Trastuzumab and docetaxel in a preclinical organotypic breast cancer model using tissue slices from mammary fat pad: Translational relevance

With the ever-increasing number of drugs approved to treat cancers, selection of the optimal treatment regimen for an individual patient is challenging. Breast cancer complexity requires novel predictive methods and tools. In the present study, we set up experimental conditions to obtain an 'ex vivo' organotypic culture from xenotransplanted mice aiming at recapitulating the human clinical condition. The effect of trastuzumab (large biological molecule) and docetaxel (small chemical entity) was subsequently investigated on this organotypic model and compared with in vivo and in vitro activity on tumor cells. Tissue slices of 200 µm were obtained from mammary fat pad of SCID mice xenotransplanted with human MCF-7 breast cancer cells. Viability and proliferation were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and Ki-67 immunohistochemistry,and apoptosis by cleaved caspase-3 immunohistochemistry. In vivo antitumor activity of trastuzumab and docetaxel was determined by caliper measurement of tumor volume and Ki-67 expression on explanted masses by immunohistochemistry. A Teflon support and normoxia were necessary experimental conditions to obtain high viability of excised breast cancer infiltrated mammary fat pad slices upon 48 h cultivation, as shown by MTT proliferation assay, and Ki-67 expression. Breast cancer tissue slices treated for 48 h with trastuzumab or docetaxel showed a significant dose-dependent reduction of viability by MTT assay. Consistently, both drugs down-modulated Ki-67 and increased cleaved caspase-3. Tumor masses collected from docetaxel-or trastuzumab-treated mice showed a similar reduction of proliferation markers. By contrast, MCF-7 cell cultures were significantly inhibited by docetaxel but not by trastuzumab. Tumor tissue slices represent a more predictive experimental cancer model compared to cell cultures for both small and large molecule antitumor efficacy. This observation supports the relevance of microenvironment in the overall tumor biology and response to therapeutics.


Introduction
Breast cancer is the most common type of cancer in the world with more than 1.3 million patients and a mortality rate of ~450,000 deaths/year (1). The high incidence of disease is suggestive of slow progress in the prevention programs. In the treatment of women with localized disease, mortality rates have been improved, but the median survival time in the metastatic setting is only 25 months (2). Systemic chemotherapy as treatment of choice for most cancer types and its relatively modest improvement in survival associated with significant toxicity, highlighted the need over the past decade to develop targeted therapies. Targeted drugs can restore the deregulated signalling transduction pathway in which a mutated gene and its encoded protein is involved (3). Targeted therapy represents the major hope against cancer and a substantial step towards personalized medicine, since this type of drugs is mostly active in cancer cells without affecting normal healthy cells. However, the efficacy of these single signal transduction pathway inhibitors is in most cases modest, thus the progress against cancer is slow and it is translated into a few weeks of survival prolongation. Elucidation of the mechanisms beyond intrinsic and acquired tumor resistance to therapies is hampered by high inter-patient and intratumor heterogeneity (4)(5)(6)(7). Consequently, a patient-specific experimental approach is needed to analyze individual responses to therapies. Based on comprehensive gene expression profiling, breast tumors are classified into at least three major subtypes: luminal, human epidermal growth factor receptor 2 + (HER2 + ) and basal-like (8,9), with different risk factors for incidence, response to treatment, disease progression and preferential organ sites of metastases (10)(11)(12). Antitumor drugs, presently used in the clinical practice, have been previously characterized Trastuzumab and docetaxel in a preclinical organotypic breast cancer model using tissue slices from mammary fat pad: Translational relevance in the two-dimensional in vitro culture and in vivo xenograft tumor models. A broad analysis on investigational drugs, done at the National Cancer Institute, pointed out at a poor correlation between preclinical data from in vitro models and tumor xenografts and phase II efficacy data leading to the conclusion that only compounds that are successful in a large number of different models are likely to be effective in the clinic (13). Thus, frequent failures in drug development can be explained by the fact that the existing preclinical models do not represent the complexity (heterogeneity) that is typical of human tumors. We have, therefore, recently explored tissue slice technology via the Krumdieck tissue slicer, by preserving the tissues in the three-dimensional structure, it allows the setting up of a powerful and representative ex vivo tumor model. Moreover, taking into account the notion that cancer cell lines, passaged in vitro for years, may not reflect the biology of in vivo tumors, we compared the activity of docetaxel (small molecule) and trastuzumab (large molecule), both commonly used for breast cancer therapy, in cancer cell lines and organotypic tissue slices. Tumor explantation. Mice were sacrificed, tumors explanted and immediately cut using the Vibratome VT1200 (Leica, Germany) to obtain 200 µM thick slices of the whole tumors.

Materials
Tissue slice maintaining conditions. To set the best experimental conditions, tumor slices were maintained in floating normoxic conditions for a maximum of 4 days using a modular incubator chamber 37˚C in 6-multiwell plates, using Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, a mix of penicillin-streptomycin and 1% L-glutamine as culture medium. Medium change was performed every 24 h.
Immunohistochemistry. Immunochemistry analysis was performed by employing paraffin sections. Tissue slices of 200 µm were paraffin-embedded (horizontal orientation). The sections were incubated with primary antibodies overnight. After washing, secondary antibodies were applied at 1:200 dilutions for 30 min. Images were acquired using a Nikon microscope (Eclipse 80i, Nikon, Japan) with a Nikon digital camera (DXM1200F). H&E staining was performed to examine the extent of the slice integrity.
Drug treatment of slice cultures. Slices obtained from three mice were evaluated in triplicate for MTT assay 2 or 3 days after slicing. For treatment of slices, docetaxel (Sigma-Aldrich) and trastuzumab (Herceptin) were used. The concentrations were chosen on the basis of results obtained from tumor cell growth inhibition assay. Before testing docetaxel in its final concentrations of 200-20 nM, it was dissolved in dimethyl sulfoxide (DMSO) and diluted in culture medium. Trastuzumab was tested at the concentrations of 20-0.02 µg/ml. The incubation period for treatment was up to 2 days. At the end of the treatment, tissue slices were incubated with 5 mg/ml of MTT at 37˚C for 1 h, harvested and precipitated-salt extracted by incubation with 0.1 M HCl-isopropyl alcohol at room temperature for 40 min. Viability values were determined by dividing the optical density of the formazan at 570 nm by the dry weight of the explants.
Growth inhibition tumor cell assay. MTT assay was performed for evaluation of the cell viability within 3 days of culture. Tumor cells were seeded 3,000 cells/well. upon 24 h, tumor cells were treated with different concentrations of trastuzumab (200-20 µg/ml) and docetaxel (500-0.5 nM) for 3 days. At the end of treatment, the medium was discarded, cells were washed twice with PBS, and replaced by MTT-containing medium. The plates were incubated at 37˚C for 4 h. Then the MTT solution was discarded and without washing, DMSO was added to dissolve the formazan formed. After 15 min incubation, cell plates were transferred to the microplate reader (Victor, Wallac) and the absorbance at 570 nm was measured. The results were expressed as percent of drug-treated viable cells in comparison with vehicle-treated cells. The IC 50 values were calculated using four-parameter fit by ALLFIT program.
Statistical analysis. All data are expressed as the mean value ± standard error (SE) and differences between groups were analyzed using Mann-Whitney u test. Mean values are considered significantly different at P<0.05.

Results
In vitro and in vivo antitumor activity of docetaxel and trastuzumab. In order to evaluate the in vitro and in vivo antitumor activity of docetaxel and trastuzumab, standard human MCF-7 cell culture and a tumor xenograft study in nude mice were carried out. These tumor cells were first of all analysed for ERBB2 membrane expression by FACS analysis with 97.6% of tumor cells positive for ERBB2-conjugated antibody (R&S, FAB 11299) (data not shown). Tumor cell proliferation was tested by MTT assay after 3 days of culture showing a dose-dependent inhibitory effect of docetaxel but not trastuzumab, resulting in an IC 50 of 7±0.1 nM and >200 µg/ml, respectively ( Fig. 1A and B). Consistently with in vitro data, docetaxel strongly inhibited the growth of the MCF-7 xenografts (TVI=82%; P<0.01) whereas trastuzumab exhibited a lower but significant antitumor activity (TVI=28%; P<0.05) (Fig. 1C). Reduction of Ki-67 expression correlated with both docetaxel-dependent and trastuzumab-dependent antitumor activity (Fig. 1D). Ki-67 is an important marker predicting recurrence, prognosis and overall survival in breast cancer patients (14)(15)(16). It has also been associated with positive axillary lymph nodes in most studies (17,18). Recently, Ki-67 was integrated as a prognostic factor into molecular typing in prognosis of patients with luminal B breast cancer (19). Therefore, we investigated Ki-67 expression in further tumor slice experiments.
Tumor slice preparation. The set up of ex vivo organotypic cultures of human breast carcinoma was carried out before   condition ( Fig. 2A and B). Accordingly, significantly decreased expression of Ki-67 was found by immunohistochemistry in floating slices compared to Teflon-supported slices (Fig. 2C  and D).
These results indicated that an inert support is useful to keep slices viable and tumor cells in a proliferative status, thus indicating that tissue slices represent a physiologically more relevant model compared to tissue cultures to study solid cancer. Moreover, despite the lack of blood supply the viability of Teflon-supported 200 µm thick tissue slices, maintained in normoxia, suggests an adequate nutrient diffusion from the culture medium. A schematic representation of the procedure to obtain organotypic breast cancer tissue slices from MCF-7 mammary fat pad xenotransplant is shown in Fig. 3.

Evaluation of docetaxel and trastuzumab antitumor activity on breast cancer tissue slices.
Because substantial morphological integrity of tissues, defined as preservation of general architecture including epithelial structures and their spatial relationship to stroma, was observed in tumor tissue slices up to 2 days of culture (Fig. 2). MTT assay was performed to quantify viability. Data in Fig. 4A show similar number of viable cells after 1 or 2 days dropping to ~80% after 3 days of cultivation. Further experiments were then performed cultivating the tissue slices for 2 days in the presence of different concentrations of docetaxel or trastuzumab. MTT assay revealed a significant dose-dependent loss of viable cells upon treatment with both drugs (Mann-Whitney u test; ** P<0.01) (Fig. 4B). In agreement with this result, the percentage of proliferating cells assessed by Ki-67 immunostaining significantly decreased upon docetaxel or trastuzumab exposure (Fig. 4C), paralleled by increased percentage of cleaved caspase-3 positive cells (Fig. 4D). In particular, biomarkers of proliferation and apoptosis used to quantify effects of trastuzumab and docetaxel were evaluated by counting numbers of positively stained cells (Fig. 5A-F). Taken together, these data show that tissue slices can better reflect the in vivo experimental outcome.

Discussion
In the present study we report a method to obtain organotypic breast cancer tissue slices useful to evaluate the efficacy of both small and a large molecule drugs. The advantage of the tissue slice cultures is that the complex cross-talk between matrix and cells as well as the cellular heterogeneity and susceptibility to drugs is preserved avoiding potential source of artefacts deriving from isolation of tumor cells from their biological environment (20,21). Moreover, the presence of ECM provides important differentiation cues, for example, by signalling via Toll-like receptors and integrins (22)(23)(24), thus further supporting the use of tissue slice method for screening of therapeutics. Organotypic tissue slice cultures offer a more attractive option compared to cell line cultures or subcutaneous xenografts both lacking the complexity of a tumor nested in a mammary gland (25)(26)(27). Tissue slice samples with a thickness between 400-800 µm, cultured 24 h were previously used to evaluate the transcriptional effects of 1,25(OH) 2 D 3 in breast cancer benefitting from the heterogeneous combination of epithelial and stromal cells that secrete a variety factors affecting the overall response of tumor cells to the vitamin (28). Tissue slices of 250 µm from human primary invasive ductal breast tumors were also employed to test tamoxifen and doxorubicin activity, confirming the complexity and heterogeneity of breast cancer among patients (29). Primary tissue slices of 400 µm were cultured in the presence of rapamycin, showing that such a culture method preserves the tumor AKT/mTOR pathway activity (30). Moreover, slice cultures of 350 µm from head and neck squamous cell carcinoma from patients were also used to test cisplatin, docetaxel and cetuximab allowing the design of personalized therapies and investigation of mechanisms of tumor resistance (31,32). It is becoming increasingly evident that the development of cancer and the response to anti-cancer drugs not only depend on genetic alterations but also on specific interactions between tumor cells and surrounding tissue components. In invasive breast carcinoma, differentiated myoepithelial cells and intact basement membranes are lost and tumor cells are in direct contact with an activated collagenous tumor stroma (27). To simulate such conditions either three-dimensional tissue cultures using several biomatrices or co-culture experiments with tumor fibroblasts have been performed. However, these systems cannot mimic the complex tissue architecture and the high degree of variability seen in individual tumors. Therefore, to evaluate the activity of docetaxel and trastuzumab antitumor drugs we used tissue slice cultures starting from orthotopic human breast cancer xenografts in mice and compared results with in vivo efficacy. Our data support the use of the patient's tissue slices as predictive translational model for personalized therapies.
So far, neither in vitro nor ex vivo testing technologies have gained a significant impact in predicting clinical efficacy of therapeutic treatments. Present data can be framed within a widespread effort to identify testing conditions useful to increase the success rate of drug investigation, thus facilitating translational medicine.
Here we provide evidence that breast cancer tissue slice is a more useful model than cell cultures to predict antitumor efficacy of both small and large molecules. Sensitivity of breast tumors to anticancer drugs depends upon dynamic interactions between epithelial tumor cells and their microenvironment including stromal cells and extracellular matrix. Moreover, in addition to cancer cell viability, this model improves the understanding of the impact of a given treatment on stromal and endothelial cells and the distribution of a biologic, a small molecule or viral vectors within tumor microenvironment. Another potential application may include the ex vivo addition of immune cells for OncoImmunology studies.
In conclusion, tissue slices represent an optimal tool to investigate therapeutics and further studies to evaluate this model for novel investigational drugs are warranted.