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. Author manuscript; available in PMC: 2021 Sep 8.
Published in final edited form as: Nat Cancer. 2021 Feb 18;2(2):218–232. doi: 10.1038/s43018-021-00173-0

Figure 4. C3 and C3aR deficiency are associated with an M1-like TAM phenotype.

Figure 4.

(a) C3aR mRNA expression in MN/MCA1 (left panel) (n=3 independent experiments) or 3-MCA derived (right panel) (n=9 independent experiments) tumor cells and in leukocytes sorted from MN/MCA1 (left panel) or 3-MCA-induced (right panel) tumors of wt mice (n=5 or 6 mice; mean ± SEM). (b) Immunofluorescence analysis of C3aR expression in wt peritoneal macrophages, used as positive control, and MN/MCA1 tumor cells. One experiment performed with two technical replicates for each cell culture, two-three fields have been acquired for each replicate. Scale bar: 10μm. (c) Immunofluorescence analysis of C3aR expression in tumor infiltrating macrophages (Iba-1+ cells) in wt derived MN/MCA1 tumor tissues. One experiment performed (n=5 mice), two-three fields have been acquired for each mouse. Scale bar: 50μm. (d, e) Analysis by FACS of macrophage (F4/80+) (d) and monocyte (Ly6C+) (e) frequency among living cells (Aqua−) in MN/MCA1 tumors in wt and C3−/− (n=9 wt, n=8 ko) or C3aR1−/− (n=6 wt, n=8 ko) mice sacrificed 21 days after tumor cell injection. (f-i) Analysis by FACS of M2 macrophage frequency (F4/80+/CD206+) (f, h) and of the expression of selected M1 markers (CD11c, MHC II, CD80 and CD86) gated on total macrophages (F4/80+) (g, i) in wt and C3−/− (f, g, n=9 wt, n=8 ko) or C3aR−/− (h, i, n=6 wt, n=8 ko) mice sacrificed 21 days after tumor cell injection. (j-l) Frequency of macrophages (F4/80+) and monocytes (Ly6C+) (j), M2 macrophages (F4/80+/CD206+) (k) and expression of M1 markers (CD11c, MHC II, CD80 and CD86) gated on total macrophages (F4/80+) (l) in MN/MCA1 tumors in wt and C3aR−/− (n=15 in each group) mice sacrificed at similar tumor volume (2cm3). d and e left panels, f and g: two experiments performed; d and e right panels, h and i: three experiments performed; j and l: one experiment performed. d-l: mean is shown. Exact p values are reported, unpaired two-tailed Student’s t test (d; e; g: CD11c, CD80 and CD86 panels; h; i; j: F4/80+ panel; l:CD11c, MHCII and CD86 panels) or two-tailed Mann Whitney test (f, g: MHCII panel; j: Ly6C+ panel; k; l: CD80 panel).