Fig. 2

Knockdown of CXCR4 abrogates trastuzumab resistance. CXCR4 was silenced by specific shRNA in HCC1419 cells (“Materials and methods” section). The puromycin-resistant stable colonies were pooled together and named shCXCR4. A pool of cells infected with the lentivirus containing a non-effective vector (shRNA-NE) was selected and used as the control. A Western blot analysis was used to confirm the reduction in CXCR4 expression. B Illustration of co-cultures. C. BCAFs and HMFs were cultured in Dulbecco modified Eagle medium/nutrient mixture/F-12 supplemented with 10% FBS for 72 h. The culture supernatant was collected and tested for SDF-1α using ELISA following the manufacturer’s instructions. The medium used for the cell culture was used as the negative control. The data were analyzed with one-way ANOVA. D, E CXCR4-knockdown cells or non-silent control cells were co-cultured with BCAFs (D) or with BCAFs and PBMCs (E) in 3D, followed by treatment with trastuzumab as illustrated in B. At the endpoint of the study, relative cell viability was quantitatively analyzed using CellTiter-Glo 3D viability assay kit. The data were analyzed with one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001 compared with the non-silent control cells). F, G CXCR4-knockdown cells or non-silent control cells were used for trastuzumab-induced antibody-dependent cellular cytotoxicity (detail in “Materials and methods” section). The cells were stained with propidium iodide and analyzed by flow cytometry (F). Data were analyzed using t-test analysis of variance and are reported as the mean ± SD of triplicates (G)