Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The immunotoxin targeting PRLR increases tamoxifen sensitivity and enhances the efficacy of chemotherapy in breast cancer

doi: 10.1186/s13046-024-03099-4

Figure Lengend Snippet: Analyzing bioactivity of monoclonal anti-PRLR antibodies. ( A ) Binding of mAbs on recombinant PRLR was determined by ELISA. The ELISA plate was pre-coated with recombinant PRLR protein. Serial diluted mAbs were added to the wells as primary antibodies. ( B ) Binding of mAbs on T47D was determined by flowcytometry. Indicated mAbs were added to the cells as primary antibodies. After that, antibodies bound on cell membranes were detected by anti-human Fc-APC antibody. ( C ) Flowcytometry was used to determine internalization of PRLR-targeting mAbs. The cells were cultured with indicated antibody on ice for 60 min to saturate the cell membranes with antibody. Subsequently, cells were transferred to 37℃ to start the internalization. Antibodies left on cell membranes under 37℃ were detected by anti-human-Fc-APC at 0 h and 1 h. Internalization (%) was calculated by [MFI (0 h) – MFI (1 h)]/MFI (0 h). ( D ) Western blot detecting p-ERK (T202/T204), ERK, p-ERα (Ser118), ERα, p-STAT3 (Y705), STAT3, p-STAT5 (Y694), STAT5 and β-actin (loading control) in T47D cells stimulated by PRL in the presence of indicated PRLR-targeting mAbs. Cells were starved in DMEM devoid of FBS for 24 h prior to activation of PRL for 15 min. ( E ) Cell viability of T47D cells was determined by CCK8 in the presence of PRL with indicated PRLR-targeting mAbs. Cells were cultured for 72 h before viability was tested. Viability of cells without any treatment was set as 100%. ( F ) Viability of T47D spheroid was determined by Celltiter-glo in the presence of PRL or N8 mAb. Left: the image of spheroids in indicated groups. Right: Curve of the viability of T47D spheroids to tamoxifen concentration. Viability of T47D spheroid treated without any treatment (PBS/IgG/0µM tamoxifen) was set as 100%. ( G ) Cell viability of MCF7-TAMR cells was determined when overexpression of PRL was induced (Left) or N8 mAb was present (Right). Cell viability of MCF7-TAMR cells without any treatment (0µM tamoxifen/0µg/ml N8) was set as 100%

Article Snippet: Then, the tumor sections or microarrays were blocked with 3% bovine serum albumin in PBS for 30 min and incubated with anti-human PRLR rabbit antibody (ab170935, Abcam, England) or anti-ki67 rabbit antibody (ab15580, Abcam, England) at 4 °C for overnight.

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Activation Assay, Concentration Assay, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The immunotoxin targeting PRLR increases tamoxifen sensitivity and enhances the efficacy of chemotherapy in breast cancer

doi: 10.1186/s13046-024-03099-4

Figure Lengend Snippet: N8-PE24 combined with tamoxifen efficiently inhibited tamoxifen-resistant breast cancer cell growth. ( A ) Cell viability of MCF7 (A left) or T47D (A right) was analyzed after treatment of N8-PE24 with or without tamoxifen. Viability of cells treated without any reagents (0µM tamoxifen and 0 µg/ml N8-PE24) was set as 100%. ( B ) Cell viability of MCF7-TAMR and T47D-TAMR was analyzed after treatment of N8-PE24 with or without tamoxifen. Viability of cells treated without any reagents (0µM tamoxifen and 0 µg/ml N8-PE24) was set as 100%. ( C ) Cell viability of MCF7 or MCF7-TAMR was analyzed after treatment of serial-diluted tamoxifen with or without N8-PE24. Viability of MCF7 or MCF7-TAMR treated without any reagents (0µM tamoxifen and 0 µg/ml N8-PE24) was set as 100%. ( D ) Dosage and treatment schedule of MCF7-TAMR xenograft model treated with N8-PE24 combined with tamoxifen. Female SPF grade NOD/SCID mice aged 6 weeks were implanted s.c with ten million cells on day 0. When the tumor reached a volume of 100 mm 3 , treatment began. ( E ) Tumor growth curve of MCF7-TAMR xenografts treated with indicated drugs. ( F ) Tumor image of MCF7-TAMR xenografts treated with indicated drugs. Tumor volume was calculated as (long diameter (mm) × short diameter × short diameter (mm))/2. ( G ) IHC analysis of Ki67 and PRLR of MCF7-TAMR xenografts in different groups

Article Snippet: Then, the tumor sections or microarrays were blocked with 3% bovine serum albumin in PBS for 30 min and incubated with anti-human PRLR rabbit antibody (ab170935, Abcam, England) or anti-ki67 rabbit antibody (ab15580, Abcam, England) at 4 °C for overnight.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The immunotoxin targeting PRLR increases tamoxifen sensitivity and enhances the efficacy of chemotherapy in breast cancer

doi: 10.1186/s13046-024-03099-4

Figure Lengend Snippet: N8-PE24 combined with tamoxifen or paclitaxel could inhibit 231-PRLR breast cancer xenograft. ( A ) PRLR IHC analysis of tumor and adjacent normal tissues from a TNBC patient. ( B ) Flowcytometry analysis of PRLR on MDA-MB-231 cells treated with or without tamoxifen. 2.5µM tamoxifen was added to cells for 2 days before analysis. Anti-PRLR-APC antibody was used for staining. ( C ) Flowcytometry analysis of PRLR on 231-PRLR cells. Isotype-APC (red) and anti-PRLR-APC antibody (blue) were used for staining. ( D ) Western blot determining PRLR protein level in 231-PRLR cells when tamoxifen was present or not. 2.5µM tamoxifen was added to cells for 2 days before cell were collected and lysed. Cell lysates were then probed by anti-PRLR antibody, ( E ) Evaluation of cell viability by CCK8 assay to determine inhibition effect of N8-PE24 when tamoxifen was present or not on 231-PRLR BC. Viability of 231-PRLR cells treated without any reagents (0µM tamoxifen and 0 µg/ml N8-PE24) was set as 100%. ( F ) Dosage and treatment schedule for 231-PRLR xenograft model. Female SPF grade Balb/c nude mice aged 6 weeks were implanted s.c with five million cells on day 0. When the tumor reached a volume of 100 mm 3 , treatment began. ( G ) Tumor growth curve of 231-PRLR xenografts treated with indicated drugs on nude mice. Tumor volume was calculated as (long diameter (mm) × short diameter (mm) × short diameter (mm))/2. ( H ) Tumor image of 231-PRLR xenografts treated with indicated drugs. ( I ) IHC analysis of Ki67 and PRLR of 231-PRLR xenografts in different groups

Article Snippet: Then, the tumor sections or microarrays were blocked with 3% bovine serum albumin in PBS for 30 min and incubated with anti-human PRLR rabbit antibody (ab170935, Abcam, England) or anti-ki67 rabbit antibody (ab15580, Abcam, England) at 4 °C for overnight.

Techniques: Staining, Western Blot, CCK-8 Assay, Inhibition