Fig 2: The effects of pentraxin 3 (PTX3) antibodies on triple‐negative breast cancer (TNBC) cells. (A) PTX3 antibodies (Ab‐10 or Ab‐49) inhibit the interaction between PTX3 and CD44. The abilities of PTX3‐ and PTX3 pre‐incubated with Ab‐10 or Ab‐49 to bind immobilised CD44 were assessed using a competitive binding assay. (B) Epitope identification by ELISA using the indicated PTX3 antibodies and immobilised PTX3 or various shortened PTX3 peptides; the PTX3 amino acids 200–236 (RI37), 320–359 (GI40), 209–217 (AD9) and 352–360 (GR9) were examined for recognition by Ab‐10 or Ab‐49. (C) The suppressive effect of PTX3 antibodies on PTX3‐activated signalling pathways in MDA‐MB‐231 cells. MDA‐MB‐231 cell lysates were incubated with isotype antibody (IgG1κ), Ab‐10 or Ab‐49 and harvested for immunoblotting with the indicated antibodies. (D) Quantitative analysis of the protein activity of p‐AKT, p‐p65 and p‐ERK1/2 in MDA‐MB‐231 cells. (E) The suppressive effect of PTX3 antibodies on PTX3‐activated gene transcription in MDA‐MB‐231 cells. The total RNA of MDA‐MB‐231 cells that were incubated with IgG1κ, Ab‐10 or Ab‐49 was harvested for real‐time reverse transcription polymerase chain reaction (RT‐PCR). (F) The migration and invasion of IgG1κ‐, Ab‐10‐ and Ab‐49‐treated MDA‐MB‐231 cells with or without PTX3 treatment were assessed by Transwell assay. (G) An in vitro sphere formation assay was performed with IgG1κ‐, Ab‐10‐ and Ab‐49‐treated MDA‐MB‐231 cells with or without PTX3 treatment. (H) IgG1κ, PTX3 antibody (Ab‐10) and paclitaxel (Taxol) in the indicated groups of orthotopically allografted 4T1‐Luc2‐bearing mice by intraperitoneal injection. (I) The effects of IgG1κ, Ab‐10 and Taxol on the growth of 4T1‐Luc2 tumours in BALB/c mice were measured using external calipers. (J) Representative in vivo bioluminescence images, (K) metastasis quantification of 4T1‐Luc2 tumours in lung and brain and (L) Kaplan–Meier survival curves after administering IgG1κ, PTX3 antibody (Ab‐10) or paclitaxel (Taxol) in the indicated groups of 4T1‐Luc2‐bearing mice. All data are expressed as the mean ± SEM. Differences among groups were analysed using one‐way ANOVA followed by Tukey's multiple comparison test. *p < .05, **p < .01, ***p < .001, ns: no significance

Fig 3: Pentraxin 3 (PTX3) interacts with CD44 and regulates metastatic and stemness gene expression through the ERK1/2, AKT and NF‐κB pathways in MDA‐MB‐231 cells. (A) MDA‐MB‐231 cells were pre‐infected with control (shCtl) or shCD44#1 lentivirus and then treated with PTX3 without serum in the medium. Lysates from the experimental cells were harvested for microwestern array analysis. The quantitative results of the microwestern array analysis were obtained and are presented as the ratio of protein levels in PTX3‐treated shCD44 MDA‐MB‐231 cells/PTX3‐treated shCtl MDA‐MB‐231 cells. Expression of the indicated proteins was normalised to the average of α‐tubulin first and then individually themselves, and the increased or decreased protein levels are represented as red or green, respectively. (B) MDA‐MB‐231 cells were pre‐infected with lentivirus bearing shCtl or shCD44#1 expression vector and were then treated with PTX3 for 30 min. Lysates from the experimental cells were harvested for Western blot analysis, and specific antibodies were applied, as indicated. Relative protein expression was normalised to α‐tubulin. (C) MDA‐MB‐231 cells were pre‐infected with lentivirus bearing shCtl or shCD44#1 vector and were then treated with PTX3 for 24 h. (D) MDA‐MB‐231 cells were pre‐treated with or without wortmannin (100 nM), BAY 11‐7085 (2 μM) or PD98059 (10 μM) for 1 h and were then treated in the presence or absence of PTX3 for 24 h. mRNA levels of RANKL, MMP2, TWIST2 and NANOG were measured by real‐time reverse transcription polymerase chain reaction (RT‐PCR). GAPDH was used as an internal control. (E) The indicated signalling inhibitors were pre‐treated for 1 h and then treated in the presence or absence of PTX3 for 24 h. Total cell lysates were prepared and immunoblotted for p‐AKT, p‐p65 and p‐ERK1/2 and were normalised to the respective non‐phosphorylated protein levels. (F) A highly simplified diagram of the PTX3/CD44 signalling pathway. All data are expressed as the mean ± SEM. Differences among groups were analysed using one‐way ANOVA followed by Tukey's multiple comparison test. *p < .05, **p < .01, ***p < .001

Fig 4: A combination of pentraxin 3 (PTX3) peptide and paclitaxel enhances the suppressive effect on tumour growth and metastasis in a triple‐negative breast cancer (TNBC) mouse model. (A and B) The activity of various biotinylated truncated PTX3 peptides to bind immobilised CD44 was determined using ELISA. (C and D) The migration of various biotinylated truncated PTX3 peptide‐treated luciferase‐expressing 4T1 cells (4T1‐Luc2) with or without PTX3 treatment was assessed using the Transwell assay. 4T1‐Luc2 cells were assessed for migration ability by measuring luciferase activity. Protein remodelling predicted the interaction of (E) AD9 and (F) GR9 with the CD44 dimer. (G) The stability of various biotinylated and PEGylated retro‐inverso narrowed down PTX3 peptides AD9 (P2rdAD9) or GR9 (P12rdGR9) was assessed by incubation with serum, and their stability was analysed at the indicated times by ELISA. Peptide levels were calculated relative to the quantities determined at time point zero. (H) The CD44 binding activity of P2rdAD9 or P12rdGR9 was assessed by incubation with CD44, and their interaction was analysed by ELISA. (I) The effect of P2rdAD9 or P12rdGR9 on the migration of PTX3‐treated 4T1‐Luc2 cells was assessed using a Transwell assay, as indicated. (J) The effect of P2rdAD9 or P12rdGR9 on the cytotoxicity of anticancer drug‐treated 4T1 cells was assessed using 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Paclitaxel (Taxol), doxorubicin (Dox) and cisplatin (CDDP). (K) An experimental scheme for evaluating orthotopically allografted 4T1‐Luc2 tumours in BALB/c mice. Once tumours reached an average volume of 50 mm3, the mice were administered P2rdAD9 or P12rdGR9, with or without Taxol. (L and M) The effect of P2rdAD9, P12rdGR9 or Taxol on the growth of 4T1‐Luc2 tumours in BALB/c mice was measured using external calipers. All data are expressed as the mean ± SEM. Differences among groups were analysed using one‐way ANOVA followed by Tukey's multiple comparison test or unpaired two‐tailed t‐test. *p < .05, **p < .01, ***p < .001, ns: no significance