Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A, B) C3 (A) and C3aR1 (B) mRNA levels in the parahippocampal gyrus of NCI (n=32), MCI (n=34), or AD (Dementia, n=160). Spearman’s correlation, ρ (C3)=0.36, p<2.6E-08; ρ (C3aR)=0.33, p<3.4E-07.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques:

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A)Representative GFAP and Iba1 immunostainings with DAPI in the hippocampus of 9 month-old WT, C3aR KO, PS19, and PS19/C3aR KO mice. Scale bar: 50 μM.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques:

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A) Representative Western blots of PHF1- and CP13-positive phospho-tau and total tau in PS19 and PS19/C3aR KO mouse brains at 9 months. γ-tubulin is used as a loading control.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques: Western Blot

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A) Representative synaptophysin (Syp) and Syp/NeuN co-immunostaining in area CA3 of hippocampus of WT, C3aR KO, PS19, and PS19/C3aR KO mice at 9 months. Scale bar: 50 μM.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques: Immunostaining

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A) Unsupervised clustering gene expression heatmap from RNAseq analysis of hippocampal tissues of 9 month-old WT, C3aR KO, PS19, and PS19/C3aR KO mice. 1726 DEGs were identified by comparing the PS19 vs WT group (1155 genes upregulated, red, and 571 downregulated, blue). n=4-5/genotype, adjusted p<0.01.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques: Expressing

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A) Gene-set enrichment analyses (GSEA) of the microglia sensome gene list in PS19 vs WT (left, NES=2.81, FDR<0.001) and in PS19/C3aR KO vs PS19 (right, NES= −3.0, FDR<0.001) comparisons. NES: normalized enrichment score; FDR: false discovery rate.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques:

Journal: Neuron

Article Title: Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer’s disease

doi: 10.1016/j.neuron.2018.10.031

Figure Lengend Snippet: (A, B) Total STAT3 (A) and Y705-phospho-STAT3 (B) levels in 9 month-old WT (n=10), C3aR KO (KO, n=10), PS19 (PS, n=7), and PS19/C3aR KO (PS/KO, n=7) mice measured by RPPA (mean ± s.e.m). The asterisks above PS19 are for PS19 vs WT comparison; those above PS19/C3aR KO are for PS19/C3aR KO vs PS19 comparison. Student｀s t-test. *p<0.05; **p<0.01.

Article Snippet: After washing in TBS, sections or cultured cells on coverslips were blocked with TBS-T (0.4% Triton X-100) containing 2% donkey serum for 30 min, and then incubated in primary antibody diluted in blocking solution overnight at 4°C (mouse anti-GFAP (Millipore, AB_94844); rabbit anti- Iba1 (Wako, AB_839504); mouse anti-NeuN (Millipore, AB_2298772), rabbit anti-synaptophysin (Abcam, AB_2286949); rat anti-C3 (Hycult, AB_10129042); rat anti-C3aR (Hycult, AB_10130173); rabbit anti-pSTAT3 (Tyr705) (CST, AB_2491009)).

Techniques:

Journal: Nature cancer

Article Title: Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression

doi: 10.1038/s43018-021-00173-0

Figure Lengend Snippet: (a-h) 3-MCA-induced sarcoma incidence in C3−/− (a, n=20 wt, n=18 ko), MBL1/2−/− (b, n=10 wt, n=14 ko), C4−/− (c, n=10 mice in each group), C1q−/− (d, n=7 wt, n= 8 ko), fB−/− (e, n=9 wt, n=12 ko), C5aR1−/− (f, n=10 wt, n=19 ko), C5aR2−/− (g, n=14 wt, n=10 ko) and C3aR−/− (h, n=14 wt, n=16 ko) mice. One representative experiment out of three (a), two (b, d, e and f) or one (c, g and h) performed is shown. Experiment of panel d was performed using wt littermates. Exact p values are reported, two-tailed Wilcoxon matched-pairs signed rank test.

Article Snippet: The following primary antibodies and final concentrations were used: anti-mouse C3aR (Hycult Biotech, clone 14D4, 1 μg/ml), anti-mouse C3b/iC3b/C3c (Hycult Biotech, clone 2/11, 5 μg/ml), anti-CD31 (Millipore, clone 2H8, 1 μg/ml), anti-CD45 (Abcam, rabbit polyclonal, 5 μg/ml), anti-Iba-1 (FUJIFILM Wako Pure Chemical Corporation, rabbit polyclonal, 2 μg/ml), and anti-NG2 (Millipore, rabbit polyclonal, 5 μg/ml).

Techniques: Two Tailed Test

Journal: Nature cancer

Article Title: Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression

doi: 10.1038/s43018-021-00173-0

Figure Lengend Snippet: (a-h) 3-MCA-induced sarcoma growth curves (left panels) or mean tumor volume (± SEM, right panels) in C3−/− (a, n=20 wt, n=18 ko), MBL1/2−/− (b, n=10 wt, n=14 ko), C4−/− (c, n=10 mice in each group), C1q−/− (d, n=7 wt, n=8 ko), fB−/− (e, n=9 wt, n=12 ko), C5aR1−/− (f, n=10 wt, n=19 ko), C5aR2−/− (g, n=14 wt, n=10 ko) and C3aR−/− (h, n=14 wt, n=16 ko) mice. Experiment of panel d was performed using wt littermates. (i) Representative H&E staining of 3-MCA tumor tissues of wt, C3−/−, MBL1/2−/−, C4−/− and C3aR−/− mice. One experiment performed, three representative fields have been acquired for each group (n=4 mice in each group). Scale bar: 100μm. (j-q) FS6 tumor incidences in C3−/− (j, n= 4 wt, n=8 ko); MBL1/2−/− (k, n=10 wt, n=9 ko); C4-/ (l, n=11 wt, n=10 ko); C3aR−/− (m, n=15 wt, n=12 ko); C1q−/− (n, n=15 wt, n=8 ko); fB−/− (o, n=9 wt, n=4 ko); C5aR1−/− (p, n=12 wt, n=5 ko) and C5aR2−/− (q, n=12 wt, n=8 ko) mice. (r-u) FS6 tumor volumes (mean ± SEM) (r, t) and incidences (s, u) in MBL1/2±/− (r-s, n=12 he, n=8 ko) and C3aR±/− (t-u, n=21 he, n=12 ko) littermates. One representative experiment out of three (a), two (b, d, e, f, j and k) or one (c, g, h, l, n, p, q, r, s, t and u) performed is shown. o, m: two pooled experiments. The same wt mice were used simultaneously as control mice of experiments of panels p, q and one out of the two pooled experiments of panel m. Two-tailed Mann Whitney test (r) or unpaired two-tailed Student’s t test (t). Exact p values are reported, two-tailed Wilcoxon matched-pairs signed rank test (j-q, s and u).

Article Snippet: The following primary antibodies and final concentrations were used: anti-mouse C3aR (Hycult Biotech, clone 14D4, 1 μg/ml), anti-mouse C3b/iC3b/C3c (Hycult Biotech, clone 2/11, 5 μg/ml), anti-CD31 (Millipore, clone 2H8, 1 μg/ml), anti-CD45 (Abcam, rabbit polyclonal, 5 μg/ml), anti-Iba-1 (FUJIFILM Wako Pure Chemical Corporation, rabbit polyclonal, 2 μg/ml), and anti-NG2 (Millipore, rabbit polyclonal, 5 μg/ml).

Techniques: Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature cancer

Article Title: Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression

doi: 10.1038/s43018-021-00173-0

Figure Lengend Snippet: (a-p) MN/MCA1 and FS6 tumor volumes (mean ± SEM) in C3−/− (a, n=5 mice in each group) and (b, n=14 wt, n=8 ko); C4−/− (c, n=10 wt, n=9 ko) and (d, n=11 wt, n=10 ko); C1q−/− (e, n=9 mice in each group) and (f, n=15 wt, n=8 ko); fB−/− (g, n=6 wt; n=4 ko) and (h, n=9 wt, n=4 ko); MBL1/2−/− (i, n=7 mice in each group) and (j, n=10 wt, n=9 ko); C3aR−/− (k, n=9 wt, n=9 he, n=8 ko) and (l, n=15 wt, n=12 ko); C5aR1−/− (m, n=7 wt, n=6 ko) and (n, n=12 wt, n=5 ko); C5aR2−/− (o, n=7 wt, n=4 ko) and (p, n=12 wt, n=8 ko) mice. (q) Primary tumor volume and number of lung metastasis 27 days after MN/MCA1 tumor cell im injection in wt and C3−/− (mean ± SEM, n=10 wt, n=7 ko) mice. One representative experiment out of 15 (a), 8 (k), two (b, c, g, i, j, m and q) or one (d, f, n, o and p) performed is shown. e: Four pooled experiments performed with co-housed wt and heterozygous littermates. h, l: two pooled experiments. k: one out of 8 experiments was performed with co-housed littermates. The same wt mice were used simultaneously as control mice of experiments of panels i and m or for panels n, p and one out of the two pooled experiments of panel l. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Exact p values at the day of sacrifice are: a: p=0.0003; b: p=0.0014; c: p=0.025; d: p=0.0033; e: p=0.0218; j: p=0.0008; k: p<0.0001; l: p=0.0215; q left: p=0.0097; q right: p=0.0104. Unpaired two-tailed Student’s t test (a: 16 and 21 days; c: 18 and 21 days; e; f; g; h; k: 16 days; m; o; q: 20 and 26 days) or two-tailed Mann Whitney test (a: 12 and 19 days; c: 15 days; d; i; j; k: 19 and 21 days; l; n; p; q: 24 days).

Article Snippet: The following primary antibodies and final concentrations were used: anti-mouse C3aR (Hycult Biotech, clone 14D4, 1 μg/ml), anti-mouse C3b/iC3b/C3c (Hycult Biotech, clone 2/11, 5 μg/ml), anti-CD31 (Millipore, clone 2H8, 1 μg/ml), anti-CD45 (Abcam, rabbit polyclonal, 5 μg/ml), anti-Iba-1 (FUJIFILM Wako Pure Chemical Corporation, rabbit polyclonal, 2 μg/ml), and anti-NG2 (Millipore, rabbit polyclonal, 5 μg/ml).

Techniques: Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature cancer

Article Title: Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression

doi: 10.1038/s43018-021-00173-0

Figure Lengend Snippet: (a-b) Correlation plot between C3AR1 expression level and macrophage (a) or monocyte (b) quantification scores. Blue line: linear model interpolating curve; darker bands: linear model confidence intervals (n = 263 patients). (c-d) Kaplan-Meier survival curves of TCGA sarcoma patients divided based on the median enrichment scores of C3 deficiency-associated signatures of macrophages (n=60 increasing genes) (c) and monocytes (n=85 increasing genes) (d) (n=262 patients in total, n = 131 patients in each group, the patient corresponding to the median value has been excluded by the analysis). Shaded areas of Kaplan-Meier survival curves represent 95% upper and lower confidence intervals. (e-f) Correlation plot between C3 deficiency-associated signature of macrophages (e) or monocytes (f) and a M1-like macrophage signature (n=263 patients). Blue line: linear model interpolating curve; darker bands: linear model confidence intervals. (a-f) Exact p value of two-sided Log-rank test (for survival curves) and correlation test (Spearman correlation for scatterplots) are reported.

Article Snippet: The following primary antibodies and final concentrations were used: anti-mouse C3aR (Hycult Biotech, clone 14D4, 1 μg/ml), anti-mouse C3b/iC3b/C3c (Hycult Biotech, clone 2/11, 5 μg/ml), anti-CD31 (Millipore, clone 2H8, 1 μg/ml), anti-CD45 (Abcam, rabbit polyclonal, 5 μg/ml), anti-Iba-1 (FUJIFILM Wako Pure Chemical Corporation, rabbit polyclonal, 2 μg/ml), and anti-NG2 (Millipore, rabbit polyclonal, 5 μg/ml).

Techniques: Expressing

Journal: Nature cancer

Article Title: Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression

doi: 10.1038/s43018-021-00173-0

Figure Lengend Snippet: (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).

Article Snippet: The following primary antibodies and final concentrations were used: anti-mouse C3aR (Hycult Biotech, clone 14D4, 1 μg/ml), anti-mouse C3b/iC3b/C3c (Hycult Biotech, clone 2/11, 5 μg/ml), anti-CD31 (Millipore, clone 2H8, 1 μg/ml), anti-CD45 (Abcam, rabbit polyclonal, 5 μg/ml), anti-Iba-1 (FUJIFILM Wako Pure Chemical Corporation, rabbit polyclonal, 2 μg/ml), and anti-NG2 (Millipore, rabbit polyclonal, 5 μg/ml).

Techniques: Expressing, Derivative Assay, Immunofluorescence, Positive Control, Cell Culture, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature cancer

Article Title: Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression

doi: 10.1038/s43018-021-00173-0

Figure Lengend Snippet: (a) Analysis by FACS of M2 macrophage frequency (F4/80+CD206+) and of selected M1 marker (CD11c, MHC II, measured as MFI) expression gated on total macrophages (F4/80+) in wt and C3−/− mice sacrificed 27 days after im MN/MCA1 tumor cell injection (n=6 wt mice, n=5 ko mice, mean ± SEM). (b) Analysis by FACS of monocyte (Ly6C+), macrophage (F4/80+), M2 macrophage frequency (F4/80+CD206+) and of CD86 (measured as MFI) expression gated on total macrophages (F4/80+) in wt and C3aR−/− mice sacrificed 34 days after FS6 tumor cell injection (n=9 wt mice, n=4 ko mice; mean ± SEM). (c) Quantitation of vessel density, vascular area and vascular coverage by pericytes in 3-MCA-derived tumors of wt and C3−/− mice (n=7 mice in each group; mean ± SEM). (d) Frequency of CD3+CD4+, CD3+CD8+ and activated effector/effector memory T cells (CD8+CD44+CD62L−) in wt and C3aR−/− mice sacrificed 34 days after FS6 tumor cell injection (n=9 wt mice, n=4 ko mice; mean ± SEM). a-d: one experiment performed. Exact p values are reported, unpaired two-tailed Student’s t test or two-tailed Mann Whitney test (a-d).

Article Snippet: The following primary antibodies and final concentrations were used: anti-mouse C3aR (Hycult Biotech, clone 14D4, 1 μg/ml), anti-mouse C3b/iC3b/C3c (Hycult Biotech, clone 2/11, 5 μg/ml), anti-CD31 (Millipore, clone 2H8, 1 μg/ml), anti-CD45 (Abcam, rabbit polyclonal, 5 μg/ml), anti-Iba-1 (FUJIFILM Wako Pure Chemical Corporation, rabbit polyclonal, 2 μg/ml), and anti-NG2 (Millipore, rabbit polyclonal, 5 μg/ml).

Techniques: Injection, Marker, Expressing, Quantitation Assay, Derivative Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature cancer

Article Title: Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression

doi: 10.1038/s43018-021-00173-0

Figure Lengend Snippet: (a) FACS analysis of helper T cell (CD3+/CD4+) frequency in MN/MCA1 tumors (left, n=9 wt mice, n=8 C3−/− mice; right, n=6 wt mice, n=7 C3aR−/− mice). (b) Analysis by RT-PCR of selected Th1 markers of CD4+ T cells sorted from MN/MCA1 tumors (mean ± SEM; n=5 mice per group). (c-e) Frequency of CD4+Tbet+ and CD4+Eomes+ (c, n=9 wt mice, n=7 C3−/− mice; d, n=7 mice in each group), CD3+CD8+ (e, left, n=9 wt mice, n=8 C3−/− mice; right, n=6 wt mice, n=7 C3aR−/− mice) and activated effector/effector memory T cells (CD8+CD44+CD62L−) (f, left, n=9 wt mice, n=8 C3−/− mice; right, n=6 wt mice, n=7 C3aR−/− mice) in MN/MCA1 tumors. (g) CD3+CD4+, CD3+CD8+ and activated effector/effector memory T cell (CD8+/CD44+/CD62L−) frequency in MN/MCA1 tumors of mice sacrificed at similar tumor volume (2cm3) (n=15 mice in each group). a-g: mean is shown. (h) Local IFNγ in MN-MCA1 tumors (mean ± SEM; n=28 wt, n=16 C3−/−, n=9 C3aR−/− mice from 4 pooled experiments). (i, j) MN/MCA1 primary tumor volume in mice treated with anti-CD8 (n=12 wt mice + ctrl IgG, n=9 wt mice + anti-CD8, n=11 ko mice + ctrl IgG, n=9 ko mice + anti-CD8) (i), anti-IFNγ (n=10 wt mice + ctrl IgG, n=10 wt or ko mice + anti- IFNγ, n=9 ko mice + ctrl IgG) (j) or ctrl IgG. (k) Primary tumor volume after MN/MCA1 sc injection in mice (n=9 mice in each group) treated with anti-PD-1 or ctrl IgG. (l) MN/MCA1 primary tumor volume and lung metastasis in mice treated with anti-PD-1 or ctrl IgG, (n=6 mice in each group). (m, n) Primary tumor volume after MN/MCA1 (n=6 mice vehicle + ctrl IgG, n=7 mice vehicle + anti-PD1, n=9 mice C3aRa + ctrl IgG, n=8 mice C3aRa + anti-PD1) (m) or FS6 (n=8 mice vehicle + ctrl IgG, n=9 mice vehicle or C3aRa + anti-PD1, n=8 mice C3aRa + ctrl IgG) (n) sc injection in wt mice treated with C3aRa or vehicle, and anti-PD-1 or ctrl IgG. i-n: mean ± SEM is shown. a, e and f left panels: three experiments performed; a, e and f right panels: four experiments performed; c, d: two experiments performed; b, g, j, k, l, m and n: one experiment performed; i: two pooled experiments. Exact p values are reported, unpaired two-tailed Student’s t test (b: for Stat4, Eomes, Ifng and Tnfa; c: left panel; d; e; f: right panel; g; h) or two-tailed Mann Whitney test (a; b: for Tbet and Il2; c: right panel; f: left panel), Kruskal Wallis [p=0.0008 (i), p=0.0042 (k), p=0.0111 (l left panel), p=0.0033 (l right panel), p=0.0009 (m) and p=0.0194 (n left panel)] or Ordinary one-way Anova (p<0.0001) (j) with unpaired two-tailed Student’s t test or two-tailed Mann Whitney U-test as post-hoc tests (i-n) and two-tailed Wilcoxon matched-pairs signed rank test (n, right panel).

Article Snippet: The following primary antibodies and final concentrations were used: anti-mouse C3aR (Hycult Biotech, clone 14D4, 1 μg/ml), anti-mouse C3b/iC3b/C3c (Hycult Biotech, clone 2/11, 5 μg/ml), anti-CD31 (Millipore, clone 2H8, 1 μg/ml), anti-CD45 (Abcam, rabbit polyclonal, 5 μg/ml), anti-Iba-1 (FUJIFILM Wako Pure Chemical Corporation, rabbit polyclonal, 2 μg/ml), and anti-NG2 (Millipore, rabbit polyclonal, 5 μg/ml).

Techniques: Reverse Transcription Polymerase Chain Reaction, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature cancer

Article Title: Complement activation promoted by the lectin pathway mediates C3aR-dependent sarcoma progression and immunosuppression

doi: 10.1038/s43018-021-00173-0

Figure Lengend Snippet: (a-d) Representative magnification images (20X) of immunostaining analysis for C1q (a), C4d (b), C3c (c) and C3aR (d) in UPS tissue sections. One experiment performed (n=19 patients), 10 representative fields have been acquired for each patient. Representative images for patients showing negative (0% IRA, left panel) or positive (>0% IRA, central and right panels) staining for C3aR expression (d). Scale bar: 100μm. (e-f) Kaplan-Meier survival curves representing the DFS (e) and the metastasis-free survival (f) for patients showing negative (n=5 patients) or positive (n=14 patients) staining for C3aR expression. Exact p value of Log-rank test for survival curves, Hazard ratio (HR) and confidence intervals (CI) are indicated in the figures (e, f).

Article Snippet: The following primary antibodies and final concentrations were used: anti-mouse C3aR (Hycult Biotech, clone 14D4, 1 μg/ml), anti-mouse C3b/iC3b/C3c (Hycult Biotech, clone 2/11, 5 μg/ml), anti-CD31 (Millipore, clone 2H8, 1 μg/ml), anti-CD45 (Abcam, rabbit polyclonal, 5 μg/ml), anti-Iba-1 (FUJIFILM Wako Pure Chemical Corporation, rabbit polyclonal, 2 μg/ml), and anti-NG2 (Millipore, rabbit polyclonal, 5 μg/ml).

Techniques: Activation Assay, Expressing, Immunostaining, Staining