Short term culture of breast cancer tissues to study the activity of the anticancer drug taxol in an intact tumor environment

Abstract

Background: Sensitivity of breast tumors to anticancer drugs depends upon dynamic interactions between epithelial tumor cells and their microenvironment including stromal cells and extracellular matrix. To study drug-sensitivity within different compartments of an individual tumor ex vivo, culture models directly established from fresh tumor tissues are absolutely essential.

Methods: We prepared 0.2 mm thick tissue slices from freshly excised tumor samples and cultivated them individually in the presence or absence of taxol for 4 days. To visualize viability, cell death, and expression of surface molecules in different compartments of non-fixed primary breast cancer tissues we established a method based on confocal imaging using mitochondria- and DNA-selective dyes and fluorescent-conjugated antibodies. Proliferation and apoptosis was assessed by immunohistochemistry in sections from paraffin-embedded slices. Overall viability was also analyzed in homogenized tissue slices by a combined ATP/DNA quantification assay.

Results: We obtained a mean of 49 tissue slices from 22 breast cancer specimens allowing a wide range of experiments in each individual tumor. In our culture system, cells remained viable and proliferated for at least 4 days within their tissue environment. Viability of tissue slices decreased significantly in the presence of taxol in a dose-dependent manner. A three-color fluorescence viability assay enabled a rapid and authentic estimation of cell viability in the different tumor compartments within non-fixed tissue slices.

Conclusion: We describe a tissue culture method combined with a novel read out system for both tissue cultivation and rapid assessment of drug efficacy together with the simultaneous identification of different cell types within non-fixed breast cancer tissues. This method has potential significance for studying tumor responses to anticancer drugs in the complex environment of a primary cancer tissue.

Figure 1

Procedure of slice preparation and tissue cultivation.

Figure 2

Principle of three and two-color fluorescence viability assay: Bcr-Abl positive BaF3 cells were cultivated in the presence of 1μM Imatinibfor 8 hours. (a) Following Imatinib treatment cells were incubated with TMRM (given color: red), SYTO^®63 (given color: blue), and Picogreen (given color: green) and visualized using a confocal microscope with a 63X objective. (b) Following Imatinib treatment cells were incubated in a PBS-BSA solution containing PI (given color: red) and SYTO^®63 (given color: blue) and visualized by confocal microscopy. (c) Comparison of cell death indices determined by Annexin V-FITC staining or TMRM/Picogreen staining in Imatinib treated cells by means of FACS analysis. (d) Comparison of cell death indices determined by PI/SYTO63 staining or TMRM/Picogreen staining. Cells were visualized by confocal microscopy with a 40× objective. 10 different areas with each at least 20 cells were counted. Values are means of the ratio PI⁺ cells or Picogreen⁺ cells to total cells ± SD from 10 different areas.

Figure 3

Viability of cultivated tissue slices: Tumor slices obtained from a breast carcinoma were individually cultivated in 1 ml medium for 24 hours. Slices were then analyzed for cell viability or cultivated for additional 72 hours or 144 hours as indicated. (a) Representative example of a tissue slice stained with Picogreen, SYTO^®63, and TMRM. (b) Hematoxylin and eosin (HE) staining of a section from a paraffin embedded tissue slice after a culture period of 96 hours in comparison to that observed in a section obtained from the same tumor directly after surgery ('original' tumor sample). (c) Cell viability after an ex vivo cultivation period of 24 and 96 hours determined by TMRM, SYTO^®63, and Picogreen labeling. (d) Quantification of cell death (number of visually counted Picogreen⁺ cells in relation to the number of total cells in 3 different areas) in non fixed tumor slices from 6 patients. Values reflect means ± SEM (n = 6). (e) Ratio of luminescence based quantification of ATP to DNA content of homogenates from slices cultivated for 24, 96, or 144 hours. (Data of a representative experiment shown).

Figure 4

Influence of Taxol treatment on cell viability: (a)Three-color fluorescence viability assay (top and middle) and hematoxylin/eosin staining (bottom) of slices treated with different taxol concentrations for 72 hours (images of a representative experiment). (b) The numbers of TMRM⁺, SYTO^®63⁺, and Picogreen⁺ cells in tumor slices from (a) were evaluated by counting of at least 50 cells from three different areas of different images. Viable cells were assessed as numbers of TMRM⁺ and SYTO^®63⁺ cells in relation to total cells (left panel). Dead cells were evaluated as numbers of Picogreen⁺ cells in relation to total cells (right panel). Values reflect means ± SEM. (c) Dose dependent effect of taxol treatment on cell viability determined by quantification of TMRM⁺, SYTO^®63⁺, and Picogreen⁺ cells in non fixed tumor slices (left and middle panel) and by evaluation of ATP/DNA ratio in slice homogenates (right panel). Ratios of controls were set to 100%, and the relative ratios of the taxol treated tissues were calculated. Data represent range, median and 25 to 75% percentile of a set of 10 experiments. Kruskal-Wallis test: p < 0.0001 for all three. (d) Effect of taxol treatment on proliferation and apoptosis. Formalin-fixed and paraffin-embedded slices were stained with anti-BrdU or anti-caspase 3 antibodies by conventional immunohistochemistry and counterstained with hematoxylin.

Figure 5

Identification of different cell types within tumor slices: (a) epithelial cells were identified using an anti-HEA-125 antibody recognizing an epithelial specific adhesion molecule (Ep-CAM). The morphology of epithelial cell clusters in slices cultivated for 96 hours (left panel) was compared to that observed in sections from paraffin embedded material from the same tumor prepared immediately after surgery labeled either with anti-HEA-125 (middle panel; counterstained with hematoxylin) or anti-cytokeratin 18 (right panel; counterstained with hematoxylin). (b) To visualize the vascular network tissue slices cultured for 96 hours were directly labeled using a PE-conjugated CD34 antibody (left panel) or a FITC-conjugated Ulex europaeus Agglutinin I (UEA-1; middle panel). The morphology of this network is comparable to that observed in paraffin embedded material obtained directly after surgery and stained with CD34 antibody (right panel, counterstained with hematoxylin). (c) Simultaneous staining of epithelial cells and determination of cell viability in slices treated with or without taxol for 72 hours. Epithelial cells were identified using a FITC-conjugated anti-HEA-125 antibody (green). Cell viability was determined using the two DNA selective dyes PI (red) and SYTO^®63 (blue).