Treatment of Herceptin-resistant tumor cells using EDC Herceptin conjugate Acquired resistance is one common mechanism by which tumor cells survive treatment and cause disease recurrence. For example, the majority of patients whose tumors overexpressed Her2 achieved an initial response to Herceptin but developed resistance within 1 year [18]. Bolin Liu’s group [19] has developed a method to program tumor cells to acquire Herceptin resistance by continuously culturing BT474 cells with a gradually increasing concentration of Herceptin for 4 months. BT474- HR20 (HR20) cells have been defined as a Herceptin-resistant cell line and grow well in the presence of 20 mg/mL Herceptin [19]. In treating parental tumor cells, we found that treatment of the Her2-overexpressing (Figure 3b) but Herceptin-resistant tumor cell line HR20 with a high concentration of EDC- Herceptin conjugate suppressed tumor cell viability, whereas treatment with wild-type Herceptin at the same concentration failed to affect HR20 cell growth (Figure 3a). As determined with Herceptin Conjugate Induces Tumor Cell Necrosis a fluorescence live/dead cell staining kit, more dead cells were detected in samples treated with the Herceptin conjugate than were detected in samples treated with the wild-type Herceptin (Figure 3c). Induction of tumor cell death but inhibition of tumor cell proliferation by EDC-Herceptin conjugate To determine whether the reduction in cell survival was due to reduced cell proliferation or to increased cell death, a BrdU flow cytometric assay was performed as a marker of cell proliferation, and a cell death staining kit was used to distinguish the dead cells from the live cells. Cell proliferation was not greatly different after EDC-Herceptin conjugate treatment (Figure 4a). As we observed in HR20 cells, the EDC-Herceptin conjugate induced a great amount of cell death in OS187 cells (Figure 4b). This led us to conclude that the decreased cell survival rate after EDC-Herceptin conjugate treatment was due to the induction of cell death rather than to the inhibition of cell proliferation. Figure 3. Induction of Herceptin-resistant tumor cell death by EDC-Herceptin conjugate. Herceptin-resistant tumor cells HR20 were treated with wild-type Herceptin or EDC conjugate at low concentration (20 mg/mL) or high concentration (80 mg/mL) for 72 h, and cell survival rates were analyzed using MTT assay. (a) Cell survival rate at the end of the described treatment. (b) Her2 expression level analyzed by flow cytometry as described in Figure 1. (c) Detection of cell death frequency following different treatments. Cells were treated with sham, wt low (wild-type Herceptin 20 mg/mL), EDC low (EDC-Herceptin conjugate 20 mg/mL), wt high (wild-type Herceptin 80 mg/mL), or EDC high (EDC-Herceptin conjugate 80 mg/ mL). Live and dead cells were analyzed using a live/dead cell staining kit (see detail in Methods). Green fluorescence stained cells, live cells; Red florescence stained cells, dead and dying cells. ***, P,0.001. doi:10.1371/journal.pone.0023270.g003 PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23270