Fig 1: The expression of PTX3 is suppressed in colon cancer cells due to a regulation of epigenetic silencing. A, B The mRNA expression and secreted protein levels of PTX3 in CCD-18Co human colon fibroblasts, and 7 human colon cancer cell lines. C, D The mRNA expression and secreted protein levels of PTX3 in mouse embryonic fibroblasts (MEFs) and the mouse colon cancer cell line MC38. The conditioned medium was harvested for ELISA assay (n = 3). E–H PTX3 mRNA and protein in CCD-18Co cells, MEFs, SW620 cells, and MC38 cells were measured by real-time PCR and western blot, respectively. The experimental cells were treated with IL-1β or TNF-α for 48 h. I–L PTX3 mRNA and protein in HCT116, HT29, SW620, and MC38 cells were measured by real-time PCR and western blot, respectively. The experimental cells were treated with 5-azacytidine for 4 days and stimulated with 20 ng/ml IL-1β or TNF-α for 24 h beginning on the 3rd day. GAPDH as internal control. p values were calculated by two-tailed unpaired Student’s t test or one-way ANOVA. ns represents nonsignificant difference. *represents a p value of < 0.05. **represents a p value of < 0.01. ***represents a p value of < 0.001. Values on the plots are presented as the means ± SEMs. Data are combined from 3 to 5 independent experiments
Fig 2: PTX3 promotes M2-like macrophage polarization. A, B The mRNA expression of M2 and M1 markers in THP-1 macrophages treated with or without 50 ng/ml PTX3. C The mRNA expression of M2 markers in PTX3-treated THP-1 macrophages with 250 ng/ml IgG1κ or WHC-001 (αPTX3Ab). D The mRNA expression of Arg1, Cd206, and Il1b in bone marrow-derived macrophages treated with or without 100 ng/ml PTX3. E Flow cytometric analysis of M1- and M2-like macrophages in BMDMs co-cultured with MEFs-shVoid, -shPtx3#915,or shPtx3#916 for 48 h. The CD45 + CD11b + F4/80 + cells were characterized by M1-like (CD86 + CD206−) and M2-like (CD206 + CD86−) macrophages through flow cytometric analysis. F The mRNA expression of Ptx3, Arg1, and Cd206 in tamoxifen-induced Ptx3fl/fl or Ptx3fl/fl; Ubc-Cre bone marrow-derived macrophages (M-CSF-primed M0 BMDMs) following stimulation with 20 ng/ml mIL-13 and 20 ng/ml mIL-4 (M2 polarization). G The mRNA expression of IFNG and TNFA in activated Jurkat cells (stimulated with PMA + ionomycin) incubated with conditioned medium from untreated THP-1 macrophages (Ctrl-CM) or PTX3-treated THP-1 macrophages (PTX3-CM). H Angiogenesis of HUVECs incubated in serum-free medium (No cell-CM), conditioned-medium from untreated THP-1 macrophages (Ctrl-CM) or conditioned medium from PTX3-treated THP-1 macrophages (PTX3-CM). The relative angiogenic ability was determined by the number of branch sites/nodes of tubes per field of view. I Western blot analysis of recombinant wild-type and N220A mutant PTX3 proteins treated with or without PNGase. J The mRNA expression of M2 markers in THP-1 macrophages treated with or without 50 ng/ml recombinant N220A mutant PTX3 protein. p values were calculated by two-tailed paired Student’s t test, one-way ANOVA, or two-way ANOVA. ns represents nonsignificant difference,*represents a p value of < 0.05, **represents a p value of < 0.01, and ***represents a p value of < 0.001. Values on the plots are presented as the means ± SEMs. Data are combined from 3 to 5 independent experiments
Fig 3: PTX3 contributes to M2 polarization through activation of CREB1 signaling. A RNA sequencing was performed in unstimulated THP-1 macrophages and PTX3-stimulated THP-1 macrophages. The top 200 upregulated and downregulated genes were selected for Connectivity Map analysis. Forward (overexpression) and reverse (knockdown) perturbation candidate genes were identified. B Western blot analysis of p-CREB1 and CREB1 in THP-1 macrophages treated with or without 50 ng/ml PTX3. C Western blot analysis of p-CREB1 and CREB1 in BMDM streated with or without 100 ng/ml PTX3. D The protein level and E mRNA level of CEBPB in THP-1 macrophages treated with or without 50 ng/ml PTX3. F, G The protein levels of p-CREB1, CREB1, and CEBPB in THP-1 macrophages treated with 100 ng/ml PTX3 plus either 500 ng/ml IgG1κ or WHC-001 (αPTX3Ab). H Western blot analysis of p-CREB1 and CREB1 in HEK293 cells transiently transfected with pcDNA3.1, CREB1-wt, or CREB1-S133A. I Illustration of the luciferase promoter constructs of CEBPB (− 890/ + 13), VEGF (− 1347/− 46), and ARG1 (− 919/ + 30). The CREB1 binding site is indicated in red and the CEBPB binding site is indicated in black. The promoter activity of the pGL3-CEBPB (− 890/ + 13), pGL3-VEGF (− 1347/− 46), and pGL3-ARG1 (− 919/ + 30) constructs in HEK293 cells cotransfected with pcDNA3.1, CREB1-wt, or CREB1-S133A is shown. J The promoter activity of pGL3-CEBPB (− 890/ + 13) in THP-1 macrophages treated with or without 50 ng/ml PTX3. K The promoter activity of pGL3-CEBPB (− 890/ + 13) in THP-1 macrophages cotransfected with CREB1-wt or CREB1-S133A following treatment with or without 50 ng/ml PTX3. L Schematic model showing that PTX3 contributes to stroma-mediated immunosuppression by promoting M2-like macrophage polarization through activation of the CEBPB/CREB1 axis in colon cancer. p values were calculated by two-tailed paired Student’s t test or one-way ANOVA. ns represents nonsignificant difference, *represents a p value of < 0.05, **represents a p value of < 0.01, and ***represents a p value of < 0.001. Values on the plots are presented as the means ± SEMs. Data are combined from 3 to 5 independent experiments
Fig 4: PTX3 expression is positively correlated with stromal and fibroblast signatures in colon cancer. A total of 287 colon adenocarcinoma samples represented in the TCGA dataset were divided into the PTX3high (n = 143) and PTX3low (n = 144) groups by the median cutoff value. A KEGG pathways related to cellular processes were selected for gene set enrichment analysis (GSEA), and the enriched signaling pathways (blue, inflammation-related signaling; and black, and stromal activation-related signaling) in PTX3high tumors are shown. The threshold criteria were p value of < 0.05 and FDR < 0.25. B GSEA of inflammatory response signature in the PTX3high (n = 143) and PTX3low (n = 144) groups. The threshold criteria were NES > 1.5, p value of < 0.05, and FDR < 0.25. C The stromal scores of 191 colon adenocarcinoma samples from the TCGA dataset were extracted via the ESTIMATE web tool and used for Pearson correlation analysis with PTX3 expression levels (log2). D, E Fibroblast scores in 283 colorectal tumor samples from the TCGA dataset were extracted from the xCell database. The samples were divided into the PTX3high (n = 142) and PTX3low (n = 141) expression groups or the fibroblasthigh (n = 142) and fibroblastlow (n = 141) score groups by the median cutoff value. F, G The survival index of 203 colorectal adenocarcinoma patients was obtained via the SurvExpress web-tool. The samples were divided into the fibroblasthigh (n = 114) and fibroblastlow (n = 89) group by the best cutoff value, and each of these groups was subdivided into a PTX3high group and a PTX3low group by the median cutoff value. H PTX3 expression and I fibroblast scores in different stages in the TCGA COAD dataset are shown. Spearman correlation analysis of PTX3 expression and the fibroblast score in stage I (J), stage II (K), stage III (L), and stage IV (M) tumors. p values were calculated by two-tailed unpaired Student's t test, one-way ANOVA, two-sided log-rank test, HR hazard ratio, or CI confidence interval. ns represents nonsignificant difference. ***represents a p value of < 0.001. Values on the plots are presented as the means ± SEMs
Fig 5: Inhibition of PTX3 reduces stroma-mediated tumor growth in vivo. Conditional knockout mice (Ptx3fl/fl or Ptx3fl/fl; Ubc-Cre) were given tamoxifen (2 mg/mice, i.p) to drive Cre expression and A colon tissues were harvested for genotyping by detecting the recombined fragment (Ptx3fl/fl, n = 2 and Ptx3fl/fl; Ubc-Cre, n = 5). B Conditional knockout mice were given tamoxifen (2 mg/mouse, i.p) daily for 5 days, and after another 5 days, were treated with LPS (0.3 mg/kg, i.p) for 6 h. Lung, liver, colon, and kidney tissues were harvested to measure the Ptx3 protein levels (Ptx3fl/fl, n = 1 and Ptx3fl/fl; Ubc-Cre, n = 1). C The experimental design for the in vivo tumor growth of MC38 by latter knockout of Ptx3 in conditional knockout mice (Ptx3fl/fl, n = 4 and Ptx3fl/fl; Ubc-Cre, n = 6). Tamoxifen was given 5 days after MC38 cell inoculation, and D the plasma Ptx3 level was measured and E tumor growth curves were generated. F, G Illustration and growth curves of subcutaneous tumors formed from 5 × 104 MC38 cells (n = 5), 1 × 105 MEFs (n = 3), or 5 × 104 MC38 cells mixed with 1 × 105 MEFs (n = 5) in C57BL/6 mice. H, I Intracellular and secretory Ptx3 protein levels in shVoid, shPtx3#915, and shPtx3#916 MEFs. Conditioned medium was harvested for ELISA assay (n = 3). J The growth curves of subcutaneous tumors formed from MC38 cells (n = 5) and MC38 cells mixed with MEFs-shVoid (n = 6), -shPtx3#915 (n = 6), or -shPtx3#916 (n = 6). K–M Illustration and growth curves of subcutaneous tumors in the MC38 cell/MEF tumor model. Intratumoral administration of IgG1κ (n = 4) or WHC-001 (αPTX3 Ab, n = 4) (100 μg) was performed every 3 days for a total of 4 treatments beginning when the tumor volume reached to 80 mm3. Tumor volume (mm3) = (L × W2)/2. p values were calculated by two-tailed unpaired Student’s t test or one-way ANOVA. *represents a p value of < 0.05. **represents a p value of < 0.01. ***represents a p value of < 0.01. Values on the plots are presented as the means ± SEMs
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