immune pressure. (B) Principal-component analysis (PCA) of the RNA-seq from EpRas cells isolated from three organ (lung, liver, and brain) metastatic samples in IC and ID hosts. (C) Unsupervised hierarchical clustering from lung, liver and brain metastasis ( Z score) in ID and IC hosts ( n = 3 IC and n = 3 ID independent biological replicates). (D) Gene ontology enrichment analysis of top 50 upregulated genes in all organs from IC mice. (E) GSEA of indicated gene lists with the ranked gene expression list of IC vs. ID in all organ samples. (F) Volcano plot of gene expression in all organs comparing IC and ID mice samples ( n = 3 independent biological replicates). (G) Dot plot representing TIM3 expression of human breast cancer cell lines from the CCLE. Primary tumor-derived cells (gray) and metastasis-derived cells (pink). (H) Dot plot representing TIM3 protein levels measured by flow cytometry of mouse breast cancer cell lines with metastatic potential in experimental models. Color code as (G). (I) TIM3 immunofluorescence of liver tissue metastasis derived from 4T07 intracardiac injection in ID and IC mice. Representative image of TIM3 immunofluorescence. Scale bars, 100 μm. Dashed line delineates metastasis tissue. Boxplot quantification of TIM3 staining mean fluorescent intensity in from 5 independent mice ( n = 5 independent biological replicates). Data represent mean ± SEM. Statistical significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by unpaired Student’s t test. Also see and .

Article Snippet: Then, 5 μg/mL anti-mouse IL-1β blocking antibody or control mouse IgG (sc-2025, Santa Cruz Biotechnology) were added to the media when indicated.

Techniques: RNA Sequencing, Isolation, Gene Expression, Expressing, Derivative Assay, Flow Cytometry, Immunofluorescence, Injection, Staining

Journal: Cancer Cell

Article Title: TIM3 + breast cancer cells license immune evasion during micrometastasis outbreak

doi: 10.1016/j.ccell.2025.06.015

Figure Lengend Snippet: TIM3 drives breast cancer metastasis (A) Kaplan-Meier survival plot after intracardiac injection of 4T07 control cells versus Tim3 -KD cells in ID (NSG) and IC (Balb/c) mice with the indicated conditions. Statistical analysis using Log rank (Mantel-Cox) test. (B) Relative photon flux BLI quantification of metastatic organs at day 16 after i.c. injection of 4T07 Ctrl and Tim3 -KD cells. Data represents mean + SEM; dots represent independent biological replicates. (C) Relative photon flux BLI quantification of whole-body metastasis of 4T07 Ctrl and Tim3 -KD in IC mice. Data represents mean + SEM. n = 22 independent biological replicates. (D) Hematoxylin-eosin staining of metastatic livers from unlabeled 4T07-Ctrl and - Tim3 -KD cells. Arrows indicate metastatic lesions. (E) Quantification of micro- and macro-metastatic lesions from (D). (F) Mammary fat pad (MFP) injection of 4T1-Ctrl and - Tim3 -KD cells in Balb/c mice. Data represents tumor growth by mean + SEM of n = 16 independent biological replicates. (G) Incidence of spontaneous metastasis at day 40 after primary tumor resection (day 20) of 4T1-Crtl and 4T1- Tim3 -KD MFP injected mice. Individual BLI images from upper body. n = 8 Ctrl and n = 6 KD mice followed after resection. Statistical significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by Chi-square test. (H) Representative immunofluorescence of breast tumor cell marker Mamaglobulin-1 (MGB1) in red and TIM3 in green in human breast cancer tissue. Scale bars, 30 μm. Representative immunohistochemistry image of TIM3 showing tumor-epithelial cell staining. Dash line delineates the tumor areas. Scale bars, 100 μm. (I) Percentage of tumor TIM3-positive samples from primary (P) and metastatic (M) matched clinical samples (ConvertHER cohort). TIM3 score percentage in primary and paired-metastatic samples (right panel). n = 75 for each condition P and M. Data represented as mean ± SEM. Statistical significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by two-tailed Student’s t test in (B), (C), (E), and (I). Also see .

Article Snippet: Then, 5 μg/mL anti-mouse IL-1β blocking antibody or control mouse IgG (sc-2025, Santa Cruz Biotechnology) were added to the media when indicated.

Techniques: Injection, Control, Staining, Immunofluorescence, Marker, Immunohistochemistry, Two Tailed Test

Journal: Cancer Cell

Article Title: TIM3 + breast cancer cells license immune evasion during micrometastasis outbreak

doi: 10.1016/j.ccell.2025.06.015

Figure Lengend Snippet: TIM3 spatiotemporal dynamics in metastasis (A) Dual reporter system designed to track bulk tumor metastasis (Firefly luciferase [Fluc]-eGFP) and Tim3 promoter activity (mCherry-Nanoluciferase [Nluc]). (B) Representative BLI images of Fluc and Nluc signal in IC mice (Balb/c) upon i.c. injection of 4T07 cells experimental metastasis. BLI monitorization of metastatic growth: metastasis curve (blue line) and Tim3 reporter curve (orange line). Bar plot shows the BLI signal ratio of NLuc/FLuc representing the intensity of the Tim3 reporter signal versus the overall bulk tumor metastasis at micrometastasis and macrometastasis time points. n = 8 mice. (C) BLI ratio of NLuc/FLuc of liver metastasis. n = 8 mice. Statistical significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by one-tailed Student’s t test. (D) BLI ratio curve of NLuc/FLuc metastasis along days of experiment. Each line represents individual mice. (E) Flow cytometry of 4T07- Tim3 reporter positivity measured by mCherry intensity of liver metastatic digested tissues from ID and IC at micrometastasis and macrometastasis time points. Representative plot of n = 3 individual mice per time point. (F) IF images from liver micrometastasis and macrometastasis. Luciferase (red) and TIM3 (pink) stainings. Dash line delineates the metastatic tissue. Scale bars, 100 μm. (G) Schematic representation of the lineage tracing system introduced in 4T07 cells (see for details). Tim3 - cancer cells have red fluorescence of dsRed. Tim3 + cancer cells have green fluorescence of eGFP. (H) In vitro lineage tracing test of Tim3 + and Tim3 - 4T07 cells. Induction by 4-hydroxy-tamoxifen (4-OHT) O/N at 1 μM. Flow cytometry plots represent dsRed and GFP positive events in no induced cells (top) and 4-OHT induced cells (bottom). (I) Metastasis lineage tracing using 4T07 cells after intracardiac injection in IC mice. Short TAM induction during the first 3 days of metastatic seeding (see ). Bar plots quantifications of all lesions (113) present in livers of 6 independent experiments. Representative immunofluorescence images of liver sections showing TIM3 + (green) and TIM3 - (red) metastasis in (I) and (J). (J) Long-term TAM induction during 21 days after intracardiac injection of 4T07 cells in IC and ID mice. Bar plots quantifications of all lesions (61 IC and 37 ID) present in livers of 4 independent experiments. Also see .

Article Snippet: Then, 5 μg/mL anti-mouse IL-1β blocking antibody or control mouse IgG (sc-2025, Santa Cruz Biotechnology) were added to the media when indicated.

Techniques: Luciferase, Activity Assay, Injection, One-tailed Test, Flow Cytometry, Fluorescence, In Vitro, Immunofluorescence

Journal: Cancer Cell

Article Title: TIM3 + breast cancer cells license immune evasion during micrometastasis outbreak

doi: 10.1016/j.ccell.2025.06.015

Figure Lengend Snippet: Functional metastasis assessment of γδ T cells, CD8 T cells, and IL-1β in TIM3-mediated immunosuppression (A) Blocking antibody scheme; intraperitoneal administration and doses indicated. Seven days before 4T07 tumor cell i.c. injection and, weekly reminder of 250 μg of antibody during the experiment. (B) Representative BLI images of 4T07 whole body metastasis for the indicated immune blocking conditions. (C) Boxplot quantification of liver metastasis at micro (day 3) and macro (day 14) time points upon IgG2b, CD8, γδ TCR, and double (CD8, γδ TCR) cell depletion. Each dot represents independent mice. (D) Boxplot quantification of whole-body metastasis at micro (day 3) and macro (day 14) time points upon IgG2b, CD8, γδ TCR, and double (CD8, γδ TCR) cell depletion. Each dot represents independent mice. (E) In vitro proliferation and co-culture of γδ T cells with 4T07 tumor cells and flow cytometry. See for details. (F) Flow cytometry quantification of IL-17 γδ T cell levels after co-culture in non-treated (NT) conditions and upon anti-IL1β treatment. Data represents mean ± SEM, n = 6 (NT) and n = 4 (anti-IL1β) independent biological replicates. (G) Whole-body metastasis assays after systemic delivery of 4T07-IL-1β - KO cells compared to 4T07 TIM3 + (Ctrl) cells. Representative BLI images of metastasis at the indicated conditions. BLI metastasis growth curves. Data represents mean ± SEM, n = 10 independent mice. (H) Liver metastatic lesions at day 3 of metastatic seeding of 4T07-IL-1β-KO cells compared to 4T07 TIM3 + (Ctrl) cells. (I) Representative BLI images of ex vivo metastatic livers at day 14 after i.c. injection of 4T07-Tim3 + (Ctrl) and 4T07-IL-1β-KO cells. Statistical significance respect to the Ctrl; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by two-tailed Student’s t test in (C), (D), (F), and (G); unpaired Student’s t test in (H). Also see .

Article Snippet: Then, 5 μg/mL anti-mouse IL-1β blocking antibody or control mouse IgG (sc-2025, Santa Cruz Biotechnology) were added to the media when indicated.

Techniques: Functional Assay, Blocking Assay, Injection, In Vitro, Co-Culture Assay, Flow Cytometry, Ex Vivo, Two Tailed Test

Journal: Cancer Cell

Article Title: TIM3 + breast cancer cells license immune evasion during micrometastasis outbreak

doi: 10.1016/j.ccell.2025.06.015

Figure Lengend Snippet: Clinical and preclinical evaluation of TIM3 blockade for breast cancer metastasis (A) Disease free survival (DFS) and overall survival (OS) Kaplan-Meier curves of IHC epithelial-tumor TIM3 + and TIM3 - breast cancer primary tumor samples. Data obtained from Tissue microarrays (TMAs) with 257 breast cancer primary tumors from all subtypes. Statistical significance calculated by Log Rank (Mantel-Cox) for Chi-square and p value. (B) DFS Kaplan-Meier curves of IHC intratumoral tumor infiltrating lymphocytes (TILs) TIM3 + and TIM3 - breast cancer primary tumor samples. TMAs with 252 breast cancer primary tumors from all subtypes. Statistical significance calculated by Log Rank (Mantel-Cox) for Chi-square and p value. (C) Violin plot of TIM3 score in patients stratified by relapse ( n = 42) and non-relapse ( n = 215) from breast cancer primary tumor samples. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by two-tailed Student’s t test. (D) Multivariate Cox regression analysis of TIM3 IHC from previous samples including p value and hazard ratio (HR) with confidence interval. Factor names: TIM3 in tumor epithelial cells (eTIM3), intratumoral TILs (iTIM3), and fibroblasts (fTIM3). Estrogen-receptor positivity (ER), HER2 positivity (HER2), patient stage-II BC (pT2), patient stage-III BC (pT3), patient lymph node-1 (pN1), patient lymph node-2 (pN2), and tumor infiltrating lymphocytes (TILs). (E) Intraportal 4T07 cell injection and anti-TIM3 treatment. Representative images of liver metastasis of anti-IgG2a and anti-TIM3 conditions. Boxplots representing liver metastatic growth by BLI measurements during early time points of the experiment. Each dot represents an individual mouse. (F) Spontaneous metastasis assay using 4T1 MFP injection in Balb/c mice. At 4 × 4 mm size, anti-Tim3 treatment starts. On the left, the graph represents the primary tumor volume, and the scheme of neoadjuvant/adjuvant treatment regime of TIM3-blockade therapy (250 μg). On the right, bar plot quantification of spontaneous metastatic incidence at day 40 and take rate of metastasis incidence. Met (metastasis detection) or No-Met (no metastasis detection). n = 14 and n = 15 mice per condition. Statistical significance; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001, by Chi-square test. (G) Graphical abstract of TIM3 + tumor cells from early seeding to macrometastasis in the liver. Also see .

Article Snippet: Then, 5 μg/mL anti-mouse IL-1β blocking antibody or control mouse IgG (sc-2025, Santa Cruz Biotechnology) were added to the media when indicated.

Techniques: Two Tailed Test, Injection, Adjuvant

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: In mice, treatment with angiotensin II (Ang II) and β-aminopropionitrile (BAPN) causes acute inflammation before aortic dissection occurs. ( A ) Gross morphology of aortic dissection (AD) in male C57BL/6J mice; aortas in control mice without Ang II or BAPN treatment (Control), aortas without AD after exposure to ANGII (1 µg/kg/µl) and BAPN (5 g/drinking water) (No-AD), and aortas with AD after exposure to ANGII and BAPN (AD). ( B , C ) Flow cytometric analysis showing percentages with plots after gating of CD45 + immune cells excluding doublets ( B ) and cell numbers of total CD45 + cells, neutrophils, Ly6C high monocytes, and macrophages ( C ). (n = 5 controls, n = 4 Non-AD, n = 5 AD). One-way analysis of variance with Tukey’s multiple comparison test; * P < 0.05. ( D ) Uniform manifold approximation and projection (UMAP) dimensionality reduction analysis identifying unique immune populations in male mice exposed to Ang II (1 µg/kg/µl) and BAPN (5 g/l in drinking water) for 1 week. ( E ) Dot plots displaying the signature cell gene expression markers. ( F , G ) CellChat showing the activated interaction between each cell population in the interleukin-1 (IL-1) signaling pathway. ( H ) Feature plots of IL-1β and Il1r1 expression on UMAP. CellChat; a tool that can quantitatively infer and analyze intercellular communication networks from scRNA-seq data. Macs macrophages, Monos monocytes, DCs dendritic cells, NK cells natural killer cells, ILCs Innate lymphoid cells, VSMCs vascular smooth muscle cells, ECs endothelial cells.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 μg every 3 days for 2 weeks (Fig. ).

Techniques: Dissection, Control, Comparison, Gene Expression, Expressing

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: IL-1β + inflammatory macrophages are accumulated before the onset of macroscopic aortic dissection (AD) in mice. ( A ) Re-clustering of monocytes and macrophages into three groups: mouse aortas without Ang II or BAPN (control), mouse aortas without AD after exposure to Ang II and BAPN (non-AD), and mouse aortas with AD after exposure to Ang II and BAPN (AD). ( B ) Violin plots depicting single-cell gene expression of each canonical monocyte or macrophage marker for clustering. ( C ) Proportion of each monocyte-macrophage cluster. ( D ) Heatmap of the top-5 differentially expressed genes in each monocyte and macrophage cluster. ( E ) Trajectory pseudo-time analysis in Monocle3 with Seurat cluster annotations (left) and change in the expression of IL-1β across pseudo-time for the monocyte and monocyte-derived macrophage partitions (clusters 0, 1, 2, 3, 4) in the non-AD and AD groups (right). Macs macrophages, Monos monocytes, Ang II Angiotensin II, BAPN β-aminopropionitrile.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 μg every 3 days for 2 weeks (Fig. ).

Techniques: Dissection, Control, Gene Expression, Marker, Expressing, Derivative Assay

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: Anti-IL-1β neutralizing antibody improves survival rate in mice. ( A ) Kaplan–Meier survival curve tracking death due to ruptured aortic dissection (AD) in male C57BL/6J mice exposed to Ang II (1 µg/kg/µl) and BAPN (1 g/l in drinking water) for 2 weeks after treatment with isotype control or anti-IL-1β neutralizing antibody (isotype IgG or anti-IL-1β neutralizing antibody; 200 β g i.p./mouse/every 3 days, n = 14 sham; n = 14 anti-IL-1β neutralizing antibody). Log-rank (Mantel–Cox) test, *P < 0.05. ( B ) Incidence of AD in AD model mice treated with the isotype control or anti-IL-1β antibody (n = 11/14 sham; n = 7/14 anti-IL-1β neutralizing antibody). ( C ) Blood pressure of AD model mice treated with the isotype control or anti-IL-1β neutralizing antibody (n = 8, sham; n = 9, anti-IL-1β neutralizing antibody). ( D ) Representative images of Elastica van Gieson (EVG) staining in the ascending (upper) and thoracoabdominal aorta (bottom). ( E ) Percentage of EVG-stained area per total tunica media without AD in the ascending aorta (upper) and thoracoabdominal aorta (bottom). *P < 0.05. Ang II Angiotensin II, BAPN β-aminopropionitrile.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 μg every 3 days for 2 weeks (Fig. ).

Techniques: Dissection, Control, Staining

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: Single-cell RNA sequencing reveals a characteristic immune cell landscape in the ascending aorta in patients with Stanford type A acute aortic dissection (AAD). ( A ) Representative contrast-enhanced computed tomography (CT) image of Stanford type A AAD. ( B ) Histological images stained with hematoxylin and eosin (HE) and Elastica van Gieson (EVG). ( C , D ) Uniform manifold approximation and projection (UMAP) dimensionality reduction analysis identifying a unique single-cell immune landscape in the aortas of combined ( C ) and each individual Control or AAD group ( D ) (n = 3 Controls from Li et al. ; n = 2 AAD). ( E ) Dot plots displaying signature cell gene expression markers for each immune cell cluster. ( F ) Comparison of the proportions of each cluster between controls and patients with AADs.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 μg every 3 days for 2 weeks (Fig. ).

Techniques: RNA Sequencing, Dissection, Computed Tomography, Staining, Control, Gene Expression, Comparison

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: Monocytes and inflammatory macrophages expressing IL-1β are accumulated in ascending aorta in patients with Stanford type A aortic dissection (AAD). ( A ) Sub-clustering of myeloid cells by uniform manifold approximation and projection (UMAP) in Controls and AADs (n = 3 Controls from LiY et al ; n = 2 AAD). ( B ) Dot plots displaying signature cell gene expression markers for each subcluster of myeloid cells. ( C ) Featured plots displaying characteristic gene expression of IL1B, NLRP3, and CCL2 in myeloid cells. ( D ) Pie charts showing the proportion of each myeloid cell cluster in the Control and AAD groups. Percentage of partitioned monocytes and monocyte-derived macrophages (clusters 0, 1, 2, and 3). ( E ) Gene ontology (GO) terms showing enriched biological processes (BP) (left) and molecular functions (MF) of clusters 0, 1, 2, 3, and 4. ( F ) Trajectory pseudo-time analysis in Monocle3 with Seurat cluster annotations (left) and change in the expression of IL-1β across pseudo-time for monocytes and monocyte-derived macrophage partitions (clusters 0, 1, 2, and 3) in AAD samples (right). ( G ) Histological staining with EVG, CD68 and, IL-1β. The scale bar represents 100 μm. Macs macrophages, Monos monocytes, cDC conventional dendritic cells, pDCs plasmacytoid dendritic cells.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 μg every 3 days for 2 weeks (Fig. ).

Techniques: Expressing, Dissection, Gene Expression, Control, Derivative Assay, Staining