Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: IM + anti-VEGF prolongs survival of GBM mice and is immunostimulatory (A) Schematic of the long-term therapeutic trials in the lentiviral-induced mouse model of glioma. (B) Representative images of H&E-stained tissue sections from a tumor that developed in an end-stage LVRshp53 animal. Scale bar, 30 μm. Representative of whole-slide images of three tumors. (C) Survival of tumor-bearing LVRshp53 animals subjected to the indicated treatments. Control (Ctrl) (n = 10), anti-VEGF (n = 7), IM + anti-VEGF (n = 7), IM + anti-VEGF + CDL (n = 8). (D) Normalized bioluminescence in LVRshp53 animals treated as indicated for 2 weeks. (E) Survival of PDG animals subjected to the indicated treatments. Ctrl (n = 9), IM + anti-VEGF (n = 10). (F) Representative images of CD8 (green) and DAPI nuclear staining (blue). Scale bar, 50 μm. Image is illustrative of the analysis shown in (H). (G) High-magnification images of CD8 T cells in a Ctrl versus an IM + anti-VEGF-treated tumor. Scale bar, 50 mm (H) Quantification of CD8 T cells in LVRshp53 tumors treated as indicated for 12 days. Each dot indicates the average of 8–12 immuno-stained tumor tissue sections from one mouse. (I) Flow cytometry analysis of CD8 T cells in LVRshp53 tumors treated as indicated for 12 days. Cells were gated as CD45 + CD3 + CD8 + . Ctrl (n = 15), anti-VEGF (n = 8), IM (n = 10), IM + anti-VEGF (n = 10). (J and K) Representative images (J) and quantification (K) of CD4 T cells in whole LVRshp53 tumor tissue section. Animals were treated for 12 days. Scale bar, 50 μm. Ctrl (n = 6), anti-VEGF (n = 4), IM (n = 4), IM + anti-VEGF (n = 6). (L) Assessment of the functional contributions of CD8 and CD4 T cells to survival benefit. Ctrl (n = 6), αCD8 + αCD4 (n = 4), IM + anti-VEGF (n = 5), IM + anti-VEGF + αCD8 + αCD4 (n = 5), IM + anti-VEGF + αCD8 (n = 5). (M) Normalized bioluminescence in LVRshp53 mice treated as indicated in (L). (Para break) Data in all quantitative panels are shown as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. For survival analyses, Mantel-Cox test was performed. Other analyses by Mann-Whitney or one-way ANOVA tests.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Staining, Control, Flow Cytometry, Functional Assay, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: CD8 and CD4 T cells are activated upon IM + anti-VEGF treatment (A) Flow cytometry analysis of effector T cells (CD62L-CD44 + ). Ctrl (n = 6), IM (n = 4), anti-VEGF (n = 7), and IM + anti-VEGF (n = 7). (B) FACS analysis of IFNγ intracellular staining in fixed and permeabilized CD8+T cells. Ctrl (n = 16), IM (n = 10), anti-VEGF (n = 12), IM + anti-VEGF (n = 12). (C and D) Flow cytometry analysis of GzB (C) and TNFα (D) intracellular staining in CD8 T cells. Ctrl (n = 15), IM (n = 10), anti-VEGF (n = 8), IM + anti-VEGF (n = 8). (E) Functional importance of IFNγ for the survival of LVRshp53 animals subjected to the indicated treatments. Ctrl (n = 9), anti-IFNγ (n = 5), IM + anti-VEGF (n = 7), IM + anti-VEGF + anti-IFNγ (n = 6). Statistical analysis by Mantel-Cox test. (F, G, and H) Flow cytometry analysis of Ki67 (F), pSTAT5 (G), and TCF1 (H) in CD8 T cells. Ctrl (n = 7), IM (n = 4), anti-VEGF (n = 9), and IM + anti-VEGF (n = 10). (I) Representative image of HIF-1α (red) and CD8 (green) in the anti-VEGF-treated tumor. Image is illustrative of the analysis performed in (J). Scale bar, 50 mm (J) Quantification of the proximity of CD8 T cells to hypoxic regions in the entire area of full sections of GBM tumors. The zones were divided into 0 μm (i.e., within the HIF-1α+ zone), >0 and <5 μm, and >5 μm separating T cells and HIF-1α+ regions. Ctrl (n = 11), IM (n = 4), anti-VEGF (n = 4), IM + anti-VEGF (n = 5). (K) Flow cytometry analysis of intracellular HIF-1α expression in fixed and permeabilized CD8 T cells. Ctrl (n = 5), IM (n = 4), anti-VEGF (n = 8), anti-VEGFR2 (n = 5). (L) Flow cytometry analysis of intracellular FOXP3 expression in CD4 T cells. Ctrl (n = 5), IM (n = 5), anti-VEGF (n = 4), IM + anti-VEGF (n = 8). (M) Flow cytometry analysis of intracellular TGFβ expression in CD4 T cells. Ctrl (n = 5), IM (n = 5), anti-VEGF (n = 4), IM + anti-VEGF (n = 7). (N and O) Flow cytometry analysis of SLAMF7 (N) and GzB (O) expression in CD4 T cells. Ctrl (n = 8), IM (n = 5), anti-VEGF (n = 5), IM + anti-VEGF (n = 7). (Para break) Data in all quantitative panels are shown as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. Statistical analysis by one-way ANOVA, unless otherwise indicated.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Flow Cytometry, Staining, Functional Assay, Expressing

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: Imipramine downregulates an M2-like program in TAMs (A) Western blot analysis of ARG1 and IL-10 in single tumors treated or not with IM or anti-VEGF. (B) Flow cytometry analysis of ARG1 and IL-10 expression in GBM tumors treated for 1 week. Ctrl (n = 9 tumors), IM (n = 6), anti-VEGF (n = 6), anti-VEGFR2 (n = 6), Axitinib (n = 6). Macrophages were gated as CD45 + CD11b+Ly6C-Ly6G−. (C) Expression of ARG1 and IL-10 in microglia (CD49d−) and MDMs (CD49d+) assessed by FACS in untreated tumors (n = 5). (D) Expression of MHC-II within microglia as assessed by flow cytometry. Ctrl (n = 4 tumors) and IM + anti-VEGF (n = 4). (E) Ex vivo co-cultures of tumoral CD11b cells and activated splenic CFSE-labeled CD8 or CD4 T cells. Each dot represents the average of two or three technical replicates. T cells alone (n = 4), Ctrl co-culture (n = 5), anti-VEGF (n = 3), IM (n = 4), IM + anti-VEGF (n = 4). (F) Analysis of the M2-like program in cytokine-polarized macrophages as assessed by qRT-PCR analysis of Ctrl and IM-treated M2-like BMDMs. Expression is normalized to 18S statistics by Welch’s t test. Each dot represents an individual sample. Data are representative of three independent experiments. (G) Analysis of the M1-like program in BMDMs assessed by FACS. Each dot represents an individual replicate. (H) Expression of Hrh1 mRNA normalized to 18S in ex vivo M1-and M2-polarized BMDMs, either untreated or IM treated for 24 h. Each dot represents an individual sample. Data are representative of three independent experiments. (I) mRNA expression of Hrh1 in FACS-sorted microglia or MDMs from Ctrl and IM-treated tumors. (J) mRNA expression of Arg1 , Chil3 , and Il10 in M2-polarized macrophages that were transfected with si Ctrl or two different si Hrh1 constructs. Cells were treated with 40 μm IM for 24 h. Data are representative of two independent experiments. (K) Western blot analysis of MRC1 and ARG1 expression of siRNA-transfected M2 BMDMs. Data are representative of two independent experiments. (L) CD8 and CD4 T cell proliferation during co-culture with tumoral CD11b cells isolated from untreated (n = 2) or TFP-treated tumors (n = 4). (M) mRNA expression of Hrh1 , Arg1 , and MMP2 in CD11b cells isolated from tumors treated with IM (n = 4), TFP (n = 4), or untreated Ctrl (n = 5). (N) Phagocytosis assay involving sorted microglia and MDMs from untreated or IM-treated tumors assayed with green pHrodo S. aureus bioparticles. Data presented as mean fluorescence intensity (MFI) of pHrodo/live cells. (Para break) Data in all quantitative panels are presented as mean ± SEM ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. Statistical analysis by Mann-Whitney test or one-way ANOVA, unless otherwise stated.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Western Blot, Flow Cytometry, Expressing, Ex Vivo, Labeling, Co-Culture Assay, Quantitative RT-PCR, Transfection, Construct, Isolation, Phagocytosis Assay, Fluorescence, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: Macrophage-derived CXCR3 ligands are required for the therapeutic benefit conveyed by the combinatorial regimen of IM + anti-VEGF (A) Cxcl10 and Cxcl9 expression in bulk tumors. mRNA expression is shown relative to Gapdh . Ctrl (n = 6), anti-VEGF (n = 9), IM (n = 6), IM + anti-VEGF (n = 8). (B) Representative image of CXCL10 (magenta), F4/80 (red), and DAPI (blue) staining of LVRshp53 tumors treated with IM + anti-VEGF. Scale bar, 50 μm. Images are illustrative of five to six fields in tissue sections from three different tumors. (C) CXCL9 expression in TAMs in untreated (n = 6) or tumors treated with anti-VEGF (n = 4), IM (n = 4), or IM + anti-VEGF (n = 8) revealed by flow cytometry. (D) CXCL9 expression in MDMs and microglia, evaluated as in (C). (E) mRNA Cxcl9 and Cxcl10 expression assessed in bulk tumors treated with IM + anti-VEGF (n = 10) ± αCD49d (n = 7) to selectively deplete MDMs but not microglia. Expression is normalized to Gapdh housekeeping gene. (F) Assessing the contribution of CXCR3 function to the survival of LVRshp53 animals subjected to the indicated treatments. Treatment cohorts: αCXCR3 (n = 6), IM + anti-VEGF + αCXCR3 (n = 6), IM + anti-VEGF (n = 5). (G) Representative images of CD8 T cells aimed to assess the effects of αCXCR3. Representative of whole-slide image analysis of three tumors per treatment. Scale bar, 50 mm (H) Flow cytometry analysis of CD8 T cells in tumors subjected to indicated treatments. (I) Ex vivo co-culture of tumor-derived CD11b cells and CFSE-labeled CD8 or CD4 T cells. Myeloid cells were isolated from tumors treated with IM + anti-VEGF (n = 4), IM + anti-VEGF + αCXCR3 (n = 4), or untreated Ctrl (n = 5). Each dot represents an average of two or three technical replicates. (J) Minimal effect on IFNγ secretion by CD8 T cells in tumors treated with αCXCR3, IM + anti-VEGF, or the triple combination. (K and L) No effect of αCXCR3 on (K) GzB or (L) TNFα secretion by CD8 T cells co-treated with IM + anti-VEGF. (M) Quantification of immunostaining for HEVs in tumors treated with IM + anti-VEGF (n = 8) or IM + anti-VEGF + αCXCR3 (n = 4). The data are shown as number of HEVs per square millimeter of tumor tissue. (Para break) Data in all quantitative panels are presented as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. Statistical analysis by Mann-Whitney test or one-way ANOVA, unless otherwise stated.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Derivative Assay, Expressing, Staining, Flow Cytometry, Ex Vivo, Co-Culture Assay, Labeling, Isolation, Immunostaining, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet: PD-L1 is induced in relapsing tumors and its blockade potentiates T cell function to prolong survival benefit in GBM mice (A) FACS analysis of PD-L1 in the live cell compartment of tumors treated as indicated. Ctrl (n = 4 tumors), responding to IM + anti-VEGF (n = 4), and relapsing from IM + anti-VEGF (n = 8). Responding tumors were collected after 12 days of treatment. Relapsing tumors were collected when mice became symptomatic or when tumors started to re-grow following a stable phase. (B) Representative image of immunostaining to reveal PD-L1 (red), CD45 (green), and DAPI nuclei (blue) in relapsing tumors after IM + anti-VEGF. Scale bar, 50 μm. Assessed in four relapsing tumors, n = 8–10 fields imaged per tumor. (C) Percentage of PD-L1-positive live cells comparing the CD45 − and CD45 + compartments of n = 4 relapsing GBM tumors as revealed by flow cytometry. (D) PD-L1 expression in the CD11b− and CD11b+ compartments of CD45 + cells in n = 8 tumors assessed by flow cytometry. (E) Percentage of PD-L1-positive TAMs assessed by FACS. Ctrl (n = 4), responding tumor (n = 4), relapsing tumor (n = 8). (F) Representative immunostaining to reveal PD-L1 expression in TAMs. CD206 (magenta), PD-L1 (red), and DAPI in n = 3 relapsing tumors, 8–10 images per tumor. Scale bar, 50 μm. (G) Expression of PD-L1 in MDMs and microglia of n = 8 relapsing tumors assessed by flow cytometry. (H) MHC-II expression in microglia comparing responding (n = 4) and non-responding tumors (n = 4), assessed by flow cytometry. (I and J) Representative images (I) and quantification (J) of CD8 T cells in untreated (n = 3), responding (n = 4), and relapsing (n = 3) tumors under IM + anti-VEGF treatment. CD8 (magenta) and DAPI-stained nuclei. Scale bar, 50 μm. Each dot indicates the total number of CD8 T cells in an entire tissue section from a tumor. (K) Abundance of CD8 T cells from tumors treated short term with IM + anti-VEGF (n = 5) or IM + anti-VEGF + αPD-L1 (n = 5), assessed by flow cytometry. (L–N) GzB (L), IFNγ (M), and TNFα (N) expression in CD8 T cells from tumors treated as in (K). (O) Assessment of the benefits of early versus late incorporation of anti-PD-L1. Ctrl (n = 5), IM + anti-VEGF (n = 7), IM + anti-VEGF + late anti-PD-L1 (n = 4), IM + anti-VEGF + early anti-PD-L1 (n = 7), anti-VEGF + anti-PD-L1 (n = 6). (P) The combination of a TCA (e.g., imipramine) and VEGF/VEGFR inhibitors induces autophagy in cancer cells and remodels the tumor vasculature, conveying survival benefit for mice bearing GBM. Imipramine reprograms M2-like TAMs to more pro-inflammatory phenotype, via inhibition of histamine receptor signaling. Consequent to the dual treatment, CD8 and CD4 T cells are recruited and activated to evoke their cytotoxic effects. The inclusion of anti-PD-L1 in the therapeutic regimen helps sustain the immune response and increases survival benefit. (Para break) Data in all quantitative panels are presented as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; ns, no statistical significance. Statistical analysis by Mann-Whitney test or one-way ANOVA, unless otherwise stated.

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Cell Function Assay, Immunostaining, Flow Cytometry, Expressing, Staining, Inhibition, MANN-WHITNEY

Journal: Cancer Cell

Article Title: Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity

doi: 10.1016/j.ccell.2022.08.014

Figure Lengend Snippet:

Article Snippet: InVivo MAb anti-mouse CD4 (clone GK1.5) , BioXCell , Cat #: BE0003-1; RRID: AB_1107636.

Techniques: Purification, Plasmid Preparation, Virus, Recombinant, Activation Assay, Staining, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Fluorescence, cAMP Assay, Competitive ELISA, Selection, Cell Isolation, Derivative Assay, Negative Control, shRNA, Software

Journal: Frontiers in Physiology

Article Title: Integrative Transcriptomic and microRNAomic Profiling Reveals Immune Mechanism for the Resilience to Soybean Meal Stress in Fish Gut and Liver

doi: 10.3389/fphys.2018.01154

Figure Lengend Snippet: Immunohistochemistry analysis for the main lymphocytes and key cytokines involved in SBMIE. (A) IHC results for IgM, CD4, IL17 and IL10 in the gut and liver in all time points in 40SBM group as well as 0 days in FM group (negative control) and 3 week in 70SBM group (positive control). The green signals represent the examined protein, while the blue signals (stained by Hoechst 33342) represent the nuclei. The white arrows points out typical immunohistochemistry signals. Scale bar: 30 μm. (B) The quantification of IHC signals in both the gut (left) and liver (right). In the gut, the significant ( p < 0.01) increase of signals compared to the sample at 0 days in LP or in IEL was indicated by stars or hashes. In the liver, for each protein, the signals were calculated for significant changes, indicated by lowercase letters “a, b, c, d, e”. For each protein, the same label means no difference. For comparing T cell and B cell signals, CD4 and IgM signals were calculated for p -value at each time point, the significant ( p < 0.01) more CD4 was indicated by the star, whereas the significant result for IgM ( p < 0.1) was indicated by hashes.

Article Snippet: Commercial antibodies [anti-zebrafish CD4 monoclonal antibody (mAb), Genetex] or the antibodies developed by our group, including mouse anti-grass carp IgM (GenBank: DQ417927.1 ) mAb, rabbit anti-grass carp IL17 (NCBI Accession: KC978892.1 ) polyclonal antibody (pAb), and rabbit anti-grass carp IL10 (NCBI Accession: JQ768312.1 ) pAb were used in the study.

Techniques: Immunohistochemistry, Negative Control, Positive Control, Staining

Journal: PLoS ONE

Article Title: A practical approach to pancreatic cancer immunotherapy using resected tumor lysate vaccines processed to express α-gal epitopes

doi: 10.1371/journal.pone.0184901

Figure Lengend Snippet: No infiltration of lymphocytes was observed in the α-gal(-) N-ly/α-gal(+) N-ly vaccinated groups and α-gal(-) PDAC-ly vaccinated group. However, infiltration by substantial numbers of both CD4 + and CD8 + T cells and macrophages was observed in the α-gal(+) PDAC-ly vaccinated group. H&E stained sections of the α-gal(+) PDAC-ly vaccinated group demonstrate severe destruction of PDAC cells elicited by effector cell infiltration. Representative images of five individual recipients in each group are shown. Scale bars = 100 µm.

Article Snippet: Tissue sections were incubated overnight at 4°C with either rat anti-mouse CD4 mAb (Cat. No. 103-M105, 1:500; ReliaTech GmbH , Germany) for CD4 + T cells or rat anti-mouse CD8 mAb (clone 53–6.7, Cat. No. 100701, 1:500; BioLegend, Inc. , CA, USA) for CD8 + T cells.

Techniques: Staining