Journal: Nature

Article Title: Neutrophils drive vascular occlusion, tumour necrosis and metastasis

doi: 10.1038/s41586-025-09278-3

Figure Lengend Snippet: a , Representative images (a I ) of breast cancer patient MRI images with (top) or without (bottom) necrosis in the primary tumor, as determined based on the vascular contrast signal. The percentage of recurrence-free survival (a II , p = 0.012) and overall survival (a III , p = 0.015) were higher in patients without necrosis, as was the incidence of axillary adenopathy (consistent with metastatic spread to local lymph nodes, a IV , p < 0.0001). The percentage of each molecular subtype for both categories is also shown (a V ). b , Representative image of the accumulation of neutrophils (MPO, cyan) and the absence of vasculature (CD31, green) in necrotic regions (DAPI, nuclear morphology in blue, top right, non-necrotic; bottom right, necrotic) in cleared 4T1 tumors. Representative of N = 6 cleared tumors. c , Representative immunostaining (left) and quantification (right) showing neutrophils (MPO, cyan; non-necrotic vs peri-necrotic p = 0.0002, non-necrotic vs necrotic p < 0.0001, peri-necrotic vs necrotic p < 0.0001) and NETs (DAPI + , MPO + blue, citH3 + red; non-necrotic vs peri-necrotic p = 0.25, non-necrotic vs necrotic p < 0.0001, peri-necrotic vs necrotic p < 0.0001) accumulate in necrotic regions in 4T1 tumors. N = 6 tumors. d , Representative imaging mass cytometry images of 4T1 tumors showing that necrosis-infiltrating cells (inside yellow dashed line) are bona fide neutrophils by markers. Representative of n = 16 regions from eight 4T1 tumors. e , High magnification image of DNA and NETs in necrotic regions of 4T1 tumors (another example shown in Fig. , right). Representative of n = 4 tumors. f , LLC tumors show similar pleomorphic necrosis, neutrophil infiltration, and perfused/non-perfused vessels as 4T1 tumors. Representative of n = 3 cleared tumors. g , Representative view of perfused vessels (red, i.v. lectin) in a primary LLC tumor showing intravascular neutrophil aggregates (arrowheads, MPO) upstream of non-perfused vessels (CD31). Representative of n = 3 cleared tumors. h , H&E staining of C3(1)-Tag tumors showing the presence of pleomorphic necrosis in the primary tumor of this model. Representative of n = 5 mice. i , Micrograph of cleared C3(1)-Tag tumors showing neutrophils (MPO) forming aggregates in the vasculature (CD31). Representative of n = 5 cleared tumors. j , Micrograph of cleared C3(1)-Tag tumors showing neutrophil accumulation (MPO) in necrotic regions (nuclear morphology, right) devoid of vasculature (CD31). Representative of n = 5 cleared tumors. k , Representative cleared MMTV-PyMT tumor stained for vessels (CD31), neutrophils (MPO), and nuclei (DAPI), showing the presence of smaller, non-pleomorphic central necrotic cores with fewer infiltrating neutrophils and NETs (MPO + , citH3 + ). Representative of n = 3 cleared tumors. l , Representative images and m , quantification of the number of neutrophils (p < 0.0001) and NETs (p = 0.0003) in necrotic areas (yellow dashed line) in MMTV-PyMT and 4T1 tumors. N = 3 mice per group. n , Representative view of perfused vessels (red, i.v. lectin) in a primary 4T1 tumor. Dashed lines outline a vessel (CD31, green) perfused until an intravascular neutrophil aggregate appears (yellow arrowhead). Downstream of the aggregate, perfusion is lost. Representative of n = 5 tumors. o , Micrograph showing a hypoxic region (hypoxyprobe, green) around a vessel (CD31) containing a neutrophil aggregate (yellow arrowhead, MPO). Representative of n = 4 tumors. Error bars show mean + s.e.m. ***P < 0.001, ns, not significant, as determined by one-way ANOVA with Tukey’s multiple comparison test in (c) or unpaired two-tailed Student’s t -test (m). Survival curves analyzed using log-rank (Mantel-Cox) test in (a II-III ). Axillary adenopathy data in a IV analyzed using Two-sided Fisher’s exact test.

Article Snippet: Cells were then fixed using 4% paraformaldehyde (PFA) in PBS for 10 min; blocked and permeabilized with PBS containing 0.1% Triton X-100, 25% FBS and 5% bovine serum albumin (BSA); and stained with antibodies to citH3 (Abcam) and MPO (R&D Systems) at 1:200 dilution in blocking buffer at 4 °C overnight.

Techniques: Immunostaining, Imaging, Mass Cytometry, Staining, Comparison, Two Tailed Test

Journal: Nature

Article Title: Neutrophils drive vascular occlusion, tumour necrosis and metastasis

doi: 10.1038/s41586-025-09278-3

Figure Lengend Snippet: a , Cleared 4T1 tumour showing the nuclear morphology of normal and necrotic regions: necrotic regions lack vasculature (CD31) and are enriched in neutrophils (MPO) and NETs (DAPI + , citH3 + and MPO + ). Representative of n = 5 tumours. b , Tile scan of a 4T1 tumour showing the intricate pleomorphic architecture of neutrophil-rich and NET-rich (DAPI + , citH3 + and MPO + ) necrotic tissue (left), and high magnification showing NETs (triple colocalization of DNA, MPO and citH3) in necrotic regions (right). Representative of n = 4 tumours. c , Quantification of intravascular NETs in necrotic or perinecrotic regions compared with non-necrotic regions in 4T1 tumours, normalized to the volume of vessels in each captured volume. n = 9 volumes from 5 tumours. P = 0.0022. d , Vessels adjacent to necrotic regions contain neutrophil aggregates (the dashed region magnified on the right) and NETs. Representative of n = 5 tumours. e , Representative view of perfused vessels (red, intravenous lectin) in regions adjacent to the necrotic tissue. The dashed region (zoomed in on the right) shows a vessel perfused until an intravascular neutrophil aggregate appears (arrowheads; arrows in the single channels below). Perfusion is lost downstream of the aggregate. Representative of n = 5 tumours. f , Extent of unperfused vasculature in the primary tumour correlates with the number of neutrophil aggregates in the vasculature. n = 12 volumes from 4 tumours. P < 0.0001. g , Micrograph showing intravascular accumulation of neutrophils (MPO) in the vasculature (CD31) of needle biopsies of primary tumours from patients with TNBC. Representative of n = 8 out of 20 total needle biopsies. h , Human TNBC pre-treatment needle biopsies show pleomorphic necrosis (by disrupted nuclear morphology; left) and extensive neutrophil infiltration (MPO) and NET formation (DAPI + , citH3 + and MPO + ). Representative of n = 4 pleomorphic of 20 total needle biopsies. The bars show mean + s.e.m. ** P < 0.01, as determined by unpaired, two-tailed Student’s t -test ( c ) or two-tailed Pearson correlation ( f ). The error bands show 95% confidence interval ( f ).

Article Snippet: Cells were then fixed using 4% paraformaldehyde (PFA) in PBS for 10 min; blocked and permeabilized with PBS containing 0.1% Triton X-100, 25% FBS and 5% bovine serum albumin (BSA); and stained with antibodies to citH3 (Abcam) and MPO (R&D Systems) at 1:200 dilution in blocking buffer at 4 °C overnight.

Techniques: Two Tailed Test

Journal: Nature

Article Title: Neutrophils drive vascular occlusion, tumour necrosis and metastasis

doi: 10.1038/s41586-025-09278-3

Figure Lengend Snippet: a , Representative overview of a biopsy from a TNBC patient; pre-treatment needle biopsies were stained for H&E and show pleomorphic, necrotic architecture. Representative of n = 4 evident pleomorphic, necrotic samples from 20 total needle biopsies. b , Higher magnification of necrotic regions from the tumor shown in (a). c , Representative image of a necrotic region from a needle biopsy of a TNBC tumor, showing neutrophils and NETs accumulating in the necrotic regions (nuclear morphology in the left panels). Representative of n = 4 pleomorphic, necrotic biopsies. d , Sections from the same necrotic TNBC tumors stained for NETs using either the citH3/MPO/DNA (left) or 3D9 (right) methods. Representative of n = 3 needle biopsies. e , High magnification images of NETs stained with 3D9 antibody from the TNBC shown in (d). f , Representative gross appearance and g , H&E-stained (top) or immunostained (bottom) midline sections of 4T1 tumors at indicated times after tumor implantation, showing that pleomorphic necrosis is already extensive at day 14 after tumor implantation (with necrosis emerging as early as day 7). Representative of 3 mice per timepoint. h , Quantification of 4T1 tumor weight over time. N = 3 mice per timepoint. i , Ratio of necrotic to total tissue area in the 4T1 primary tumors over time, as quantified from H&E stainings. N = 3 mice per timepoint. j , Counts of neutrophils, lymphocytes, and monocytes in circulation in 4T1 tumor-bearing mice over time. Note that neutrophils change first and the most strongly in response to the presence of the primary tumor (day 8 represents an 8-fold increase over day 0), while monocytes and lymphocytes start increasing at much later timepoints (day 18 onwards). N = 3 mice per timepoint. k , Percentage of immune cells in 4T1 primary tumors over time (time 0 represents the percentage in naïve mammary fat pads). N = 3 mice per timepoint. DC, dendritic cell. l , Quantification of NETs in 4T1 tumors over time from immunofluorescence-stained tissue sections (as shown in b). N = 3 mice per timepoint (except N = 1 for mammary fat pad at time 0, as baseline reference). m , Representative image of 4T1 tumors 7 days after tumor implantation, showing the presence of NETs in areas not yet necrotic or avascular. Representative of n = 3 mice. Error bars show mean ± s.e.m.

Article Snippet: Cells were then fixed using 4% paraformaldehyde (PFA) in PBS for 10 min; blocked and permeabilized with PBS containing 0.1% Triton X-100, 25% FBS and 5% bovine serum albumin (BSA); and stained with antibodies to citH3 (Abcam) and MPO (R&D Systems) at 1:200 dilution in blocking buffer at 4 °C overnight.

Techniques: Staining, Tumor Implantation, Immunofluorescence

Journal: Nature

Article Title: Neutrophils drive vascular occlusion, tumour necrosis and metastasis

doi: 10.1038/s41586-025-09278-3

Figure Lengend Snippet: a , Representative image of intravascular laminin (i.v. injected anti-laminin antibody) deposition in the microvasculature (CD31) of 4T1 tumors. Neutrophils (MPO) interact with the intravascular laminin patches. Representative of n = 3 mice. b , Representative image (left) and quantification (right) showing that perivascular cell-poor (defined as perivascular αSMA + cells, green) vessels (CD31, red) contain more luminally exposed laminin (i.v. injected anti-laminin antibody, cyan) than pericyte-covered vessels (vessels with high pericyte coverage). N = 6 volumes from 3 mice. p = 0.0454. c , Tumor regions surrounding necrosis also contain intravascular (i.v. lectin, red) deposits of fibrin in close association with neutrophils (MPO). Right panel: quantification of the neutrophil-fibrin distance compared to the distance to random intravascular points, showing that intravascular neutrophils are in close proximity to the fibrin deposits. N = 3 mice. d , NETs (citH3 + , MPO + ) are found in and around fibrin deposits in the tumor vasculature. Representative of n = 3 mice. e , Intravascular fibrin deposits colocalize with platelet aggregates near necrotic regions, whereas f , neither fibrin nor platelet deposits are found in non-necrotic tumor regions. Representative of n = 3 mice. g , Tile scan of a 4T1 tumor showing that necrotic regions (dashed areas) and surrounding regions contain fibrin and platelet deposits. Representative of n = 3 mice. h-i , Still images from real-time intravital imaging of the tumor microvasculature (lectin, cyan), showing neutrophils (LysM-GFP, green) and platelets (anti-CD41, red) forming intravascular aggregates in vivo in two independent tumor regions. Representative of n = 3 mice. j , Representative flow cytometry plots (left) and quantification (right) of neutrophils in our neutropenic mice (and Cre – littermate controls). N = 4 controls and 3 neutropenic mice. P = 0.0011. k , Representative H&E stainings (left) and quantification (right) of the necrotic area in LLC tumors implanted into neutropenic mice or littermate controls. N = 4 control and 3 neutropenic mice. p = 0.0303. l , Number of circulating neutrophils and platelets in 4T1 tumor-bearing mice treated with dipyridamole or vehicle control. N = 3 mice per group. m , Number of circulating neutrophils and platelets in mice treated with tirofiban or vehicle control. N = 3 mice per group. n , Representative H&E stainings (left) and quantification (right) of the necrotic area in 4T1 tumors from mice treated with dipyridamole or tirofiban. N = 20 control-, 10 dipyridamole-, and 10 tirofiban-treated mice. Control vs Tirofiban p = 0.0358. Control vs Dipyridamole p = 0.6313. Bars show mean + s.e.m. *P < 0.05, **P < 0.01, ns, not significant, as determined by unpaired two-tailed Student’s t -test (b, j-m) or one-way ANOVA with Tukey’s multiple comparison test (n).

Article Snippet: Cells were then fixed using 4% paraformaldehyde (PFA) in PBS for 10 min; blocked and permeabilized with PBS containing 0.1% Triton X-100, 25% FBS and 5% bovine serum albumin (BSA); and stained with antibodies to citH3 (Abcam) and MPO (R&D Systems) at 1:200 dilution in blocking buffer at 4 °C overnight.

Techniques: Injection, Imaging, In Vivo, Flow Cytometry, Control, Two Tailed Test, Comparison

Journal: Nature

Article Title: Neutrophils drive vascular occlusion, tumour necrosis and metastasis

doi: 10.1038/s41586-025-09278-3

Figure Lengend Snippet: a , Differentially expressed genes of the clusters shown in Fig. (human TNBC scRNA-seq), showing the genes upregulated and downregulated in Cluster 3, with selected genes noted to the right. b , GO terms of the genes upregulated in cluster 3, as shown in Fig. . c , LLC tumor growth over time in PAD4 ΔN and PAD4 WT mice. Line shows the non-linear least squares fit of the data. N = 5 mice per group. p = 0.048. d , Quantification of circulating immune cells in PAD4 ΔN and PAD4 WT mice 4 weeks after tumor implantation. N = 5 mice per group. e , Quantification of NETs (DAPI + , citH3 + , MPO + ) in tumors from PAD4 ΔN and PAD4 WT mice. N = 8 cleared tumor volumes from 4 mice per group. p < 0.0001. f , H&E staining of the lungs of PAD4 ΔN and PAD4 WT mice 2 weeks after i.v. injection of 5×10 5 LLC cells (experimental metastasis model) and, g , quantification of the metastatic area, h , number of metastatic foci, and i , mean area of metastatic foci in the lungs of PAD4 ΔN and PAD4 WT mice subjected to experimental metastasis. N = 8 PAD4 WT and 7 PAD4 ΔN mice. j , Representative micrograph of an LLC tumor 4 weeks after injection into PAD4 WT or k , PAD4 ΔN mice and stained for E-cadherin and vimentin. Representative of n = 3 mice per group. l , Quantification of circulating immune cells in mice treated with DNase I or vehicle. N = 4 mice per group. Neutrophils p = 0.0005, Monocytes p = 0.0149, % Ly6c Low p = 0.1003. m , Representative H&E images (left) and quantification of necrosis (right) showing that daily treatment with DNase I reduced 4T1 tumor necrosis (yellow dashed line). N = 10 mice per group. p = 0.0372. n , Representative H&E staining and o , quantification of metastasis as percentage of total lung area of 4T1 tumor-bearing mice treated with DNase I or vehicle control. N = 9 control and 10 DNase I treated mice. p = 0.0441. Bars show mean + s.e.m. *P < 0.05, ***P < 0.001, as determined by unpaired two-tailed Student’s t -test in (d, e, g, h, i, l, m, o) or two-way ANOVA in (c). P-value in (b) calculated using a Fisher’s cumulative hypergeometric probability with multiple testing correction (using gProfiler).

Article Snippet: Cells were then fixed using 4% paraformaldehyde (PFA) in PBS for 10 min; blocked and permeabilized with PBS containing 0.1% Triton X-100, 25% FBS and 5% bovine serum albumin (BSA); and stained with antibodies to citH3 (Abcam) and MPO (R&D Systems) at 1:200 dilution in blocking buffer at 4 °C overnight.

Techniques: Tumor Implantation, Staining, Injection, Control, Two Tailed Test

Journal: Nature

Article Title: Neutrophils drive vascular occlusion, tumour necrosis and metastasis

doi: 10.1038/s41586-025-09278-3

Figure Lengend Snippet: a , Representative images (left) of cleared LLC tumours from PAD4 WT (able to form citrullinated NETs) and PAD4 ΔN mice (unable to form citrullinated NETs), showing lack of citrullinated NETs (DAPI + , citH3 + and MPO + ) and no loss of vasculature (CD31; quantified on the right) in neutrophil-rich (MPO) areas in tumours from PAD4 ΔN compared with PAD4 WT mice (the arrow points to neutrophil-rich, NET-rich, avascular necrotic region). n = 8 volumes from 4 mice per group. P = 0.0042. b , Examples of gross appearance of LLC tumours from PAD4 WT and PAD4 ΔN mice, showing that necrosis depends on NET formation. c , Representative midline sections of LLC tumours from PAD4 WT or PAD4 ΔN mice stained with H&E. d , Quantification of the necrotic area of the H&E-stained tissues. n = 8 PAD4 WT and n = 9 PAD4 ΔN mice. P = 0.0025. e – g , Quantification of lung metastasis showing reduced metastatic area ( e ; P = 0.0095), absolute number of metastatic foci ( f ; P = 0.0144) and no differences in mean area of individual metastatic foci ( g ) in PAD4 WT and PAD4 ΔN LLC tumour-bearing mice. n = 19 mice per group. h , Representative H&E staining of the lungs quantified in panels e – g . i , Heatmap (bottom left) of the genes upregulated in cancer cells from necrotic PAD4 WT mice compared with non-necrotic PAD4 ΔN mice and Gene Ontology terms analysis (right) of those genes, showing that cancer cells in necrotic tumours upregulate pathways related to haematopoiesis, EMT, metabolism, migration and necrosis. The bars show mean + s.e.m. * P < 0.05 and ** P < 0.01, as determined by unpaired, two-tailed Student’s t -test ( a , d – g ) or Fisher’s cumulative hypergeometric probability with multiple testing correction ( i ).

Article Snippet: Cells were then fixed using 4% paraformaldehyde (PFA) in PBS for 10 min; blocked and permeabilized with PBS containing 0.1% Triton X-100, 25% FBS and 5% bovine serum albumin (BSA); and stained with antibodies to citH3 (Abcam) and MPO (R&D Systems) at 1:200 dilution in blocking buffer at 4 °C overnight.

Techniques: Staining, Migration, Two Tailed Test

Journal: bioRxiv

Article Title: Hepatocyte expression of fetal insulin receptor isoform contributes to the promotion of liver cancer through non-cell autonomous mechanisms

doi: 10.1101/2025.06.02.655477

Figure Lengend Snippet: Apc lox/lox mice were injected with recombinant AAV8 encoding saCas9 and sgRNA against Rosa26 (sgRosa/APC ΔHep , n=6) or Insr (sgInsr/APC ΔHep , n=5) locus. One month later, mice were injected with an adenovirus encoding the Cre recombinase (AdenoCre) and sacrificed 50 days later. A. Schematic illustration of the protocol. B. Liver-to-body weight ratio (n=5-6). C. Representative images of H&E staining showing microvacuolar steatosis, glycogenated nuclei, apoptotic bodies and inflammatory cells in sgInsr/APC Δhep livers. Scale bar: 50 μM. D. Representative images of myeloperoxidase (MPO) IHC staining from sgRosa/APC Δhep and sgInsr/APC Δhep livers ( left panel , scale bar: 120 μM); Quantification of MPO staining ( right panel ; n=5-6). E. Representative images of F4/80 IHC staining from sgRosa/APC Δhep and sgInsr/APC Δhep livers ( left panel , scale bar: 100 μM); Quantification of F4/80-positive macrophages ( right panel ; n=5-6). F. Representative images of proteome profiler cytokine/chemokine arrays performed on whole-cell lysates from sgRosa/APC Δhep and sgInsr/Apc Δhep livers (n=2). G. Expression of transcripts encoding chemokines/cytokines evaluated by RT-qPCR (n=5-6). H. Representative images of TUNEL assay from sgRosa/APC Δhep and sgInsr/APC Δhep livers ( left panel ); Quantification of TUNEL-positive cells ( right panel , n=5-6). Data are median ± IQR.

Article Snippet: For the assessment of liver steatosis, 10-μm liver frozen sections were fixed in 10% formalin for 10 min, rinsed in 60% isopropyl alcohol, and then incubated in Oil Red O solution (Sigma) for 15 min. IHC staining for myeloperoxidase (#22225-1-AP, Proteintech), glutamine synthetase (GS) (#610518, BD Transduction Laboratories), Ki67 (#MA5-14520, SP6, ThermoFisher Scientific), cleaved caspase-3 (#9661, D175) and F4/80 (#70076, D2S9R, Cell Signaling) was performed according to the provider instructions.

Techniques: Injection, Recombinant, Staining, Immunohistochemistry, Expressing, Quantitative RT-PCR, TUNEL Assay

Journal: Journal of advanced research

Article Title: Sedanolide alleviates DSS-induced colitis by modulating the intestinal FXR-SMPD3 pathway in mice.

doi: 10.1016/j.jare.2024.03.026

Figure Lengend Snippet: Fig. 6. Gut microbiota played a vital role in the protective effect of sedanolide against DSS-induced colitis in mice. (A) Experimental scheme. (B) Percent of initial weight of mice after DSS administration (left) and the weight of mice at the last day (right). (C) DAI score of mice after DSS administration. (D) The level of IL-6 and TNF-a in the serum. (E) Colon length of mice at the last day. (F) The level of FITC-dextran in serum. (G) Representative images of H&E staining of colon tissues (scale bar: 100 lm) and representative images of immunohistochemical staining for MPO, Occludin and ZO-1 in colon tissues (scale bar: 50 lm). Data are shown as mean ± SEM. *p < 0.05, **p < 0.01.

Article Snippet: Immunohistochemical staining for myeloperoxidase (MPO) was performed using an anti-MPO antibody (Proteintech, Chicago, USA) according to the standard procedure.

Techniques: Staining, Immunohistochemical staining