Journal: bioRxiv

Article Title: Anti-PD-1/PD-L1 Therapy Triggers Cognitive Deficits and Anxiety-Like Behaviors Through Tumor-Initiated Neuroinflammatory Niches in Male Mice

doi: 10.1101/2025.09.03.673981

Figure Lengend Snippet: a. In the upper panel, CD206 (green), MHCII (magenta) and DAPI (grey) immunoreactivity in B16, B16-Ova and MC38 tumor slices (30 μm). The boxed areas represent magnification of clusters of MHCII + and CD206 + cells. Scale bar: 500 μm and 100 μm. In the lower panel, GR1+ (magenta), CD11b+ (blue) and DAPI (grey) immunoreactivity in B16F10, B16F10-Ova and MC38 tumor slices. The boxed areas represent magnification of clusters of GR1 + , CD11b + and GR1 + CD11b + cells. Scale bar: 500 μm and 100 μm. On the right, bar of quantifications of CD206 + or MHCII + TAMS, monocytes (CD11b + GR1 - ) and MDSCs (CD11b + GR1 + ) density in B16F10, B16F10-Ova and MC38 tumor slices. Scale bars: 1 mm, zoom 100µm. b. On the left, representative immunolabeling of collagen IV + (green) and MECA-79 + (magenta) vessels in B16F10, B16F10-Ova and MC38 tumor slices. Boxed areas show magnification of HEVs (MECA79 + CollagenIV + ). On the left, bars of quantification of MECA-79 + coverage, expressed as the percentage of total area, in B16F10, B16F10-Ova and MC38 tumor slices. Scale bars: 200 µm, zoom 100µm. On the right, representative microphotographs of DAPI (grey) and PD-L1 (cyan) immunolabeling in B16F10, B16F10-Ova and MC38 tumor slices with bars of quantification of PD-L1 + cells, expressed as percentage of total cells. Below, DAPI (grey), and CD8+ (cyan) immunoreactivity in B16F10, B16F10-Ova and MC38 tumor slices with bars of quantifications of CD8 + cytotoxic TL density. The boxed areas represent magnification of DAPI (grey) nuclear staining with CD8+ (cyan) TL overlapping. Scale bars: 50 µm, zoom 10 µm. Statistical analysis was performed by using one-way ANOVA with Bonferroni correction for multiple comparisons (n=5). Data are represented as bars with symbols for individual data points and they are expressed by mean ± SEM, * p<0.05, ** p <0.01, *** p <0.001. c. Chart illustrating the immunoscore (left) and the immunosuppressive score (right) calculation methods in B16, B16-Ova and MC38 tumor slices. Densities of CD8 + TL, Meca-79 + HEVs, CD11b + monocytes and MHCII + anti-tumoral TAMs in tumor slices were converted into percentile values. Percentiles values were then converted into score values from 0 attributed to [0-25%] percentile, to 3 for [75-100%] percentile. The mean of each score was then calculated to generate immunoscore. The staining intensity values of CD206 + TAMs, CD11b + GR1 + MDSCs and percentage of PD-L1 + cells in tumor slices were converted into percentile values. Percentiles values were then converted into score values with score 0 for [0-25%] to 3 [75-100%] percentiles. The mean of each score was then calculated to generate immune-suppressive score. d. Correlation map between immunoscore, immune-suppressive score and behavioral results. From the heatmap of Kendall correlation coefficients, only significant correlations for each B16F10, B16F10-Ova and MC38 mice are displayed (adjusted p-value < 0.05). Blue circle indicates a positive correlation and red circle, a negative correlation. Color intensity is proportional to the amplitude of the correlation coefficient in a range of 0 to 1/-1.

Article Snippet: The following anti-mouse antibodies were used, with clone, fluorophore, and reference indicated in parenthesis: CD45 (REA737, VioGreen, 130-110-803, Miltenyi Biotec), CD11b (M1/70, FITC, 101206, Biolegend), CD11c (N418, PerCP, 117328, Biolegend), MHCII (M5/114.15.2, BV605, 107639, Biolegend), CD206 (APC, FAB2535A, R&D systems), Ly6G (1A8, BV421, 1238135, Sony Biotechnology), Ly6C (REA796, Vioblue, 130-111-921, Miltenyi Biotec), CD3 (17A2, BV421, 100228, Biolegend), CD4 (REA1211, PE, 130-123-206, Miltenyi Biotec), CD8 (53-6.7, FITC, 11-0081-82, Invitrogen), NK1.1 (PK136, PerCP, 1143630, Sony Biotechnology), CD19 (6D5, AF700, 115527, Biolegend), FoxP3 (MF-14, AF647, 1232040, Sony Biotechnology), RORγt (Q31-378, BV786, BD Biosciences) and TCRγδ (GL3, BV605, 118129, Biolegend).

Techniques: Immunolabeling, Staining

Journal: bioRxiv

Article Title: Anti-PD-1/PD-L1 Therapy Triggers Cognitive Deficits and Anxiety-Like Behaviors Through Tumor-Initiated Neuroinflammatory Niches in Male Mice

doi: 10.1101/2025.09.03.673981

Figure Lengend Snippet: a. On the left, schematic representation of velum interpositum localized below the hippocampus and near lateral ventricles in mouse brain. In the middle, representative images of nuclei staining by DAPI (grey) and leptomeninges (ERTR7 + , magenta) immunostaining showing positions of dentate gyrus and ERTR7 + velum interpositum on whole cerebral slices of MC38-bearing mice. Scale bar: 1 mm. On the right, representative micrographs and statistical quantification of macrophages (F4/80 + , magenta) density (n/mm 2 ) and nuclei (DAPI, blue) immunolabeling in velum interpositum (Lectin + , grey) and dentate gyrus vessels (Lectin + , green) of PBS and B16F10- and MC38-bearing mice. Scale bars: 100 µm. b. Representative images and quantification of pro-tumoral macrophages (CD206 + , green) and anti-tumoral (MHCII + , magenta) immunolabeling in velum interpositum (Lectin + , grey) of PBS and B16F10- and MC38-bearing mice. Scale bar: 100 µm. c. Schematic diagram showing the timeline for bromodeoxyuridine (BrdU) administration in B16F10- (light blue) and MC38- (pink) bearing mice. After tumors inoculation (D0), B16F10 and MC38 mice received BrdU (50 mg/kg) by intraperitoneal injection (i.p.) once a day for 2 days before sacrifice. At D9 (B16F10) or D17 (MC38) mice were sacrificed, and brain were collected for immunofluorescence assays. Below, representative micrographs of neuronal precursors (BrdU + , green) and mature neurons (NeuN + , grey) in the dentate gyrus of the hippocampus of PBS and B16F10-, MC38-bearing mice. The white squares below the images correspond to magnification showing absence of co-localization of neuronal precursors (BrdU + , green) with mature neurons (NeuN + , grey) in the dentate gyrus (DG). On the right, histogram showing the variation in the number of BrdU + neuronal precursors (density, n/mm 2 ) in the dentate gyrus of PBS mice compared with B16F10- and MC38-bearing mice. Scale bar: 100 µm, zoom: 10 µm. d. Representative images of reactive (CD68 + , magenta) microglia (Iba1 + , grey) immunolabeling in dentate gyrus of PBS-mice and B16F10- and MC38-bearing mice. On the right, histogram showing the variation in the number of CD68 + Iba1 + reactive microglial cells (density, n/mm 2 ) in dentate gyrus of B16F10- and MC38-bearing mice compared to PBS-mice. Scale bar: 100 µm. Statistical analyses were performed by using one-way ANOVA or Kruskal-Wallis test with Bonferroni or Dunn’s correction for multiple comparisons. Data are represented as bars with symbols for individual data points and they are expressed by mean ± SEM, n=4-5, * p<0.05, *** p <0.001. f . Representative example of photomicrographs used for the morphological analysis of microglial cells (Iba1 + , magenta). The boxed areas represent magnification of isolated Iba1 + microglial cells (bottom) and corresponding binary image (top) from the dentate gyrus of PBS, B16F10- and MC38-bearing mice. Analysis of the morphology of the microglial cells was performed on 8 cells of dentate gyrus of PBS-, B16F10- and MC38-bearing mice (n=4). Scale bar: 50 μm, zoom 10 μm. Below, quantification of fractal dimension coefficient and area of the soma of microglial cells of PBS-, B16F10- and MC38-bearing mice. Data are represented as violin-plots with individual points representing individual microglial cells (n of cells=32, n of mice = 4 for experimental group). On the right, comparative Radar Plot Chart of morphological parameters (number of processes, length of processes, area of the soma, fractal dimension, lacunarity and span ratio) analyzed on microglia cells in dentate gyrus of PBS-, B16F10- and MC38-bearing mice. Statistical analyses were performed by using one-way ANOVA or Kruskal-Wallis test with Bonferroni or Dunn’s correction for multiple comparisons, * p < 0.05, ** p <0.01, *** p <0.001. BrdU: Bromodeoxyuridine, NeuN: neuronal nuclei antigen, CD206: cluster of differentiation 206, CD68: cluster of differentiation, DAPI: 4’,6-diamidino-2-phenylindol, Iba-1: ionized calcium-binding adapter molecule 1, MHCII: major histocompatibility complex 2.

Article Snippet: The following anti-mouse antibodies were used, with clone, fluorophore, and reference indicated in parenthesis: CD45 (REA737, VioGreen, 130-110-803, Miltenyi Biotec), CD11b (M1/70, FITC, 101206, Biolegend), CD11c (N418, PerCP, 117328, Biolegend), MHCII (M5/114.15.2, BV605, 107639, Biolegend), CD206 (APC, FAB2535A, R&D systems), Ly6G (1A8, BV421, 1238135, Sony Biotechnology), Ly6C (REA796, Vioblue, 130-111-921, Miltenyi Biotec), CD3 (17A2, BV421, 100228, Biolegend), CD4 (REA1211, PE, 130-123-206, Miltenyi Biotec), CD8 (53-6.7, FITC, 11-0081-82, Invitrogen), NK1.1 (PK136, PerCP, 1143630, Sony Biotechnology), CD19 (6D5, AF700, 115527, Biolegend), FoxP3 (MF-14, AF647, 1232040, Sony Biotechnology), RORγt (Q31-378, BV786, BD Biosciences) and TCRγδ (GL3, BV605, 118129, Biolegend).

Techniques: Staining, Immunostaining, Immunolabeling, Injection, Immunofluorescence, Isolation, Binding Assay, Immunopeptidomics

Journal: bioRxiv

Article Title: Anti-PD-1/PD-L1 Therapy Triggers Cognitive Deficits and Anxiety-Like Behaviors Through Tumor-Initiated Neuroinflammatory Niches in Male Mice

doi: 10.1101/2025.09.03.673981

Figure Lengend Snippet: Representative images and statistical quantification of density (n/mm 2 ) of pro-tumoral (CD206 + ) and anti-tumoral (MHCII + ) myeloid cells staining in Laminin + (grey) leptomeninges (upper panel), Willis polygon (middle panel) and perivascular space of parenchyma vessels (lower panel) of B16F10- and MC38-bearing mice compared to PBS-controls. Scale bar: 100 µm. Statistical analyses were performed by using one-way ANOVA or Kruskal-Wallis test with Bonferroni or Dunn’s correction for multiple comparisons. Data are represented as bars with symbols for individual data points and they are expressed by mean ± SEM, n=4-5, * p<0.05. b. Schematic diagram showing the timeline of the in vivo assay of blood brain barrier permeability in B16- (blue) and MC38- (pink) bearing mice. After tumor cells inoculation (D0), mice received 100 µl of Dextran 488 (3kD, i.p) at D9 for B16-bearing mice and at D17 for MC38-bearing mice. Post-injection (15 min), after mouse sacrifice by intracardiac perfusion, brains and blood were collected. The permeability index represents the fluorescence signal of cerebral tissue normalized to the weight of the tissue and to the plasma fluorescence signal. On the left, bars represent statistical comparison of permeability index of B16- and MC38-mice compared with PBS mice. Statistical analyses were performed by using one-way ANOVA test with Bonferroni correction for multiple comparisons. Data are represented as bars with symbols for individual data points and they are expressed by mean ± SEM, n=4-5, * p<0.05, ** p <0.01. c. Representative micrographs showing Dextran 488 (green) extravasation from Podocalyxin + (grey) vessels in the DC (upper panel, left), cerebellum (upper panel right), frontal cortex (lower panel, left) and somatomotor cortex (lower panel, right), and Meningeal dura mater (lower left panel) of PBS and B16- and MC38-bearing mice. Scale bar: 50 μm. CD206: cluster of differentiation 206, D: day, i.p.: intraperitoneal injection, MHCII: major histocompatibility complex 2.

Article Snippet: The following anti-mouse antibodies were used, with clone, fluorophore, and reference indicated in parenthesis: CD45 (REA737, VioGreen, 130-110-803, Miltenyi Biotec), CD11b (M1/70, FITC, 101206, Biolegend), CD11c (N418, PerCP, 117328, Biolegend), MHCII (M5/114.15.2, BV605, 107639, Biolegend), CD206 (APC, FAB2535A, R&D systems), Ly6G (1A8, BV421, 1238135, Sony Biotechnology), Ly6C (REA796, Vioblue, 130-111-921, Miltenyi Biotec), CD3 (17A2, BV421, 100228, Biolegend), CD4 (REA1211, PE, 130-123-206, Miltenyi Biotec), CD8 (53-6.7, FITC, 11-0081-82, Invitrogen), NK1.1 (PK136, PerCP, 1143630, Sony Biotechnology), CD19 (6D5, AF700, 115527, Biolegend), FoxP3 (MF-14, AF647, 1232040, Sony Biotechnology), RORγt (Q31-378, BV786, BD Biosciences) and TCRγδ (GL3, BV605, 118129, Biolegend).

Techniques: Staining, In Vivo, Permeability, Injection, Fluorescence, Clinical Proteomics, Comparison, Immunopeptidomics

Journal: bioRxiv

Article Title: Anti-PD-1/PD-L1 Therapy Triggers Cognitive Deficits and Anxiety-Like Behaviors Through Tumor-Initiated Neuroinflammatory Niches in Male Mice

doi: 10.1101/2025.09.03.673981

Figure Lengend Snippet: a. Histograms representing statistical comparison of the number (n/ml) of myeloid (left) and lymphocyte (right) immune cells populations from whole blood collected from B16 or MC38-bearing mice treated with anti-PD1, anti-PD-L1or IgG. Data are represented as bars with symbols for individual data points and they are expressed by mean ± SEM, (n=4-5), * p<0.05, ** p <0.01. Statistical analyses were performed by one-way ANOVA or Kruskal-Wallis test with Bonferroni or Dunn’s correction for multiple comparisons (B16F10-IgG vs B16F10-PD-1 or B16F10-PD-L1 and MC38-IgG vs MC38-PD-1 or MC38-PD-L1 and PBS vs B16F10-IgG or MC38-IgG). b. Schematic timeline of immuno-neutralization of γδ T lymphocytes in B16- and MC38-bearing mice treated with anti-PD-L1 and IgG. B16F10 mice were injected (i.p.) with anti-PD-L1 or IgG on day 2-4-7 and with an anti-TCRγδ on days 4-7. MC38 mice were injected (i.p.) with anti-PD-L1 or IgG on days 6-10-13 and with anti-TCRγδ on days 6-10-13. Depending on the kinetics of tumor growth (B16: D5-D8, MC38: D13-D16), mice behavior was evaluated by means of OFT, NOR, EPM and TST tests, then sacrificed the day after the end of the behavioral session (B16: D9, MC38: D17) and brains, blood and tumors were collected for further analysis. c . Curves of tumoral volumes represented as mean ± SEM (n = 10) of B16-IgG/B16-PD-L1 and MC38-IgG/MC38-PD-L1 mice receiving or not anti-TCRγδ at indicated time points. Statistical comparison was performed by two-way ANOVA with Bonferroni test for multiple comparisons (MC38 IgG vs. MC38 IgG-TCRγδ, MC38 IgG vs. MC38 PD-L1, MC38 IgG vs. MC38 PD-L1-TCRγδ, MC38 IgG-TCRγδ vs. MC38 PD-L1, MC38 IgG-TCRγδ vs. MC38 PD-L1-TCRγδ, MC38 PD-L1 vs. MC38 PD-L1-TCRγδ, *** p<0.001 (IgG vs PD-L1), $ p<0.05, $$$ p<0.001 (IgG vs IgG-TCRγδ). d. Impact of anti-TCRγδ treatment on the proportion of circulating CD3 + TCRγδ + lymphocytes in blood of B16- and MC38-bearing mice treated with anti-PD-L1 and IgG. Statistical comparison was performed by one-way ANOVA test with Bonferroni correction for multiple comparisons (B16F10 IgG vs. B16F10 PD-L1, B16F10 IgG-TCRγδ vs. B16F10 PD-L1-TCRγδ, B16F10 PD-L1 vs B16F10 PD-L1-TCRγδ, B16F10 IgG vs B16F10 IgG-TCRγδ, MC38 IgG vs. MC38 PD-L1, MC38 IgG-TCRγδ vs. MC38 PDL1-TCRγδ, MC38 PD-L1 vs. MC38 PD-L1-TCRγδ, MC38 IgG vs. MC38 IgG-TCRγδ). Data are represented as bars with symbols for individual data points and they are expressed by mean ± SEM, * p<0.05, ** p<0.01. e. Impact of anti-TCRγδ treatment on anxiety-like behaviors in EPM and on short-term memory in NORT in B16- and MC38-bearing mice treated with anti-PD-L1 and IgG. Statistical analyses were performed (n=10) using one-way ANOVA or Kruskal-Wallis with Bonferroni or Dunn’s correction for multiple comparisons (B16F10 IgG vs. B16F10 PD-L1, B16F10 IgG-TCRγδ vs. B16F10 PD-L1-TCRγδ, B16F10 PD-L1 vs B16F10 PD-L1-TCRγδ, B16F10 IgG vs B16F10 IgG-TCRγδ, MC38 IgG vs. MC38 PD-L1, MC38 IgG-TCRγδ vs. MC38 PDL1-TCRγδ, MC38 PD-L1 vs. MC38 PD-L1-TCRγδ, MC38 IgG vs. MC38 IgG-TCRγδ). Data are represented as violin plot with symbols for individual data points and they are expressed by mean ± SEM, * p<0.05, ** p <0.01. CD: cluster of differentiation, CD11b: integrin alpha M subunit, CD11c: complement component 3 receptor 4 subunit, CD206: cluster of differentiation 206, CD45: leukocyte common antigen, D: day, DAPI: 4’,6-diamidino-2-phenylindol, EPM: elevated plus maze, FoxP3: forkhead box P3, IgG: immunoglobulin G, i.p.: intraperitoneal injection, Ly6C: lymphocyte antigen 6 family member C, Ly6G: lymphocyte antigen 6 family member G, MHCII: major histocompatibility complex 2, M-MDSC: monocytic-derived myeloid derived suppressor cells, NeuN: neuronal nuclei antigen, NK: natural killer, NOR: novel object recognition test, OFT: open field test, PD-1: programmed cell death 1, PD-L1: programmed cell death ligand 1, PMN-MDSC: polymorphonuclear myeloid derived suppressor cells, ROR: Retinoic acid-related Orphan Receptors, SSC: side scattering gating, TCR: T cell receptor, TST: tail suspension test.

Article Snippet: The following anti-mouse antibodies were used, with clone, fluorophore, and reference indicated in parenthesis: CD45 (REA737, VioGreen, 130-110-803, Miltenyi Biotec), CD11b (M1/70, FITC, 101206, Biolegend), CD11c (N418, PerCP, 117328, Biolegend), MHCII (M5/114.15.2, BV605, 107639, Biolegend), CD206 (APC, FAB2535A, R&D systems), Ly6G (1A8, BV421, 1238135, Sony Biotechnology), Ly6C (REA796, Vioblue, 130-111-921, Miltenyi Biotec), CD3 (17A2, BV421, 100228, Biolegend), CD4 (REA1211, PE, 130-123-206, Miltenyi Biotec), CD8 (53-6.7, FITC, 11-0081-82, Invitrogen), NK1.1 (PK136, PerCP, 1143630, Sony Biotechnology), CD19 (6D5, AF700, 115527, Biolegend), FoxP3 (MF-14, AF647, 1232040, Sony Biotechnology), RORγt (Q31-378, BV786, BD Biosciences) and TCRγδ (GL3, BV605, 118129, Biolegend).

Techniques: Comparison, Neutralization, Injection, Immunopeptidomics, Derivative Assay, Suspension

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: EV-dead induces TNBC lung metastasis and promotes TAM infiltration in vivo. A Diagram of the EV-dead and EV-alive separation procedures and their representative transmission electron microscopy (TEM) images. Scale bar: 100 nm. The sizes of the EVs were detected using a Flow NanoAnalyzer, and their protein markers were identified by Western blotting analysis. B NTA analysis was conducted to detect the relative secretion content of EV-dead and EV-alive; n = 3. C Representative images of zebrafish breast cancer xenotransplantation model assay. The effects of EV-alive (100 μg/ml) and EV-dead (50–100 μg/ml) on the metastasis of 4 T1 cells in the presence or absence of M0 co-injection were investigated; n = 6. D Schematic diagram of the animal assay and representative pictures of the in vivo imaging assay and tumors. Peritumoral injection with EV-dead (200 μg/20 g weight, q3d) promoted tumor growth of 4 T1-Luc xenografts, as evidenced by increases in both tumor volume and weight, but had no significant effect on the weight of the mice; n = 8. Scale bar: 1 cm. E Representative images of the lungs and the lung HE staining assay. Metastatic foci were identified by HE staining of the lung sections. Scale bar: 100 μm; n = 3. F Infiltration levels of CD45 + /F4/80 + /CD206 + TAMs in mammary tumors; n = 3. G Expression levels of ARG1 and iNOS in mammary tumor tissues; n = 3. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C.

Techniques: In Vivo, Transmission Assay, Electron Microscopy, Western Blot, Injection, In Vivo Imaging, Staining, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: CXCL1 EV-dead promotes TNBC growth and lung metastasis in vivo by activating TAM/PD-L1 signaling. A The successful generation of 4 T1/rCXCL1 Flag cells was validated by immunoblotting and immunofluorescence assays. Scale bar: 5 μm. B The difference in CXCL1 content between EV-dead and EV-dead rCXCL1-Flag was compared by ELISA. EV-dead rCXCL1-Flag was isolated from the supernatants of apoptotic 4 T1/rCXCL1 Flag cells induced by paclitaxel treatment; n = 3. C , D Peritumoral injection with EV-dead rCXCL1 (200 μg/20 g weight, q3d) significantly accelerated TNBC growth (C) and lung metastasis (D) compared to that of the EV-dead group (200 μg/20 g weight, q3d); Tumor volume: n = 6; Number of metastatic lesions, n = 3; K-M curves of lung metastasis time, n = 10. Scale bar: 1 cm. E Tumor tissue immunofluorescence experiment showed that flag-tagged CXCL1 (red) from EV-dead rCXCL1-Flag was predominantly phagocytosed by CD206 + macrophages (green) in the TME. Scale bar: 5 μm. F The infiltration levels of CD45 + /F4/80 + /CD206 + TAMs (up) and CD45 + /F4/80 + /PD-L1 + TAMs (down) in the TME of mice following treatment with saline, EV-dead, or EV-dead rCXCL1-Flag ; n = 3. G QPCR assay was conducted to investigate the CTC quantity in the peripheral blood of mice following treatment with saline, EV-dead, or EV-dead rCXCL1-Flag ; n = 3. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C.

Techniques: In Vivo, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Isolation, Injection, Saline

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: CXCL1 knockdown in EV-dead or macrophage depletion inhibits EV-dead-induced TNBC growth and lung metastasis in vivo . A The successful generation of 4 T1/shCXCL1 cells was verified by western blotting assay. The difference in CXCL1 content between EV-dead and EV-dead shCXCL1 was compared by ELISA. EV-dead shCXCL1 was isolated from the supernatants of apoptotic 4 T1/shCXCL1 cells induced by paclitaxel treatment; n = 3. B Representative images of the tumors ( n = 7) and the in vivo imaging assay ( n = 3), and mouse weight and tumor volume curves ( n = 7). Clodronate liposomes (CL) were used to deplete macrophages in the TME of the 4 T1-Luc xenograft model. Scale bar: 2 cm. C Representative images of the lungs ( n = 3) and lung HE assay ( n = 3) as well as the K-M curves of lung metastasis time ( n = 10). Scale bar: 100 μm. D , E The infiltration levels of CD45 + /F4/80 + /CD206 + TAMs (D) and CD45 + /F4/80 + /PD-L1 + TAMs (E) in the TME of mice following treatment with EV-dead, EV-dead shCXCL1 , or the combination of EV-dead and CL; n = 3. F CD206 (green) and PD-L1 (red) expression levels in the TME. Arrows indicate PD-L1 expression in TAMs. Scale bar: 10 μm. G The quantity of CTCs in the peripheral blood of mice treated as indicated; n = 3. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C.

Techniques: Knockdown, In Vivo, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, In Vivo Imaging, Liposomes, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

doi: 10.1186/s13046-024-03050-7

Figure Lengend Snippet: TPCA-1 chemosensitizes TNBC to paclitaxel and inhibits CXCL1 EV-dead -induced TNBC growth and lung metastasis in vivo . A Representative images of tumors and the tumor volume curves ( n = 6). TPCA-1 (10 mg/kg/d) and paclitaxel (10 mg/kg/3d) were administered. B Representative images of the in vivo imaging assay and lung HE staining assay; n = 3. Scale bar: 100 μm. C , D The infiltration levels of CD45 + /F4/80 + /CD206 + TAMs and CD45 + /F4/80 + /PD-L1 + TAMs in the TME of mice following treatment with TPCA-1, paclitaxel, or the combination of TPCA-1 and paclitaxel; n = 3. E The CTC quantity in the peripheral blood of mice when they were treated as indicated; n = 3. F Representative images of tumors and the tumor volume curves ( n = 6). EV-dead and EV-dead rCXCL1 (200 μg/20 g weight, q3d) were administered by peritumoral injection. G Representative images of the in vivo imaging assay and lung HE staining assay; n = 3. Scale bar: 100 μm. H , I The infiltration levels of CD45 + /F4/80 + /CD206 + TAMs ( n = 3) and CD45 + /F4/80 + /PD-L1 + TAMs ( n = 6) in the TME of mice when they were treated as indicated. J The CTC quantity in the peripheral blood of mice when they were treated as indicated; n = 3. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: Then, Raw264.7 cells were harvested and incubated with FITC-conjugated F4/80 antibody (SC-71085, Santa Cruz, CA, USA), PE-conjugated CD206 antibody (141,705, Biolegend, CA, USA), or PE-Cy7-conjugated CD206 antibody (E-AB-F1135H, Elabscience, Houston, TX, USA) for 30 min at 37 °C.

Techniques: In Vivo, In Vivo Imaging, Staining, Injection

Journal: STAR Protocols

Article Title: CyTOF protocol for immune monitoring of solid tumors from mouse models

doi: 10.1016/j.xpro.2022.101949

Figure Lengend Snippet:

Article Snippet: Anti-Mouse CD206-169Tm dilution 1:100 , Standard BioTools , 3169021B.

Techniques: Blocking Assay, Purification, Recombinant, Staining, Sterility, Antibody Labeling, Software, Transferring, Microscopy