Breast tumor cells isolated from in vitro resistance to trastuzumab remain sensitive to trastuzumab anti-tumor effects in vivo and to ADCC killing

Abstract

An understanding of model systems of trastuzumab (Herceptin) resistance is of great importance since the humanized monoclonal antibody is now used as first line therapy with paclitaxel in patients with metastatic Her2 overexpressing breast cancer, and the majority of their tumors has innate resistance or develops acquired resistance to the treatment. Previously, we selected trastuzumab-resistant clonal cell lines in vitro from trastuzumab-sensitive parental BT-474 cells and showed that cloned trastuzumab-resistant cell lines maintain similar levels of the extracellular Her2 receptor, bind trastuzumab as efficiently as the parental cells, but continue to grow in the presence of trastuzumab and display cell cycle profiles and growth rates comparable to parental cells grown in the absence of trastuzumab (Kute et al. in Cytometry A 57:86-93, 2004). We now show that trastuzumab-resistant and trastuzumab-sensitive cells both surprisingly display trastuzumab-mediated growth inhibition in athymic nude mice. This demonstrates that resistance developed in vitro is not predictive of resistance in vivo. The observation that in vitro resistant cells are sensitive to trastuzumab in vivo could be explained by antibody dependent cellular cytotoxicity (ADCC). Therefore, both parental and trastuzumab-resistant cells were assayed for ADCC in real time on electroplates with and without trastuzumab in the presence of a natural killer cell line (NK-92), and granulocyte or mononuclear cellular fractions isolated from human peripheral blood. Mononuclear cells and NK-92 cells were more effective in killing both parental and trastuzumab-resistant cells in the presence of trastuzumab. Both trastuzumab-resistant cells and trastuzumab-sensitive cells showed similar susceptibility to ADCC despite displaying divergent growth responses to trastuzumab. The granulocyte fraction was able to kill these cells with equal efficacy in the presence or absence of trastuzumab. These results support a model of trastuzumab tumor cell killing in vivo mediated primarily by ADCC from the mononuclear fraction of innate immune cells and suggest that in the clinical setting not only should changes in signaling transduction pathways be studied in acquired tumor resistance to trastuzumab, but also mechanisms by which tumors impede immune function should be evaluated.

Fig. 1

HER2 is maintained at similar levels in parental BT-474 cells and in a trastuzumab resistant clonal cell line. Flow Cytometry expression of HER2 on BT-474 cells and on a BT-474 trastuzumab resistant clone. Panel a–c BT-474 cells treated with no primary antibody (a), 10 μg/ml of trastuzumab (b) or 10 μg/ml of pertuzumab (c). a, b, c were then treated with goat anti human IGG-PE labeled. Panel d–f BT-474 trastuzumab-resistant clone treated with no primary antibody (d), 10 μg/ml of trastuzumab (e) or 10 μg/ml of pertuzumab (f). d, e, f were then treated with goat anti human IGG-PE labeled

Fig. 2

Both parental BT-474 cells and in vitro selected trastuzumab resistant cells are growth inhibited in nude mice treated with trastuzumab. Growth of BT-474 cells and BT-474 trastuzumab resistant clone in athymic nude mice. Animals were given a subcutaneous injection of either cell line. Tumors were allowed to grow until they reached approximately 50–60 mm³. Animals were divided equally based on size into treated or non-treated groups. On day 21, the animals were treated with an i.p. injection (arrows) of trastuzumab at 10 mg/kg. The animals were given subsequent trastuzumab treatments (10 mg/kg) every 7–10 days (arrows). Tumor growth was followed for an additional 45 days. Open inverted triangle indicates BT-474 no trastuzumab treatment, open circle indicates BT-474 Resistant Clone no trastuzumab, dark filled inverted triangle indicates BT-474 plus trastuzumab, dark filled circle indicates BT-474 Resistant Clone plus trastuzumab

Fig. 3

Trastuzumab growth inhibition curves for BT-474 cells and BT-474 trastuzumab-resistant clone under various conditions using the RT-CES system. BT-474 cells (panel a) or BT-474 trastuzumab-resistant clone (panel b) were plated onto a 96-well E-Plate and allowed to grow for 48 h. Cells were grown in media alone (black) or were treated with trastuzumab (see arrow) at 10 μg/ml (red), 1 μg/ml (blue), or 0.1 μg/ml (green). BT-474 cells (panel c) or BT-474 trastuzumab-resistant clone (panel d) were grown similar to panel a and b but were treated with 0.1 μg/ml trastuzumab (red), NK-92 cells at a 1:1 effector to target ratio (green), or trastuzumab (0.1 μg/ml) and NK-92 cells at an effector to target ratio of 1:1 (blue). Cell indices of triplicate wells were measured every 30 min. Quantitation of these results by measuring the change in area under the curves in relationship to the non-treated controls are presented in panel e and f for the sensitive and resistant cell lines, respectively

Fig. 4

Parental BT-474 and trastuzumab-resistant cells are killed by ADCC with near equal efficacy. ADCC killing curves using human white blood cells against BT-474 and BT-474 trastuzumab-resistant clone cells measured using the RT-CES system. BT474 cells (panel a) or BT474 trastuzumab-resistant clones (panel b) were plated onto a 96-well E-Plate and allowed to grow for 24 h prior to treatment. The cells were grown in media alone (black) or treated with isolated mononuclear cells at an effector to target ratio of 2:1 (blue, red) or granulocytes at an effector to target ratio of 1:3 (green, purple). Treatment with trastuzumab (0.1 μg/ml) alone (gray) or in combination of effector cells (blue = MNC) or (purple = PMN) is shown with dashed lines. Cell indices of triplicate wells were measured every 30 min

Fig. 5

Mononuclear cells and granulocytes isolated from 14 individuals show large variability of tratuzumab ADCC activity. Summary of ADCC killing by mononuclear cells or granulocytes from 14 individuals. BT-474 cells were added to a 96-well E-Plate and allowed to grow for 24–48 h. Blood collected from 14 different individuals was separated into mononuclear (MNC) or granulocyte (PMN) cell fractions. BT-474 cells were treated with media alone, trastuzumab (0.1 μg/ml), effector cells+/− trastuzumab. a MNC at 6:1 effector to target ratio without (open bar) or with trastuzumab (filled bar) (0.1 μg/ml). b MNC at 2:1 effector to target ratio without (open bar) or with trastuzumab (filled bar) (0.1 μg/ml). c PMN at 1:3 effector to target without (open bar) or with trastuzumab (filled bar) (0.1 μg/ml). The upper part of each figure gives the AUC difference due to the addition of trastuzumab (ADCC). The cell indices were monitored in triplicate and the average curves analyzed for relative area under the curve for cell killing activity as described in “Materials and methods”