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