Figure 6

Synergy and antagonism between anti-HER2 antibodies and tumor necrosis factor-α. Anti-Her2 antibodies and TNF-α acted synergistically in SK-BR-3 cells (top), but antagonistically in BT-474 cells (bottom). Growth effects observed under antibody (white columns), TNF-α (striped columns), and antibody + 1000 TNF-α (black columns) were obtained as follows. Cells were plated onto 96-well plates and incubated at 37°C with 5% CO₂. After 2 h of pre-incubation, antibodies (5 μg/ml) were added, and cells were incubated for 6 days to study the effects of antibodies alone (white columns). The effects TNF-α (1000 U/ml) alone (striped columns) were studied by adding this cytokine into cell culture medium after 4 h of pre-incubation. For combined treatments, antibodies (5 μg/ml) and TNF-α (1000 U/ml) were added after 2 h and 4 h of pre-incubations, respectively (black columns). Cells amount was determined by Sulforhodamine B assay after 6 days of treatment. Growth level under a drug condition was defined as the growth under that condition normalized by growth under no drug condition. Expected growth level under no interaction (gray columns) was calculated by multiplying the growth levels under each individual drug. The observed growth level for each combination was divided by the expected growth level to find an interaction score according to Bliss Independence Model for drug interactions (displayed under each antibody label). The combination of TNF-α with isotype control antibody (IgG1) or Trastuzumab (Tzm) provided scores near 1, meaning no interaction or independence in both cell lines. New anti-HER2 antibodies provided scores less than 0.57 in SK-BR-3 cells, meaning synergy. In contrast their interaction scores were more than 1.40 in BT-474 cells, meaning antagonism.