DNA Ploidy with Tissue Sections and Cadherins in HT Imaging
There is growing demand for obtaining information from tissue slices collected during research, clinical, or pathological studies. Information on DNA content per cell (ploidy) is of particular interest with regards to oncogenesis and analysis of tumor progression. An additional area of high interest concerns the degree of expression of cadherins, which are cellular adhesion molecules common downregulated in tumors; downregulation of cadherin increases mitotic potential of the cells and likely promotes metastasis (Haass et al., 2004; Cowin et al., 2005). While analyses of ploidy or protein expression are often performed on cells in suspension via flow cytometry, this is sometimes impractical for archived tissue samples. Furthermore, the information obtained with image cytometry maintains the context of each cell in the sample, providing information on the local tissue complexity, which can be very relevant to clinical analyses (Wohlrab et al., 2005). To address this critical need, Vala Sciences Inc, has added the ability to do ploidy and protein expression analysis of images derived from tissue sections to our CyteSeer® image analysis platform software, which can be used with any microscope or computer platform.
Methods
Tissue slices obtained from control and tumor samples were stained for cytoplasmic markers, utilizing a fluorescent red-conjugated secondary antibody, and for DNA utilizing DAPI. Images were obtained with an epi-fluorescence robotic microscopy workstation (Beckman IC100) equipped with a 40X objective. Images obtained with this system (*.bmp 8bit format) were then imported into CyteSeer for analysis, and Excel-compatible reports were exported from CyteSeer corresponding providing image segmentation and analysis for the cytoplasmic and nuclear channels, for each cell identified every field of view. The precise question we were addressing is whether or not the expression of the cytoplasmic marker, in each context, was associated with increased ploidy.
Figure 1
A tumor section stained for DAPI and tumor marker protein (cytoplasmic). Vala Sciences CyteSeer® software was utilized to quantify DNA content for cells that were stained vs unstained for the cytoplasmic marker.
Results
Of the 26 fields of view that were analyzed for the tumor histological slice, data was obtained for 5558 individual cells. The average cytoplasmic intensity for the red cytoplasmic tumor marker averaged 50 fluorescence units/pixel, with minimal and maximal values of 19 and 189, respectively (out of range of 1 to 255), indicating that the images were not saturated in the red channel. Cells displaying an ACI of 32 appeared essentially “unstained”, whereas cells with ACI>70 were brightly stained. Accordingly, gates were established so that ploidy was graphed for two cell populations, those with corresponding to 0<ACI<32 (unstained, the dimmest 18% of the cell population) vs. the cell population defined by 70<ACI<255 (highly stained, upper 14 % of the cell population). The ploidy histogram for the dim cells peaked at a TNI of approximately 120,000 fluorescence units (Figure 2). In contrast, the histogram for cells that stained brightly in the cytoplasmic channel was right-shifted compared to the dim cells. Proportionally fewer cytoplasmic bright cells displayed TNI below 120,000 (the left portion of Figure 2), and the peak TNI for the cytyoplasmic bright population was about 240,000 fluorescence units. There were proportionally greater cytoplasmic bright cells than dim cells, for every bin greater than 200,000. The results suggest that the cells that stained brightly for the cytoplasmic marker in the section obtained from the tumor have dramatically increased DNA content compared to the unstained cells.
Figure 2
Ploidy analysis of brightly stained vs unstained cells in a tumor section.
Figure 3
Ploidy analysis of brightly stained vs unstained cells in a sectin of control tissue. Unstained cells were cells displaying ACI<35, whereas bright cells displayed ACI>70.
For the analogous procedure carried out on control tissue, the ploidy histograms for dim vs brightly stained cells were much more overlapping. Figure 3 displays the data binned in exactly the same manner as Figure 2, with identical bin dimensions. The dim cells displayed a population peak for the TNI bin at a max of 40,000 fluorescence units. The brightly cytoplasmic stained cells were slightly right-shifted compared to the dim cells, in that the population peak occurred in the 80,000 TNI bin. However, neither the dim nor bright cells displayed significant fractions of the population with at bins greater than 200,000 fluorescence units. Overall, the TNI averaged143116 FU for the dim cells (n=732), vs 221242 for the bright cells (n=752, a 50% increase).
Figure 4
Ploidy analysis of control tissue with narrow bins.
It is possible that the staining for the control tissue slice was generally less bright in the DAPI channel than the tumor section for reasons unrelated to DNA content. This could be due to slightly different incubation times, or DAPI concentration, between the two staining procedures, for example. To address this possibility, we examined the population distribution utilizing narrower bins for TNI for the control tissue section (Figure 4). As shown, the bright cells were somewhat right-shifted, compared to the dim cells, however, the significance of this is unclear. Overall, the TNI averaged 91904 FU for the dim cells (n=1075 cells), vs 62200 for the bright cells (n=617). Thus, the DAPI channel fluorescence for the bright cells actually averaged 32% less compared to the cytoplasmic dim cells.
Figure 5. Co-analysis of tumor marker, cadherin expression, and DNA content.
A) An image is shown of a murine dermal tissue slice co stained for nuclei with DAPI, for cadherins (green channel), and for a skin tumor marker (red channel). B) The membrane mask derived from the pan-cadherin channel is shown overlaid on the pan-cadherin image. C) The average pan-cadherin total membrane intensity (TMI) is shown for cells that are dim vs. cells that are bright for the tumor marker. Each bar represents the mean ± SD for n= 35 to 36 cells. ** p < .0001 by Student’s T test. D) A histogram is shown for TNI of the DAPI channel for cells that are dim (blue bars) vs. cells that stained brightly for the tumor marker (magenta bars). Arrows denote most populated bins for each group.
In an additional strategy to analyze the relationship between cytoplasmic marker in this skin cancer model and the expression of cadherins, a tissue section was stained and visualized in three optical channels for nuclear DNA content (with DAPI, blue fluorescence channel), the cytoplasmic tumor marker (red fluorescence channe), and for cadherin family member, utilizing an antibody raised against a protein domain that is common and definitive for the cadherin protein family (pan-cadherin, green fluorescence channel). Areas of the section that stained highly for tumor marker showed little cadherin [removed]Figure 5A), whereas surrounding tissue stained poorly for tumor marker and brightly for pan-cadherin. To quantify the results, a membrane mask was calculated for the image by CyteSeer, utilizing the image of pan-cadherin staining (Figure 5B). As shown, the plasma membrane algorithm successfully identified cell boundaries for the normal cells bordering the tumor. The algorithm also identified cell boundaries within the tumor, itself, even though the pan-cadherin signal was relatively weak.
Calculation of the pan-cadherin mask allowed us to quantify the relationship between tumor marker and cadherin expression. To do this, the total membrane intensity (TMI) value for the cadherin channel for each cell was calculated and the the cell population was sorted on the basis of tumor marker [removed]utilizing similar criteria as for the other samples (see above)) Cells with low expression of tumor marker featured approximately 5-fold greater staining for pan-cadherin than did the cells that stained brightly for the tumor marker (Figure 5C), and this difference was highly significant. These results show that the plasma membrane algorithm can be used to quantify staining in tissue slices, on a cell-by-cell basis, in a manner similar to that which was obtained for cultured cells. Note that DNA content was also quantified on the basis of tumor marker expression, and, as with other tissue samples from this model system (see Figure 2), cells that stained brightly for the tumor marker exhibited a “right-shifted” DNA histogram (Figure 5D).
Discussion
The analysis of DAPI staining of tumor or non-transformed tissue mounted on a slide can be utilized to estimate the DNA content of the cells in the tissue, in a non-destructive manner, and this can be compared to staining in another channel (e.g., tumor markers) via Vala Sciences CyteSeer® software. The above data further demonstrate that CyteSeer can be utilized to compare expression levels of two proteins visualized in separate optical channels for a tissue slice sample. Additionally, staining for cadherins can provide an effective means of visualizing cell borders in this context, which allows accurate cell-by-cell segmentation of the tissue-slice-derived images. The application of Vala Sciences CyteSeer® ‘s image analysis capabilities to tissue samples may be widespread interest to academic, pharmaceutical, and clinical researchers wishing to compare the cellular DNA content or protein expression levels.
References
Cowin, P., Rowlands, T. M., Hatsell, S. J. 2005. Cadherins and catenins in breast cancer. Current Opinion in Cell Biology 17:499-508.
Haass, N. K., Smalley, K. S. M., Herlyn, M. 2004. The role of altered cell-cell communication in melanoma progression. J. Molecular Histol. 35:309-318.
Wohlrab, D., Klapperstuck, T., Holzhausen, H. J., Held, A., Hein, W. 2005. DNA image cytometry on sections compared with flow cytometry in human bone metastases. Oncol. Rep. 14:1005-1012.
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