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. 2020 Aug 20;9(1):1803530. doi: 10.1080/2162402X.2020.1803530

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© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Immunogenicity of cancer stem-like cells results from lower CD47 expression and increased macrophage phagocytosis. (a) CD47 expression levels quantified by quantitative PCR demonstrated 3-fold lower expression of CD47 on SCA-1⁺ ID8 cells (p < .01, bulk vs SCA-1⁺ ID8, left). Histogram in flow cytometry showed lower CD47 protein level on SCA-1⁺ ID8 cells (right, ns: no staining). Similarly, expression of CD47 is lower on CD133⁺ HM-1 cells comparing to the expression on bulk population (p < .001, value in folds). Histogram in flow cytometry also showed lower CD47 protein level on CD133⁺ HM-1 cells (right, ns: no staining). (b) CFSE-pulsed bulk and SCA-1⁺ ID8 cells were incubated with syngeneic primary CD11c⁺ cells for 1 hour. Increased phagocytosis (indicated by CD11c⁺ gated CFSE-stained cells) was observed on both live (p < .01) and irradiated (IR) (p < .05) SCA-1⁺ ID8 cells (bar data). (c) Analysis on RAW 264.7 cell revealed better phagocytosis on SCA-1⁺ ID8 cells than on bulk ID8 cells. SCA-1⁺ ID8 cells were stably transfected with either pcDNA3.1 empty vector (SCA-1⁺ ID8 vehicle) or pcDNA3.1-CD47 (SCA-1⁺ ID8 CD47). Suppression of CD47 on bulk ID8 cells by anti-CD47 antibody markedly enhances the phagocytic activities, while forced expression of CD47 on SCA-1⁺ ID8 cells (SCA-1⁺ ID8 CD47) hampered the phagocytosis. (d) Cross-presentation of phagocytic antigens to a SCA-1⁺ ID8-specific T lymphocyte line developed from SCA-1⁺ ID8 immunized mice showed the same pattern. To a lesser degree, anti-CD47 in bulk ID8 cells increased the number of activated CD8⁺ T cells, while forced expression of CD47 decreased the number activated T cells. (e) Image study showed that tumor rejection of SCA-1⁺ ID8 cells was abrogated by forced expression of CD47. Both SCA-1⁺ ID8 (vehicle) and SCA-1⁺ ID8 CD47 (started from 10000 cells) grew on 6^th day image but the former was mostly cleared on 10^th day while the latter retained their growth longer. (f) Representative analysis of peritoneal lavaged cells showed highest percentage of CD3⁺CD8⁺ cells in mice with SCA-1⁺ ID8 vehicle cells (day 20). Mice with SCA-1⁺ID8 CD47 cells had less CD3⁺CD8⁺ and F4/80 cells (p < .01). However, these mice exhibited significantly higher percentage of CD45⁺CD68⁺ cells in peritoneal lavage than mice with SCA-1⁺ID8 vehicle did (p < .001). (g) Significantly less activated CD8⁺IFN-γ⁺ splenocytes were seen from mice immunized with SCA-1⁺ ID8 CD47 cells compared to those from mice immunized with SCA-1⁺ ID8 vehicle (p < .001). *p < .01, **p < .001, NS: not significant.