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Image-based assessment of growth and signaling changes in cancer cells mediated by direct cell-cell contact.

Lapan P, Zhang J, Hill A, Zhang Y, Martinez R, Haney S - PLoS ONE (2009)

Bottom Line: Reduced WWOX signaling has been associated with reduced activation or expression of JNK and p73.We find that p73 levels are also reduced in LNCaP cells grown in contact with HUVECs, but we did not observe such a change in JNK levels.Ironically, a potential challenge to the method is its high level of sensitivity is capable of classifying events as statistically significant (due to the high number cells evaluated individually), when the biological effect may be less clear.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Technologies, Wyeth Research, Cambridge, Massachusetts, USA.

ABSTRACT

Background: Many important biological processes are controlled through cell-cell interactions, including the colonization of metastatic tumor cells and the control of differentiation of stem cells within their niche. Despite the crucial importance of the cellular environment in regulating cellular signaling, in vitro methods for the study of such interactions are difficult and/or indirect.

Methodology/principal findings: We report on the development of an image-based method for distinguishing two cell types grown in coculture. Furthermore, cells of one type that are in direct contact with cells of a second type (adjacent cells) can be analyzed separately from cells that are not within a single well. Changes are evaluated using population statistics, which are useful in detecting subtle changes across two populations. We have used this system to characterize changes in the LNCaP prostate carcinoma cell line when grown in contact with human vascular endothelial cells (HUVECs). We find that the expression and phosphorylation of WWOX is reduced in LNCaP cells when grown in direct contact with HUVECs. Reduced WWOX signaling has been associated with reduced activation or expression of JNK and p73. We find that p73 levels are also reduced in LNCaP cells grown in contact with HUVECs, but we did not observe such a change in JNK levels.

Conclusions/significance: We find that the method described is statistically robust and can be adapted to a wide variety of studies where cell function or signaling are affected by heterotypic cell-cell contact. Ironically, a potential challenge to the method is its high level of sensitivity is capable of classifying events as statistically significant (due to the high number cells evaluated individually), when the biological effect may be less clear. The methodology would be best used in conjunction with additional methods to evaluate the biological role of potentially subtle differences between populations. However, many important events, such as the establishment of a metastatic tumor, occur through rare but important changes, and methods such as we describe here can be used to identify and characterize the contribution of the environment to these changes.

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Related in: MedlinePlus

Identification of adjacent and non-adjacent endothelial cells in culture with prostate carcinoma cells.Coculture of LNCaP cells and HUVECs, LNCaP cells are labeled with anti-CDw75 antibodies and shown in red, HUVECs are labeled with anti-CD44 antibodies, in green. A. An example field image. B. Quantitation of CDw75 fluorescence used in characterizing cells as either LNCaP (high intensity, red) or HUVEC (low intensity, green). C. Mapping LNCaP cells and HUVECs. Locations can be compared to cells in panel A. Cells are identified as adjacent LNCaP in yellow, non-adjacent LNCaP cells in red and HUVECs in green.
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pone-0006822-g001: Identification of adjacent and non-adjacent endothelial cells in culture with prostate carcinoma cells.Coculture of LNCaP cells and HUVECs, LNCaP cells are labeled with anti-CDw75 antibodies and shown in red, HUVECs are labeled with anti-CD44 antibodies, in green. A. An example field image. B. Quantitation of CDw75 fluorescence used in characterizing cells as either LNCaP (high intensity, red) or HUVEC (low intensity, green). C. Mapping LNCaP cells and HUVECs. Locations can be compared to cells in panel A. Cells are identified as adjacent LNCaP in yellow, non-adjacent LNCaP cells in red and HUVECs in green.

Mentions: The algorithm has been used to identify adjacent and non-adjacent LNCaP cells, relative to HUVEC cells, as shown in Figure 1. In Figure 1A, LNCaP cells, labeled with an anti-CDw75 antibody in red, are seeded with HUVECs, stained with an anti-CD44 antibody in green. The intensity of the CDw75-based fluorescence is recorded for each cell in the field. Each cell is numbered, and the intensity for each cell is shown in Figure 1B. From this measurement, cells can be categorized as either cancer or endothelial cells. In addition to the CDw75 staining, other properties associated with cancer cells can be used at this stage, including DNA content (many cancer cell lines, including LNCaP are aneuploid and have significantly higher DNA content than primary cells). These data can be included with the antigen intensity to score cells as cancerous or not, or in some cases can be used as a single parameter for this determination (data not shown). Once the cells have been identified, their spatial information is used to map each cell to its location. Having identified each cell as LNCaP or HUVEC, each cell is then scored for the cells that are near it, the boundary distance being set by the average diameter of the cells. Cells of one type, LNCaP in this example, are therefore further defined as adjacent to HUVEC or not. Cells identified as adjacent are shown schematically in Figure 1C in yellow, or non-adjacent, in red. HUVEC cells are shown as green. From this point forward, quantitative data may be extracted and analyzed for the two subsets of LNCaP cells. Differences between these subsets are indicative of a response to direct contact with HUVEC cells.


Image-based assessment of growth and signaling changes in cancer cells mediated by direct cell-cell contact.

Lapan P, Zhang J, Hill A, Zhang Y, Martinez R, Haney S - PLoS ONE (2009)

Identification of adjacent and non-adjacent endothelial cells in culture with prostate carcinoma cells.Coculture of LNCaP cells and HUVECs, LNCaP cells are labeled with anti-CDw75 antibodies and shown in red, HUVECs are labeled with anti-CD44 antibodies, in green. A. An example field image. B. Quantitation of CDw75 fluorescence used in characterizing cells as either LNCaP (high intensity, red) or HUVEC (low intensity, green). C. Mapping LNCaP cells and HUVECs. Locations can be compared to cells in panel A. Cells are identified as adjacent LNCaP in yellow, non-adjacent LNCaP cells in red and HUVECs in green.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747553&req=5

pone-0006822-g001: Identification of adjacent and non-adjacent endothelial cells in culture with prostate carcinoma cells.Coculture of LNCaP cells and HUVECs, LNCaP cells are labeled with anti-CDw75 antibodies and shown in red, HUVECs are labeled with anti-CD44 antibodies, in green. A. An example field image. B. Quantitation of CDw75 fluorescence used in characterizing cells as either LNCaP (high intensity, red) or HUVEC (low intensity, green). C. Mapping LNCaP cells and HUVECs. Locations can be compared to cells in panel A. Cells are identified as adjacent LNCaP in yellow, non-adjacent LNCaP cells in red and HUVECs in green.
Mentions: The algorithm has been used to identify adjacent and non-adjacent LNCaP cells, relative to HUVEC cells, as shown in Figure 1. In Figure 1A, LNCaP cells, labeled with an anti-CDw75 antibody in red, are seeded with HUVECs, stained with an anti-CD44 antibody in green. The intensity of the CDw75-based fluorescence is recorded for each cell in the field. Each cell is numbered, and the intensity for each cell is shown in Figure 1B. From this measurement, cells can be categorized as either cancer or endothelial cells. In addition to the CDw75 staining, other properties associated with cancer cells can be used at this stage, including DNA content (many cancer cell lines, including LNCaP are aneuploid and have significantly higher DNA content than primary cells). These data can be included with the antigen intensity to score cells as cancerous or not, or in some cases can be used as a single parameter for this determination (data not shown). Once the cells have been identified, their spatial information is used to map each cell to its location. Having identified each cell as LNCaP or HUVEC, each cell is then scored for the cells that are near it, the boundary distance being set by the average diameter of the cells. Cells of one type, LNCaP in this example, are therefore further defined as adjacent to HUVEC or not. Cells identified as adjacent are shown schematically in Figure 1C in yellow, or non-adjacent, in red. HUVEC cells are shown as green. From this point forward, quantitative data may be extracted and analyzed for the two subsets of LNCaP cells. Differences between these subsets are indicative of a response to direct contact with HUVEC cells.

Bottom Line: Reduced WWOX signaling has been associated with reduced activation or expression of JNK and p73.We find that p73 levels are also reduced in LNCaP cells grown in contact with HUVECs, but we did not observe such a change in JNK levels.Ironically, a potential challenge to the method is its high level of sensitivity is capable of classifying events as statistically significant (due to the high number cells evaluated individually), when the biological effect may be less clear.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Technologies, Wyeth Research, Cambridge, Massachusetts, USA.

ABSTRACT

Background: Many important biological processes are controlled through cell-cell interactions, including the colonization of metastatic tumor cells and the control of differentiation of stem cells within their niche. Despite the crucial importance of the cellular environment in regulating cellular signaling, in vitro methods for the study of such interactions are difficult and/or indirect.

Methodology/principal findings: We report on the development of an image-based method for distinguishing two cell types grown in coculture. Furthermore, cells of one type that are in direct contact with cells of a second type (adjacent cells) can be analyzed separately from cells that are not within a single well. Changes are evaluated using population statistics, which are useful in detecting subtle changes across two populations. We have used this system to characterize changes in the LNCaP prostate carcinoma cell line when grown in contact with human vascular endothelial cells (HUVECs). We find that the expression and phosphorylation of WWOX is reduced in LNCaP cells when grown in direct contact with HUVECs. Reduced WWOX signaling has been associated with reduced activation or expression of JNK and p73. We find that p73 levels are also reduced in LNCaP cells grown in contact with HUVECs, but we did not observe such a change in JNK levels.

Conclusions/significance: We find that the method described is statistically robust and can be adapted to a wide variety of studies where cell function or signaling are affected by heterotypic cell-cell contact. Ironically, a potential challenge to the method is its high level of sensitivity is capable of classifying events as statistically significant (due to the high number cells evaluated individually), when the biological effect may be less clear. The methodology would be best used in conjunction with additional methods to evaluate the biological role of potentially subtle differences between populations. However, many important events, such as the establishment of a metastatic tumor, occur through rare but important changes, and methods such as we describe here can be used to identify and characterize the contribution of the environment to these changes.

Show MeSH
Related in: MedlinePlus