Journal: Journal of Experimental & Clinical Cancer Research : CR
Article Title: Cell therapy using ex vivo reprogrammed macrophages enhances antitumor immune responses in melanoma
doi: 10.1186/s13046-024-03182-w
Figure Lengend Snippet: HDAC6 inhibition regulated M2 macrophage phenotype. ( A ) Tumor growth chart of syngeneic SM1 murine melanoma tumors in C57BL/6 mice treated with intraperitoneal administration of 25 mg/kg HDAC6 inhibitor, NexturastatA (NextA) or vehicle. ( B ) Tumor-associated M1 (F480 + Cd80+) and M2 (F4/80 + Cd206+) macrophages as a % of live cells, respectively, and M1/M2 ratio in vehicle or NextA treated mice bearing SM1 murine melanoma tumors. Tumors were collected on day 20 with the tumor size endpoint as 20 mm diameter. ( C ) Polarization efficiency of murine bone marrow-derived macrophages (BMDMs) to M1 and M2 phenotypes after treatment with HDAC6i, NextA (5µM) determined by flow cytometry. ( D ) Volcano plot showing fold-change and p-value for comparing vehicle-treated M1 versus M0. M0 are naïve macrophages derived from the mouse bone marrow. ( E ) Volcano plot showing fold-change and p-value for the comparisons and vehicle-treated M2 versus M0 macrophages. The significance level was determined by log2 fold changes ≥ 1.5 (upregulation/increased) or ≤-1.5 (downregulation/decreased) and p-value < 0.05. Differentially expressed genes are depicted in blue and red, where M1 and M2 markers are labeled in figures ( D ) and ( E ), respectively. ( F ) Heatmap of known markers for classically activated M1 and M2-like macrophages. Differential expression for HDAC6 inhibition versus vehicle was performed, and M1 markers (black) and M2 markers (purple) were represented using the log 2 transformed fold changes relative to vehicle-treated M0. ( G ) We analyzed about 659 M0, 407 M1, and 450 M2 macrophages treated with vehicle; 424 M1 and 378 M2 macrophages treated with NextA on mouse innate immune IsoCode chips on the Isoplexis platform. Uniform manifold approximation and projection (UMAP) dimensionality reduction analysis of BMDMs at a single cell resolution separated them into defined clusters based on their secretome profile. ( H ) A polyfunctionality heatmap representing each macrophage phenotype secretes more than one cytokine/chemokine. The intensity of orange squares in the heatmap represents the percentage of cells secreting the cytokine/chemokine indicated by corresponding black dots below. (I) 2-D tsne plots where each cell is represented as a colored dot. As shown in the intensity scale, blue indicates low expression, and red indicates high expression. Bar graphs of respective cytokines are represented as signal intensity. Proinflammatory cytokines Tnfa, and T-cell recruiting chemokine Ip-10 (Cxcl10) are elevated in M1 + NextA, whereas growth factors Egf and Pdgf secreted by M2 phenotype are decreased with NextA treatment
Article Snippet: Isolated T-cells were labeled with CellTrace™ Violet Cell Proliferation Kit (ThermoFisher, Cat # C34557) and subsequently activated with Dynabeads™ Mouse T-Activator CD3/CD28 for T-Cell Expansion and Activation (ThermoFisher, Cat # 11452D) in RPMI complete media supplemented with 100U/mL of mouse recombinant Il-2 cytokine (ThermoFisher, Cat # 212 − 12) as per the manufacturer’s recommendations.
Techniques: Inhibition, Derivative Assay, Flow Cytometry, Labeling, Expressing, Transformation Assay
