1. Volume 160, Issue 4, Pages 700-714 (February 2015)
PTX3 Is an Extrinsic Oncosuppressor Regulating Complement-Dependent Inflammation in Cancer 
Eduardo Bonavita, Stefania Gentile, Marcello Rubino, Virginia Maina, Roberto Papait, Paolo Kunderfranco, Carolina Greco, Francesca Feruglio, Martina Molgora, Ilaria Laface, Silvia Tartari, Andrea Doni, Fabio Pasqualini, Elisa Barbati, Gianluca Basso, Maria Rosaria Galdiero, Manuela Nebuloni, Massimo Roncalli, Piergiuseppe Colombo, Luigi Laghi, John D. Lambris, Sébastien Jaillon, Cecilia Garlanda, Alberto Mantovani 
Cell 
Volume 160, Issue 4, Pages (February 2015)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 PTX3 Deficiency Increases Susceptibility to Carcinogenesis
(A and B) Incidence (A) and tumor volume (B) of 3-MCA-induced sarcoma in Ptx3−/− and Ptx3+/+ mice. In (B), the volume of each tumor is shown. One experiment with 20 mice/group out of ten performed with similar results is shown.
(C) Effect of recombinant PTX3 (9 μg/day, s.c. by osmotic pump) administered from day 0 to 50,–100, or from 0–100 on tumor incidence.
(D and E) Incidence (D) and number of lesions (mean ± SEM) (E) of DMBA/TPA-induced skin papillomas in Ptx3−/− and Ptx3+/+ mice. One experiment with ten mice/group out of five performed with similar results is shown.
(F) Histological classification of lesions developed in Ptx3−/− and Ptx3+/+ mice. ∗∗p < 0.01, Chi-square test. Two pooled experiments.
(A–E) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < paired Student's t test. See also Figure S1.
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4. Figure 2
Analysis of the Cellular Source of PTX3 and of Its Role in 3-MCA-Induced Carcinogenesis
(A) Systemic (upper panel) and local (lower panel) PTX3 production at different time points upon vehicle or 3-MCA injection in wild-type and Il-1r1−/− mice (mean ± SEM). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired Student’s t test.
(B) Immunohistochemical analysis of PTX3 expression in 3-MCA-induced sarcoma. Arrows show vessels (upper panel), infiltrating leukocytes (central panel), and interstitial stroma (lower panel). Arrowheads show tumor cells (upper and central panels).
(C) Immunofluorescence analysis of PTX3 expression in blood vessels (CD31+ cells), macrophages (F4/80+ cells), and neutrophils (Ly6G+ cells). Scale bar represents 150 μm.
(D) 3-MCA-induced sarcoma incidence in PTX3-chimeric mice (Donor → Recipient). ∗∗p < 0.01, paired Student’s t test.
See also Figure S2.
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5. Figure 3 PTX3 Deficiency Is Associated to Increased CRI
(A) Histological and immunohistochemical analysis of macrophage (CD68+) and neutrophil (Ly6G+) infiltration in Ptx3−/− and Ptx3+/+ 3-MCA-injection site at 7 days.
(B) MPO levels in 3-MCA-injection site at 7 days (mean ± SEM). See also Table S1.
(C) Analysis of the leukocyte infiltrate in 3-MCA-sarcoma by FACS. Total leukocytes (CD45+), macrophages (F4/80+/CD11b+), monocytes (CD11b+/Ly6C+), neutrophils (CD11b+/Ly6G+), and lymphocytes (CD3+) were analyzed (mean ± SEM).
(D–F) Analysis of macrophages (F4/80+ immunoreactive area, IRA), angiogenesis (CD31+ IRA), and lymphocytes (CD3+ cells) in 3-MCA-sarcoma by immunohistochemistry (each dot represents the average of ten fields, mean ± SEM).
(G) Analysis by FACS of the macrophage infiltrate (mean ± SEM) in 3-MCA-sarcoma of Ptx3−/− and Ptx3+/+ mice, after treatment with recombinant PTX3 (from day 0 to 100). ∗p < 0.05, ∗∗p < 0.01, unpaired Student’s t test.
See also Figure S3.
Cell  , DOI: ( /j.cell )
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6. Figure 4
PTX3 Deficiency Is Associated to Increased Complement Deposition in 3-MCA-Sarcoma
(A–C) Analysis by confocal microscopy of C3 deposition in the injection site (∗) at 7 days (A) or in tumors (B and C) of Ptx3−/− and Ptx3+/+ mice, after treatment with recombinant PTX3 (from day 0 to 100) (C). Correlation analysis between C3 and PTX3 expression was performed in wild-type tumors (B).
(D) Analysis by ELISA of C5a concentrations, (mean ± SEM, two pooled experiments), in 3-MCA-tumors.
(E and F) Analysis by confocal microscopy of C5b-9 (E) and Factor H (F) in 3-MCA-tumors. Correlation analysis between Factor H and PTX3 expression was performed in wild-type tumors.
(G) Incidence of 3-MCA-sarcoma in C3−/− and/or Ptx3−/− and Ptx3+/+ mice (n = 10–15).
(H) Analysis of the macrophage (F4/80+/CD11b+) infiltrate in 3-MCA-sarcoma by FACS in C3−/− and/or Ptx3−/− and Ptx3+/+ mice.
(I) 3-MCA-sarcoma incidence in Ptx3−/− and Ptx3+/+ mice treated with the C5aR antagonist PMX53 (n = 7).
(J) Immunofluorescence analysis of C3 deposition on Ptx3+/+ and Ptx3−/− cells, in the presence of recombinant PTX3, SRC19-20, or their combination. Arrows indicate faint C3 immunostaining in Ptx3+/+ cells in basal conditions or after addition of PTX3 in the assay.
(K) Quantification of C3 deposition evaluated by immunofluorescence. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired Student’s t test (A–F, H, and K), paired Student’s t test (G and I). For (A), (B), (C), (E), (F), and (K), each dot represents the average of mean fluorescence intensity of ten regions of interest (ROI) per tumor (A–C, E, and F) or well (K) (mean ± SEM). Scale bars represent 150 μm.
See also Figure S4.
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7. Figure 5
Role of CCL2 in Carcinogenesis and TAM Polarization in PTX3-Deficient Mice
(A) Incidence of 3-MCA-sarcoma in Ptx3−/− and Ptx3+/+ mice treated with anti-CCL2, or irrelevant mAb (n = 9–11) (100 μg/mouse three times/week) for the duration of the experiment. ∗∗∗p < 0.001, paired Student’s t test.
(B–D) Analysis by RT-PCR of selected M1 and M2 markers of macrophages (B and C) and monocytes (D) infiltrating 3-MCA-sarcoma in Ptx3−/− and Ptx3+/+ mice (mean ± SEM). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired Student’s t test. In (B), (C), and (D), data were relative to 18S expression and normalized versus the mean of wild-type and expressed as mean ± SEM.
See also Table S2.
Cell  , DOI: ( /j.cell )
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8. Figure 6
PTX3 Deficiency Is Associated to Increased Genetic Instability
(A and B) Number of Trp53 (A) and Kras (B) mutations (mean ± SEM) in cell lines isolated from Ptx3−/− and Ptx3+/+ tumors. See also Table S3.
(C) Analysis of the expression of p53-regulated genes by RT-PCR in cell lines isolated from Ptx3−/− and Ptx3+/+ tumors. Data were relative to GAPDH expression and represented as mean ± SEM.
(D) Immunohistochemical analysis of 8-OH-dG 15 days after 3-MCA injection in Ptx3+/+ and Ptx3−/− mice treated with AG. Left: data are reported as mean number ± SEM of 8-OH-dG+ nuclei per field. Right: representative immunofluorescence images are shown.
(E and F) Immunofluorescence analysis of γ-H2Ax (E) and 53BP1 (F) 15 days after 3-MCA injection in Ptx3+/+ and Ptx3−/− mice treated with AG (E). Left panels: data are reported as MFI of γ-H2Ax (E) and 53BP1 (F) positive foci per field and expressed as (mean ± SEM). Right panels: representative immunofluorescence images are shown.
(G) Sarcoma incidence in Ptx3+/+ and Ptx3−/− mice treated with AG in the drinking water for the entire duration of the experiment. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired (A-F) or paired (G) Student’s t test.
Cell  , DOI: ( /j.cell )
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9. Figure 7 The PTX3 Gene Is Epigenetically Modified in Human Cancers
(A) Analysis by methylated CpG island recovery assay (MIRA) of the percentage of methylation enrichment of the promoter region and of the PTX3 CpG island in human mesenchymal and epithelial tumors. CpG rich regions (iCGs) (P1, P2, transcription factor binding site [TFBS], and CpG island [iCG1-9]) and surrounding regions analyzed by MIRA are shown below the structure of PTX3 gene. Healthy epithelial tissues were from the normal counterpart of colon cancer patients (n = 3) and skin squamous carcinoma (n = 5); healthy mesenchymal tissues were from leiomyosarcoma (n = 1), angiosarcoma (n = 3), chordoma (n = 3), and desmoid tumor (n = 2) patients.
(B) Analysis by MIRA of the percentage of methylation enrichment of the same regions in healthy colon epithelium, high grade adenomas, CRC, and healthy counterparts for each CRC stage.
(C) Analysis by MIRA of the percentage of methylation enrichment of the same regions in one normal colon epithelial cell line (HcoEpic) and two CRC cell lines (RKO and HCT116) treated or not with the inhibitor of DNA methylation 5-AZA-dC.
(D) PTX3 mRNA expression and protein production by RKO and HCT116 cell lines treated or not with 5-AZA-dC in the presence of TNFα 20 ng/mL or IL-1β 20 ng/mL. Healthy colon: primary human intestinal epithelial crypt cells isolated from surgical biopsies.
(E) Analysis by ChIP of NF-κB, c-Fos, and c-Jun binding to the promoter, TFBS, and an irrelevant region in RKO cell line treated or not with 5-AZA-dC in the presence of TNFα 20 ng/ml. Data are expressed as percentage of enrichment relatively to input DNA normalized on a positive control and represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired Student’s t test.
See also Figure S5.
Cell  , DOI: ( /j.cell )
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10. Figure S1
Effect of Recombinant PTX3 on Tumor Growth, Related to Figure 1
(A) Effect of recombinant PTX3 (9 μg/day sc by osmotic pump) administered from day 0 to 50, 50 to 100 or from 0 to 100 on 3-MCA-induced sarcoma incidence in PTX3-competent mice.
(B) Effect of recombinant PTX3 on cell growth in vitro. Proliferation index: mean percentage of MTT optical density (570 nm) normalized on day 0. Left panel: HUVEC, right panel: 3-MCA sarcoma cell lines. Data are mean ± SEM of 2 experiments for HUVEC and one experiment with 6 different PTX3-competent sarcoma cell lines. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, paired Student’s t test.
Cell  , DOI: ( /j.cell )
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11. Figure S2
Analysis of the Cellular Source of PTX3 in 3-MCA-Induced Carcinogenesis, Related to Figure 2
(A) Confocal analysis of PTX3 expression in macrophages (F4/80+ cells) at the injection site and liver at different time points. Data are expressed as mean fluorescence intensity of ten regions of interest (ROI) per mouse (n = 3) analyzed. Bar: 150 μm.
(B) expression of PTX3 and γ-H2Ax (left) and their colocalization (right) at the injection site at different time points.
(C) PTX3 production by ten PTX3-competent sarcoma cell lines in basal and in the presence of inflammatory signals.
(D) PTX3 mRNA expression by monocytes, MHCIIhigh and MHCIIlow macrophages sorted from tumors, and sarcoma dell lines. Bone marrow derived macrophages (BMDM) treated or not with LPS 100 ng/mL for 4h were used as control.
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12. Figure S3
PTX3 Deficiency Is Associated to Increased CRI, Related to Figure 3
(A) Analysis of the leukocyte infiltrate in 3-MCA-sarcoma by FACS at day 100. Macrophages (F4/80+ CD11b+), neutrophils (CD11b+ Ly6G+) and lymphocytes (CD3+) were analyzed (mean ± SEM).
(B) Analysis of angiogenesis by immunohistochemistry for CD31 in mice treated with recombinant PTX3 (from day 0 to 100). ∗p < 0.05, ∗∗p < 0.01, unpaired Student’s t test.
(C) 3-MCA-induced sarcoma incidence in P-selectin- and PTX3/P-selectin-deficient mice. ∗∗∗p < 0.001, paired Student’s t test.
Cell  , DOI: ( /j.cell )
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13. Figure S4
PTX3 Deficiency Is Associated to Increased Complement Deposition in 3-MCA-Sarcomas, Related to Figure 4
(A) Deposition of C4BP in Ptx3+/+ and Ptx3−/− tumor tissues: Analysis by confocal microscopy of C4BP deposition in tumors of PTX3-deficient and PTX3-competent mice (each dot represents the average of mean fluorescent intensity from ten fields and data are expressed as mean ± SEM). Correlation analysis between C4BP and PTX3 expression was performed in wild-type tumors.
(B) Number of lesions (mean ± SEM) of DMBA/TPA-induced skin papillomas in C3- deficient or competent mice. ∗∗p < 0.01, paired Student’s t test. One experiment with 10 mice/group is shown.
(C) Analysis of circulating total IC (left panel) and C3-containing IC (right panel) in healthy and tumor-bearing mice. High salt solution was used to dissociate Complement-IC binding.
(D) ELISA assay for PTX3, Ig, C1q and C3 associated to IC.
(E) mRNA and protein expression analysis in BMDM stimulated with IC from Ptx3+/+ and Ptx3−/− healthy or tumor-bearing mice.
Cell  , DOI: ( /j.cell )
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14. Figure S5
Epigenetic Modifications of Human PTX3 in CRC Cell Lines, Related to Figure 7
(A) Analysis by MIRA of the percentage of methylation enrichment of the promoter region and the PTX3 CpG island in 2 CRC cell lines (HT29 and SW480) treated or not with 5-AZA-dC.
(B–E) PTX3 mRNA or protein expression by SW480, HT29, SW620, HUVEC or HcoEpic cells treated or not with 5-AZA-dC in the presence of TNFα or IL-1β. mRNA data were normalized on GAPDH. Data are expressed and represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired Student’s t test.
(F) Analysis of histone modifications (H3K27ac, H3K4me3, H3K9ac and H3K27me3) in the PTX3 promoter by ChIP in RKO cells in basal conditions and after treatment with TNFα and 5-AZA-dC. Data are expressed as percentage of enrichment relatively to input DNA and presented as mean ± SEM.
Cell  , DOI: ( /j.cell )
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