Search

Objective: To establish a bioluminescent MDA-MB-231 cell line which can stably express luciferase and green fluorescent protein to allow bioluminescent imaging in nude mouse models bearing human triple-negative breast cancer xenografts., Methods: The lentivirus carrying luc2, eGFP and neo fusion genes were packaged in 293T cells via calcium phosphate co-precipitation. Human triple-negative breast cancer cell line MDA-MB-231 was infected by the lentivirus, and the positive cell clones were tested for eGFP and luc2 expressions by fluorescence microscopy and Xenogen IVIS200 bioluminescent imaging system, respectively. MTT assay, transwell invasion assay and wound healing assay were performed to evaluate the changes in the proliferation, invasion and migration abilities of the infected cells. The cells were then orthotopically implanted into the right second mammary fat pat of female BALB/c nude mice. The tumor growth was monitored by the in vivo imaging system every week, and the tumor tissues were harvested to evaluate the in vivo stability and tumorigenicity of the modified cells using cryosection and HE staining., Results: The lentivirus-infected MDA-MB-231cells could stably express luc2 and eGFP, and the luciferase activity reached 9689 phontons/s/per cell. No significant changes occurred in the biological activities of the lentivirus-infected MDA-MB-231 cells. We successfully established the nude mouse model bearing orthotopically implanted human triple-negative breast cancer cells., Conclusion: The modified MDA-MB-231 cell line can be detected sensitively at the primary implantation site and distant metastasis site in nude mice, which provides a convenient and sensitive platform for the research of metastatic mechanism and new antitumor drugs of human triple-negative breast cancer. The combination of eGFP and luc2 is superior to single reporter gene.

Objective: To investigate the role of vascular endothelial growth factor (VEGF) in regulating triple-negative breast cancer (TNBC) stem cells and the possible pathways involved in this regulatory mechanism., Methods: The Oncomine database, UALCAN database and Human Protein Atlas (HPA) database were used to analyze the expression of VEGF in breast cancer and its association with the molecular subtypes and prognosis of breast cancer. Sphere formation assay was carried out to examine the effects of hVEGF 165 on sphere formation ability of TNBC MDA-MB-231 cell line; Western blotting and RT-qPCR were performed to detect the expression of the tumor stem cell markers including CD44, c-Myc, Nanog, and ALDH1 and the activation of the related pathways., Results: Data from the online databases all showed a significant increase of VEGF expression in breast cancer tissues than in the adjacent tissues ( P < 0.0001), and its expression level was associated with the molecular subtypes of breast cancer. Specifically, the expression of VEGF was markedly higher in TNBC than in other subtypes of breast cancer. Survival analysis showed that breast cancer patients with a high VEGF expression had a significantly shortened overall survival ( P < 0.0001). In the cell experiments, the sphere formation ability of MDA-MB-231 cells was significantly enhanced after treatment with hVEGF 165 ( P =0.0029). Compared with the monolayer cells, MDA-MB-231 spheres showed significantly increased expressions of VEGF, NRP-1, CD44, Nanog and c-Myc. Treatment with hVEGF 165 resulted in significant time-dependent up-regulation of the expressions of CD44, c-Myc, Nanog and ALDH1 and down-regulation of CD24 expression in the cells. The results of Western blotting demonstrated that treatment with hVEGF 165 caused significant activation of the ERK/MAPK pathway in MDA-MB-231 cells., Conclusion: VEGF promotes cancer stemness of triple-negative breast cancer possibly through the ERK/MAPK pathway.

Objective: To study the effect of death decoy receptor 3 on the prognosis of breast cancer and the invasive function of breast cancer cells in vitro . Methods: Expression of DcR3 were assessed qualitatively by Q-PCR to analyze the correlation in 115 mammary tissue samples with a 10-year median follow-up. The expression of DcR3 was examined in MCF7 and MDA-MB-231 cell lines using immunocytochemical staining and RT-PCR. DcR3 knock-down cell sub-lines were constructed. The effects of reduced DcR3 expression were observed by establishing invasion and migration models. Results: Patients were divided into the good prognosis group ( n= 81) and the poor prognosis group ( n= 26). The expression of DcR3 in the poor prognosis group (133 350+49 646 copies/50 ng RNA)was significantly higher than that in the good prognosis group (5 393+1 428 copies/50 ng RNA, P= 0.020). DcR3 transcripts were found to be increased significantly in grade 2 cancers compared to well differentiated grade 1(82 844±34 068 copies/50 ng RNA, n= 39,) vs (5 371±3 500 copies/50 ng RNA, n= 20, P= 0.029).The DcR3 gene of MCF7 cell line and MDA-MB-231 cell line were successfully knocked out and verified that DcR3 knockout. And the invasion and migration of MCF7 cells were inhibited ( P= 0.009, P= 0.001). However, no significant difference was found in these two aspects of the MDA-MB-231 cell line ( P= 0.475, P= 0.102). Conclusion: DcR3 promotes the capacity of invasion of breast cancer cells and plays an important role in the metastasis of breast cancer. DcR3 detection is helpful to the judgment about prognosis of breast cancer.

Objective: To study if programmed death-ligand 1 (PL-L1) expression in breast cancer cell activates PD-L1/PD-1 pathway in dendritic cells to inhibit dendritic cell maturation., Methods: Human monocytes were induced to differentiate into immature dendritic cells using GM-CSF and IL-4, and further to mature dendritic cells using TNF-α. PD-L1-expressing breast cancer cell line MDA-MB-231 was co-cultured in contact with the dendritic cells to observe the effects of the breast cancer cells on the maturation of the dendritic cells. A PD-L1 blocking antibody was applied to the co-culture, and the changes in the inhibitory effect of the MDA-MB-231 cells on dendritic cell maturation was observed. TNF-α-induced dendritic cells were treated with a recombinant human PD-L1 protein to study the effect of PD-L1/PD-1 pathway activation on the maturation of dendritic cells. The expression of PD-L1 in MDA-MB-231 cells and the dendritic cell maturation marker HLA-DR and CD83 were analyzed using flow cytometry., Results: MDA-MB-231 cell line showed PD-L1 positivity on the cell membrane cells at a rate as high as (99.7∓0.15)%. In mature dendritic cells, the positivity rates for HLA-DR and CD83 were (88.8∓6.96)% and (18.36∓3.07)%, respectively, but in the co-culture system, the positivity rates of the dendritic cells were significantly decreased to (42.76∓10.52)% (P<0.01) and (9.93∓2.74)% (P<0.05), respectively, indicating that MDA-MB-231 cells inhibited the maturation of dendritic cells. Following treatment with a PD-L1 antibody isotype control, the percentages of HLA-DR- and CD83-positive cells in the co-culture were (45.17∓10.19)% and (10.15∓2.54)%, which were significantly increased to (63.46∓1.72)% and (16.46∓2.58)% after treatment with PD-L1 antibody, respectively (both P<0.05). Compared with the mature dendritic cell controls, the cells treated with the recombinant human PD-L1 protein exhibited significantly lowered percentages of HLA-DR-positive [from (84.23∓4.18)% to (2.56∓2.39)%, P<0.05] and CD83-positive cells [(87.26∓1.54)% to (60.67∓1.63)%, P<0.05]., Conclusion: The effect of PD-L1 antibody therapy on triple negative breast cancer can be partially mediated by blocking PD-L1 expression on breast cancer cell membrane, which attenuates the inhibition of dendritic cell maturation in the cancer microenvironment.