Trastuzumab-DM1 causes tumour growth inhibition by mitotic catastrophe in trastuzumab-resistant breast cancer cells in vivo

The human breast cancer cell line EFM-192A was obtained from the German Resource Center for Biological Material and the cell lines BT-474, MDA-361, MDA-453, MCF-7, SK-BR-3, UACC-812, UACC-893 and ZR-75-30 were obtained from the American Type Tissue Culture Collection. The JIMT-1 cell line has been established in the laboratory of Cancer Biology, University of Tampere, Finland [35] (also available via German Resource Center for Biological Material). The cell lines were cultured according to recommended specifications.

Trastuzumab (Herceptin^®) and rituximab (Mabthera^®) were purchased from Roche Ltd. (Basel, Switzerland). Trastuzumab-DM1(T-DM1) was provided by Genentech Inc. (South San Francisco, CA, USA) through a Materials Transfer Agreement. Mouse M30 CytoDeath antibody was obtained from Roche Ltd., HercepTest staining kit was purchased from DakoCytomation (Carpinteria, CA, USA), rabbit monoclonal antibody against human HER2 (clone SP3) was obtained from NeoMarkers/Lab Vision (Fremont, CA, USA).

The effects of trastuzumab and T-DM1 on cell growth was examined by the AlamarBlue method (Invitrogen, Carlsbad, CA, USA). The cells were trypsinised and plated in 96-well, flat-bottomed, tissue culture plates. The effects of trastuzumab and T-DM1 were tested at a concentration of 0.001, 0.01, 0.1, 1, and 10 μg/ml. An MCF-7 HER2 negative breast cancer cell line with low trastuzumab binding capacity was used as a negative control. The number of viable cells was tested at 72 hours after drug exposure by adding the AlamarBlue reagent. Fluorescence was measured with excitation at 544 nm and emission at 590 nm using a Wallac Victor2 plate reader (Perkin-Elmer, Turku, Finland). Fluorescence values of samples were normalised with values of the cell culture media without cells. The results presented as the proportion of viable cells were calculated by dividing the fluorescence values of drug treated samples by the fluorescence values of untreated control samples.

ADCC was analysed by measuring the lactate dehydrogenase (LDH) released from the cancer cells as a result of ADCC activity of peripheral blood mononuclear cells (PBMC). PBMCs were separated from the heparinized blood of a single healthy donor by Ficoll density gradient centrifugation (Histopaque-1077, Sigma-Aldrich, St. Louis, MO. USA). Cancer cells (target; 5,000 to 10,000 per well) and PBMCs (effector) were co-incubated at 1:5, 1:10, 1:20, 1:40 and 1:80 target:effector ratios in 100 μL DMEM containing 5% FCS in a 96-well U-bottomed plate in quadruplicate for six hours at 37°C with trastuzumab, T-DM1 or negative control antibody, rituximab (20 μg/ml). ADCC was measured in a six-hour LDH release assay (CytoTox Non-Radioactive Cytotoxicity Assay, Promega Corporation, Madison, WI, USA). The absorbance at 490 nm was recorded by using a microplate reader (Model 680XR, Bio-Rad, Hercules, CA, USA). The negative control sample (target spontaneous) was prepared identically, contained trastuzumab or T-DM1 or rituximab, but did not contain PBMCs; effector spontaneous sample contained no target cells. Tumour cells killed by freezing at -80°C for one hour then warming up to 37°C served as positive control (target maximum). The percentage of cells killed was calculated according to the following formula: (experimental - effector spontaneous - target spontaneous)/(target maximum - target spontaneous) * 100.

The blood donor had given informed consent before for obtaining a peripheral venous blood sample for PBMC assays. These experiments were done according to the rules of the Ethical Committee of University Hospital of Tampere.

Five- to eight-week-old female SCID mice (Harlan Netherlands, Horst, Netherlands) were given a single subcutaneous injection of 5 × 10⁶ JIMT-1 cells suspended in 100 μl cell culture medium (DMEM supplemented with 7.5% FBS). Rituximab (5 mg/kg) and trastuzumab (5 mg/kg) were given intraperitoneally (i.p.) once per week, weekly T-DM1 (5 or 15 mg/kg) was given intravenously (i.v.) as it has been shown to be an effective regimen by Lewis Phillips et al. [25]. Lapatinib powder was formulated prior to dosing every day in a vehicle of 0.5% hydroxypropyl methyl cellulose and 0.1% Tween 80 and administered orally as a suspension at a dose of 100 mg/kg for 34 days on a daily schedule [36]. Tumour growth was measured with a caliper and tumour volume was calculated using the formula T_vol = π/6 × larger diameter × (smaller diameter)². Animals were euthanized by CO₂ inhalation. The experiments were done with the approval by the National Animal Experiment Board.

Samples of xenograft tumours were fixed in 4% buffered formaldehyde for 24 h, processed into paraffin, then sectioned at 5 μm. Sections were deparaffinized and stained with hematoxylin and eosin (H&E). For immunohistochemistry, tissue sections were deparaffinised followed by antigen-retrieval in Tris-EDTA buffer (0.01 M pH 9.0) at high temperature (water bath, 30 minutes at 98°C). After blocking for non-specific binding, primary antibodies (see above) were applied at optimized concentrations and incubated (30 minutes at room temperature). Standard peroxidase-polymer kit (PowerVision+ poly-HRP IHC Detection Systems, Leica Biosystems Newcastle Ltd., Newcastle, UK) was used for visualisation, with diaminobenzidine as the chromogen (Vector Laboratories Inc., Burlingame, USA). Slide scanning was done using Aperio ScanScope XT at superresolution (40×).

Cells with normal and aberrant mitotic shape were defined morphologically as described earlier [37], giant multinucleated cells (GMC) were defined as cells with more than three nuclei. Cells with normal and aberrant mitotic shape and GMCs were counted in hematoxylin and eosin stained histological sections. Apoptotic cells were counted as positive cells using CytoDeath antibody immunohistochemistry [38]. Cells with normal and aberrant mitotic shape, GMCs and apoptotic cells were counted on a minimum of 10 randomly selected × 40 high-power fields containing representative sections of tumour. Data are presented as the average of positive cells ± SD/field.

Data are expressed as the mean ± SE. The statistical significance of the differences between means were determined using Student's t test for two samples after verifying that data passed the normality test and the groups compared have equal variance. Unpaired groups were compared with the Mann-Whitney U test. Differences were statistically significant at P < 0.05.

We studied a panel of nine HER2 overexpressing breast cancer cell lines, which have been previously determined as sensitive to both trastuzumab and lapatinib (ZR-75-30, BT-474, EFM-192A, SKBR-3 UACC-812), sensitive to trastuzumab and resistant to lapatinib (MDA-361), or resistant to both trastuzumab and lapatinib (JIMT-1, UACC-893, MDA-453) [39].

After 72 h incubation, T-DM1 significantly inhibited the growth of all HER2 positive cell lines compared to trastuzumab (P < 0.05). T-DM1 produced a more pronounced effect on trastuzumab- and lapatinib-sensitive cell lines (13 to 43% of surviving cells), but inhibitory effect was seen on MDA-361 cells (50 ± 8%) as well as on cell lines which are resistant to both trastuzumab and lapatinib (MDA-453, 23 ± 2%; UACC-893, 56 ± 4%; JIMT-1, 76 ± 4%). Interestingly, the trastuzumab- and lapatinib-resistant MDA-453 cell line was the second most sensitive to T-DM1. No inhibition was seen in HER2 negative control cell line (MCF-7) (Figure 1A.). T-DM1 inhibited the growth of HER2 positive breast cancer cells in a dose-dependent manner (Figure 1B.). Concentrations of T-DM1 higher than 1 μg/ml inhibited also the growth of the HER2 negative control cell line (MCF-7), suggesting a non-specific toxic effect (data not shown).

Next we correlated the effects of trastuzumab and T-DM1 to the trastuzumab-binding capacity of each cell line [40]. No significant correlations were found (R = 0.43, P = 0.19 and R = 0.58, P = 0.06, respectively) (Figure 2A, B). No significant correlation was found between the responsiveness to trastuzumab and T-DM1 (R = 0.6, P = 0.07, Figure 2C).

Since it was previously shown that antibody-dependent cellular cytotoxicity (ADCC) has a key role in the in vivo effect of trastuzumab [10], we compared trastuzumab and T-DM1 in in vitro ADCC assay. Trastuzumab and T-DM1 evoked ADCC similarly on SKBR-3 and JIMT-1 breast cancer cells with dose-dependent cell death reaching approximately 70 to 85% killing using an effector/target ratio of 80:1. Therefore, conjugation with DM1 does seem not to alter the ability of trastuzumab to mediate ADCC (Figure 1C.).

The effects of T-DM1 were next studied in vivo using JIMT-1 xenograft models. Tumours were formed in all SCID mice inoculated with JIMT-1 cell suspension in 11 days (n = 18, mean tumour volume 64 ± 9 mm³). Thereafter, weekly treatments with trastuzumab and T-DM1 were started on Day 12 and continued until the end of the experiment. Rituximab was used as a negative control agent. While trastuzumab had no inhibitory effect on tumour growth, a partial but significant tumour growth inhibition was seen by T-DM1 from days 32 to 44 (P < 0.05) (Figure 3.).

In the next experiment, T-DM1 treatment was started at the time of JIMT-1 cell suspension inoculation to see its effect on tumour formation. The tumours in T-DM1 treated mice remained very small until Day 55 (16 ± 12 mm³). Thereafter, the tumours started to grow in four out of six mice but remained non-palpable in two (2/6). Residual cancer cells were found histologically in one of these two mice, and a complete cure was suggested in the other. Overall, the effect of T-DM1 on tumour growth was much stronger when compared to trastuzumab or lapatinib (P < 0.05) (Figure 4.). In this setting we also tested lapatinib, which had no inhibitory effect on tumour growth in comparison to the control (Figure 4.).

T-DM1 was tested also on mice treated first with trastuzumab since tumour cell inoculation (Day 0). As also shown before [11], trastuzumab slowed down the formation of tumours between days 6 and 34 (P < 0.05), probably via ADCC. For half of the mice trastuzumab was discontinued and switched to T-DM1 from Day 41 onwards. In this setting, T-DM1 was unable to inhibit tumour growth (Figure 4.).

Histological sections of the xenograft tumours were prepared and stained with hematoxylin and eosin. Enumeration of mitoses with normal morphology revealed no differences between trastuzumab and T-DM1 treated JIMT-1 xenografts (Figures 5A and 6A). In contrast, we detected a significantly higher number of cells with aberrant mitotic morphology in T-DM1-treated tumours (P < 0.05) (Figures 5B and 6B). In line with this observation, the number of giant multinucleated cells (GMCs) was increased in T-DM1 treated samples (P < 0.05) (Figures 5C and 6B, D). Aberrant mitosis and GMCs are hallmarks of mitotic catastrophe. We observed a higher number of cells with aberrant mitotic morphology and higher number of GMCs also in the tumour samples whose trastuzumab-treatment were changed to T-DM1 (P < 0.05) (Figure 5B, C.).

Apoptotic cells were detected using CytoDeath staining, which localizes a caspase-activated breakdown product of cytokeratin subtype 18. In this analysis we found significantly increased numbers of apoptotic cells in the T-DM1 treated samples in comparison to the trastuzumab treated ones (P < 0.05) (Figures 5D and 6E, F). We detected higher numbers of apoptotic cells also in the samples whose trastuzumab-treatment were changed to T-DM1 (P < 0.05) (Figure 5D.). It is also noteworthy that most of the cells with aberrant mitotic morphology were CytoDeath negative (Figure 6F).

Immunohistochemical staining localizing intracellular and extracellular epitopes of the HER2 protein was performed (using HercepTest and SP3 antibodies). No major qualitative changes in the cell membrane staining of HER2 expression were observed. Virtually all tumour cells showed strongly positive cell membrane staining reactions for both intracellular and extracellular epitopes (data not shown). However, it is noteworthy that staining positive intracytoplasmic granules were seen only in T-DM1 treated tumours. Cells undergoing mitotic catastrophe were strongly positive for cell membrane HER2 protein (Figure 6C, D).