Journal: Scientific Reports

Article Title: Endothelial Cell Phenotypes are Maintained During Angiogenesis in Cultured Microvascular Networks

doi: 10.1038/s41598-018-24081-z

Figure Lengend Snippet: Illustration of CD36 labeling in ex vivo cultured microvascular networks and stimulated in vivo microvasculature. Comparison between Day 3 ( Ex Vivo ) ( A–C ) and Day 3 ( In Vivo ) ( D–F ) microvascular networks revealed diminishing CD36 labeling along the length of the capillary sprout becoming absent at the tip. Arrows identify examples of CD36-negative capillary sprouts. Scale bars = 200 µm. ( G , H ) Capillary sprouts exhibited a substantial decrease in CD36 labeling compared to larger network vessels ( > 10 µm) in Day 3 ( Ex Vivo ) and Day 3 ( In Vivo ) tissues. White and black bars represent “Sprouts” and “Networks” respectively for Day 3 ( Ex Vivo ) and Day 3 ( In Vivo ) groups. The *** indicates a significant difference of p < 0.001 by Mann-Whitney U test.

Article Snippet: The following primary antibodies were used: UNC5b (1:100; Abcam; Cambridge, MA), VEGFR-2 (1:50; Santa Cruz Biotechnology; Dallas, TX), Alexa-568 Phalloidin (1:50; Invitrogen; Carlsbad, CA), CD36 (1:100; Novus Biologicals; Littleton, CO), and PECAM (1:200; BD Pharmingen; San Jose, CA).

Techniques: Labeling, Ex Vivo, Cell Culture, In Vivo, Comparison, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Targeting neoadjuvant chemotherapy-induced metabolic reprogramming in pancreatic cancer promotes anti-tumor immunity and chemo-response

doi: 10.1016/j.xcrm.2023.101234

Figure Lengend Snippet: Multiomics analysis supported that NAC decreased glycolysis but developed compensatory approaches (A) Transcriptome and proteome analysis revealed alterations in metabolic enzymes associated with AG treatment. The log fold-change value of transcriptome alteration is shown in yellow, while proteome alteration is shown in black. (B) Construct PDXO models from PDX mice with and without AG treatment. (C) A representative graph for PDXO in bright fields of microscope. (D) Metabolic flux experiments validated that PDXO separated from PDX mice treated with AG showed less glycolytic activity. The upper panel showsfocused isoforms of metabolites in glycolysis and TCA cycle. The heatmap reflects the relative abundance of isoforms shown in the ideographs. “m” referred to the number of C13 in the metabolite structure (n = 4). (E) Comparison of metabolic flux between the AG and control groups based on RNA-seq data (scFEA algorithm). (F) Targeted metabolomics analysis showed the differences of lactic acids, 3-PD, phosphoenolpyruvate, and alpha-ketoglutarate between PDAC samples with and without NAC, which implied NAC is associated with downregulated glycolytic activity in PDAC (n= 44) (mean with standard deviation). (G) Cell-Counting-Kit-8 (CCK-8) results showed oleic acid (50 μM) promoted the proliferation of panc-1 cells and could be blocked by targeting CD36, on the contrary (the upper panel), palmitic acid (50 μM) had no effect on the proliferation of panc-1 cells (the lower panel) (n = 5). (H and I) Oleic acid, as opposed to palmitic acid, was found to enhance the growth of PDOs according to the viability assay. The upper section of the figure displaysRepresentatives of PDOs cultured under the indicated conditions for 7 days. The lower section features a bar plot illustrating the relative viability of different groups, measured using the CellTiter-Glo 3D Cell Viability Assay (n = 5). (J) EdU assay showed oleic acid may fuel the drug resistance to AG but could be blocked by targeting CD36 (n = 4). The statistical significance shown in this figure was detected using t test.

Article Snippet: The fluorophore-conjugated antibodies and other agents used in the present study are listed as follows: For flow cytometry targeting mouse tumors, the following antibodies were used: anti-CD45 (BD, 564225); anti-CD36 (BD, 565933); anti-CD4 (BD, 550954); anti-CD8a (BD, 557654); anti-Gr-1 (BioLegend, 108412); anti-CD11B (Invitrogen, 25-0112-82); anti-CD25 (BD, 564370); and anti-FOXP3 (Invitrogen, 12-4771-82).

Techniques: Construct, Microscopy, Activity Assay, Comparison, RNA Sequencing Assay, Standard Deviation, Cell Counting, CCK-8 Assay, Viability Assay, Cell Culture, EdU Assay

Journal: Cell Reports Medicine

Article Title: Targeting neoadjuvant chemotherapy-induced metabolic reprogramming in pancreatic cancer promotes anti-tumor immunity and chemo-response

doi: 10.1016/j.xcrm.2023.101234

Figure Lengend Snippet: CD36 was systematically upregulated in tumor cells and resident and circulating immune cells (A) Immunohistochemical staining indicated that CD36 expression is increased in PDACs treated with NAC compared with UR samples (n= 54) (mean with standard deviation). Representative graphs are shown on the left. (B) Metabolic flux experiments showed that PDXOs derived from PDXs treated with AG had increased capability to uptake oleic acid (n = 3). (C) The percentages of CD36 + CD8 + T cells and CD36 + GZMB + CD8 + T cells were significantly upregulated in PDACs treated with NAC (n = 10) (mean ± SD). Representative graphs are shown on the left. (D) Representative graph by mIF showed co-localization of CD36 and TLS in PDAC. (E) Heatmap showing the correlation between CD36 expression and infiltration of immune cells, which indicated that CD36 expression was highly correlated with CD8 + T cell abundance only in PDACs treated with NAC. (F) Flow cytometry for PBMCs from PDAC patients showed CD36 was upregulated in circulating CD8 + T cells from patients treated with NAC. Left panel displays t-Distributed stochastic neighbor embedding (TSNE) analysis for labeled cell clusters (mean with standard deviation). Right panel displays the higher percentage of both CD36 + CD8 + T cells and CD36 + CD45 + immune cells in samples with NAC. (G) Flow cytometry showed that oleic acid decreased the percentage of IFN-γ + CD8 + T cells and could be rescued by CD36 blockage (n = 3). (H) T cells treated with lysates from different PDAC samples manifested distinct tumor-killing ability, which is showed by LDH-releasing experiments (n= 5). (I) T cells treated with lysates from CD36-low NAC samples showed significantly higher IFN-γ secretion compared with UR samples. (J) Caspase3/7 detection indicated that targeting CD36 synergistically enhanced the killing effect of AG based on a PDAC organoid/PBMC coculture system. Left panel shows the representative graph of caspase3/7-positive organoids at the time points 0 and 48 h. Right panel shows the percentage of apoptotic organoids in each group, which was evaluated through flow cytometry. (K) mIF showed CD36 blockage enhanced the killing effect of AG, which was showed by detecting Ki67 + organoids via mIF technology. (L) Successful construction of ovalbumin (OVA+) murine KPC organoids. (M) The OVA+organoid/OT-1-cell coculture system validated the synergistic effect of CD36 blockade on AG-mediated tumor killing (n= 3). Left panel shows the representative graph of caspase3/7-positive organoids. Right panel shows the percentage of apoptotic organoids in each group, which was evaluated through flow cytometry. The statistical significance shown in this figure was detected using t test.

Article Snippet: The fluorophore-conjugated antibodies and other agents used in the present study are listed as follows: For flow cytometry targeting mouse tumors, the following antibodies were used: anti-CD45 (BD, 564225); anti-CD36 (BD, 565933); anti-CD4 (BD, 550954); anti-CD8a (BD, 557654); anti-Gr-1 (BioLegend, 108412); anti-CD11B (Invitrogen, 25-0112-82); anti-CD25 (BD, 564370); and anti-FOXP3 (Invitrogen, 12-4771-82).

Techniques: Immunohistochemistry, Staining, Expressing, Standard Deviation, Derivative Assay, Flow Cytometry, Labeling

Journal: Cell Reports Medicine

Article Title: Targeting neoadjuvant chemotherapy-induced metabolic reprogramming in pancreatic cancer promotes anti-tumor immunity and chemo-response

doi: 10.1016/j.xcrm.2023.101234

Figure Lengend Snippet: Targeting CD36 synergistically promoted AG-mediated killing of PDAC in preclinical models (A) Visual presentation of subcutaneous xenograft murine PDAC tumor models (C57 mice) for each group. (B) Measurement of tumor volumes showed CD36 blockage synergistically promoted AG-mediated killing of PDAC in subcutaneous xenograft murine PDAC tumor models. (C) Measurement of tumor weights showed CD36 blockage synergistically promoted AG-mediated killing of PDAC in subcutaneous xenograft murine PDAC tumor models (n = 5). (D) Representative IHC staining showed Ki67 expression in subcutaneous xenografts treated with different regimens. (E) t-Distributed stochastic neighbor embedding (TSNE) analyses showed the clustering for CD36 + CD8 + T cells and GZMB + CD8 + T cells. (F) Flow cytometry revealed that more CD8 + T cells infiltrated PDAC with NAC, while the percentage of CD36 + CD8 + T cells also increased (n = 5) (mean with standard deviation). (G) ELISA results showed the combination of AG and CD36 blockade significantly improved IFN-γ and tumor necrosis factor α (TNF-α) levels intratumorally (n = 5). (H) Representative image of orthotopic murine models of PDAC. (I) Kaplan-Meier curve revealed the combination of CD36 blockade and AG significantly prolonged the survival interval of mice that received orthotopic PDAC cell transplantation (n = 10). Circle or square referred to a happened event (death or censored). Censored event means the mice is still alive at the time point that we ended follow-up. (J) CD36 blockade synergistically with AG regimens optimally narrowed the PDAC tumor size in a humanized PDX model (n = 10). (K) Representative IHC staining image of CD36-high and -low PDAC. (L) Kaplan-Meier curve showed increased CD36 expression predicted worse prognosis of PDAC patients with adjuvant AG chemotherapy. The statistical significance shown in this figure was detected using t test.

Article Snippet: The fluorophore-conjugated antibodies and other agents used in the present study are listed as follows: For flow cytometry targeting mouse tumors, the following antibodies were used: anti-CD45 (BD, 564225); anti-CD36 (BD, 565933); anti-CD4 (BD, 550954); anti-CD8a (BD, 557654); anti-Gr-1 (BioLegend, 108412); anti-CD11B (Invitrogen, 25-0112-82); anti-CD25 (BD, 564370); and anti-FOXP3 (Invitrogen, 12-4771-82).

Techniques: Immunohistochemistry, Expressing, Flow Cytometry, Standard Deviation, Enzyme-linked Immunosorbent Assay, Transplantation Assay, Adjuvant

Journal: Cell Reports Medicine

Article Title: Targeting neoadjuvant chemotherapy-induced metabolic reprogramming in pancreatic cancer promotes anti-tumor immunity and chemo-response

doi: 10.1016/j.xcrm.2023.101234

Figure Lengend Snippet:

Article Snippet: The fluorophore-conjugated antibodies and other agents used in the present study are listed as follows: For flow cytometry targeting mouse tumors, the following antibodies were used: anti-CD45 (BD, 564225); anti-CD36 (BD, 565933); anti-CD4 (BD, 550954); anti-CD8a (BD, 557654); anti-Gr-1 (BioLegend, 108412); anti-CD11B (Invitrogen, 25-0112-82); anti-CD25 (BD, 564370); and anti-FOXP3 (Invitrogen, 12-4771-82).

Techniques: Recombinant, Activation Assay, Staining, LDH Cytotoxicity Assay, Viability Assay, Isolation, Lysis, RNA Sequencing Assay, Software

Journal: Cell Death & Disease

Article Title: Microparticles from apoptotic platelets promote resident macrophage differentiation

doi: 10.1038/cddis.2011.94

Figure Lengend Snippet: Differentiation of monocytes into resident phagocytic cells induced by PM ap . ( a ) Surface spreading and actin cytoskeleton rearrangements of THP-1 cells, cultured for 7 days with vehicle buffer or PM ap . Bright-field images indicate cell spreading and filopod formation (arrows); images of FITC-phalloidin fluorescence show strands of actin filaments with PM ap (scale bars: 100 μ m, representative for three experiments). ( b ) Proliferation of THP-1 cells after 7 days of incubation with vehicle or PM ap . Data are expressed as absolute cell numbers. ( c ) Relative amount (%) of necrotic cells after incubation with PMA (10 ng/ml), PM ap , or resting platelets. ( d ) Uptake of oxLDL (labeled with DiI) by THP-1 cells, incubated for 7 days with PM ap or resting platelets (plts). ( e and f ,) Expression of phagocytic cell markers on THP-1 cells ( e ) and primary monocytes ( f ). Cells were treated for 2 or 7 days with vehicle or PM ap . Surface expression of CD36 was determined by flow cytometry, as was the intracellular expression of CD68 in permeabilized cells. Incubation conditions were as described for . Mean±S.E.M. ( n =4–5). * P <0.05 versus control

Article Snippet: THP-1 cells or monocytes in suspension were stained (30 min, 4 °C) with mouse anti-human antibodies against CD11b, CD31 (Sigma, St. Louis, MO, USA); CD14, CD16, CCR2, CCR5, CXCR4 (BD Biosciences); or CD36 (ImmunoTools, Friesoythe, Germany) and prepared as described before.

Techniques: Cell Culture, Fluorescence, Incubation, Labeling, Expressing, Flow Cytometry