Exosomes from M1-Polarized Macrophages Potentiate the Cancer Vaccine by Creating a Pro-inflammatory Microenvironment in the Lymph Node  Lifang Cheng, Yuhua Wang, Leaf Huang  Molecular Therapy  Volume 25, Issue 7, Pages 1665-1675 (July 2017) DOI: 10.1016/j.ymthe.2017.02.007 Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Molecular Therapy 2017 25, 1665-1675DOI: (10.1016/j.ymthe.2017.02.007) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 1 Isolation and Characterization of Exosomes from M1-Polarized Macrophages (A) Western blot analysis demonstrating the iNOS expression of activated M1 macrophages after induction under various conditions. (B) Representative chart of size distribution of exosomes measured using dynamic light scattering. (C) Representative chart of surface charge of exosomes determined by dynamic light scattering coupled with Laser Doppler velocimetry. (D) Representative TEM image of M1 exosomes after negatively staining with uranium acetate. Molecular Therapy 2017 25, 1665-1675DOI: (10.1016/j.ymthe.2017.02.007) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 2 Systemic and Cellular Distribution of M1 Exosomes after Subcutaneous Administration (A) Representative in vivo imaging of M1 exosome biodistribution in the lymph nodes of mice. (B) Representative flow cytometry data showing the cell populations in the lymph nodes that had taken up M1 or M2 exosomes. (C) Quantitation of the percentage of cells that had taken up exosomes of the total corresponding cell population in the lymph node. (D) Quantitation of the percentage of certain cell types that had taken up exosomes of the total cells that had taken up exosomes. Molecular Therapy 2017 25, 1665-1675DOI: (10.1016/j.ymthe.2017.02.007) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 3 Induction of Cytokine Expression by M1 or M2 Exosomes in Macrophages, DCs, and In Vivo (A) Quantitation of cytokine expression by the macrophages (RAW246.7) in vitro after exposure to the M1 or M2 exosomes for 4 hr. (B) Quantitation of cytokine expression by the dendritic cells (JAWSII) in vitro after exposure to the M1 or M2 exosomes for 4 hr. (C) Time course studies of the change of cytokine levels in the draining lymph nodes after subcutaneous administration of the M1 exosomes. Molecular Therapy 2017 25, 1665-1675DOI: (10.1016/j.ymthe.2017.02.007) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 4 M1 Exosomes Boosted Cell-Mediated Immune Response Elicited by LCP Peptide Vaccine In Vivo (A) Representative flow cytometry data showing the ratios between OVA peptide-pulsed and Trp2 peptide-pulsed splenocytes recovered from the mice immunized with different vaccine combinations. (B) Quantitation of antigen-specific killing efficiency brought about by different vaccine treatments (Student’s t test, ***p < 0.001). (C) Image of the draining lymph nodes from the injection site of the mice at the endpoint of the CTL experiment. (D) Quantitation of ELISPOT assay (Student’s t test, ***p < 0.001). Representative images of IFN-γ ELISPOT assay show M1 exosomes boosted the antigen-specific immune response, which was reflected by the higher amount of IFN-γ secretion by the immune cells when challenged with Trp2 peptide antigen. OVA peptide was used as the control. Molecular Therapy 2017 25, 1665-1675DOI: (10.1016/j.ymthe.2017.02.007) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 5 M1 Exosomes Boosted LCP Vaccine and Effectively Inhibited B16F10 Melanoma Growth on C57BL/6 Mice (A) Tumor growth inhibition study showing the anti-tumor efficacy of vaccine in the presence or absence of different adjuvants (n = 6–8; one-way ANOVA analysis, *p < 0.05 and ***p < 0.001). (B) Quantitation of TUNEL assay based on the percentage of apoptotic cells (n = 4; Student’s t test, ***p < 0.001). (C) Representative images of TUNEL assay showing apoptotic cells (green) and DAPI-stained total cells (blue). (D) Representative images of H&E staining of the tumor samples harvested at the endpoint of the experiment. Upper panel: low-magnification images of the tumor show tumor cells (large nucleus and cytoplasm) and stroma, including non-cellular structure and infiltrating immune cells (high nuclear cytoplasmic ratio). Scale bar, 200 μm. Lower panel: enlarged images of the rectangular areas in the upper panel are shown. Scale bar, 100 μm. Arrows, infiltrating immune cells. Molecular Therapy 2017 25, 1665-1675DOI: (10.1016/j.ymthe.2017.02.007) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions