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Additional file 4. Figure S4. Effect of AMD3100 on pathways. BTRT cells grown in 3D Matrigel culture were treated with vehicle, AMD3100, trastuzumab, or their combination. Cell lysates were subject to RPPA. A portion of the RPPA data were further analyzed with one-way ANOVA.

Additional file 8. Figure S8: Characteristic of the trastuzumab-resistant mouse model and effect of combined treatments on cell signaling. A five-week-old female athymic nude mice were implanted with 0.36 mg, 90-day release, 17beta-estradiol pellets. Three days later, in total 5x106 BT-T or HR6 cells in 150 μl growth factor reduced Matrigel and PBS were orthotopically injected. Once tumors reached a volume ~100 mm3, the mice were randomly grouped and received treatment with trastuzumab. Tumor sizes were measured with calipers twice weekly. Tumor volume was calculated with the formula V = lw2/2. Differences in tumor volume between groups were analyzed using two-way ANOVA. B HR-6 or BT-T tumor cells were grown in 3D Matrigel culture for 6 days, and cell lysates were analyzed with western blot. C HR6 cells were implanted into the mammary fat pad of female athymic nude mice. The mice with tumor burden were randomized to treatment with vehicle, trastuzumab, AMD3100, docetaxel, or combinations as indicated. The xenograft tumor lysates were analyzed with western blot.

Additional file 7. Figure S7: Effect of combination of AMD3100 and cisplatin or carboplatin on trastuzumab-resistant breast cancer cell growth. BTRT or SKRT cells were grown in 3D Matrigel culture and treated with AMD3100/cisplatin, or AMD3100/carboplatin. Photographs were taken on day 11. Quantitative analysis of the total acini area was performed using AlphaView SA, and the data were analyzed using one-way ANOVA (B, D, F, H: *, P < 0.0001 vs. Vehicle.

Additional file 1. Figure S1: Illustration of 3D co-culture. BTRT or SKRT cells were co-cultured with BCAFs in 96-well “U”-bottomed unattached plates. The spheres were treated with AMD3100 on day 3. For three line-co-culture, PBMCs were seeded on day 5, followed by treatment with trastuzumab. Cell viability was detected with CellTiterGlo.

Additional file 2. Figure S2. Effect of AMD3100 on cell growth in 3D co-culture. BTRT or SKRT were co-cultured with BCAFs in 96-well “U”-bottomed unattached plates and treated with AMD3100. The dynamic change of the spheres was monitored and photographed.

Additional file 3. Figure S3. BTRT cells grown in 3D Matrigel culture were treated with vehicle, AMD3100, trastuzumab, or their combination. SDF-1αwas added at the same time. Cell lysates were subject to RPPA

Additional file 5. Figure S5. Western blot. BTRT cells grown in 3D Matrigel culture were treated with vehicle, AMD3100, trastuzumab, or their combination. SDF-1α was added at the same time. A polypeptide that was designed as a CXCR4 antagonist but did not show biological function in the HER2+ breast cancer cells was used as a negative control.

Background: Although trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. We were the first to report the role of CXCR4 in trastuzumab resistance. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms. Methods: Immunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4expression. Three-dimensional co-culture (tumor cells/ breast cancer-associated fibroblasts / human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effect of CXCR4 inhibitor or trastuzumab. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms. Results: Using multiple cell lines and patient breast cancer samples we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated that the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using multiple trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel. Conclusions: Our findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.