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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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Image mosaic analysis of p73 levels in prostate carcinoma cells.Antigen intensities in LNCaP cells are clustered by proximity to HUVECs to allow overall comparisons of aggregate intensity levels between groups. Top: LNCaP cells adjacent to HUVECs; bottom: LNCaP cells non-adjacent to HUVECs.
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pone-0006822-g007: Image mosaic analysis of p73 levels in prostate carcinoma cells.Antigen intensities in LNCaP cells are clustered by proximity to HUVECs to allow overall comparisons of aggregate intensity levels between groups. Top: LNCaP cells adjacent to HUVECs; bottom: LNCaP cells non-adjacent to HUVECs.

Mentions: As noted previously, one of the major challenges to this approach is the subtly of the measurements. Specifically, changes in two groups of cells are detected using population-based methods, such as the KS statistic, and need to be reconciled with direct observation wherever possible. This is difficult to achieve by examining single fields of cells, where the variability in each sample group can make group-wide trends difficult to detect. To address this, we have grouped images of cells into mosaics, which provide a stronger visual effect based on larger groups of cells within each class. The results for the p73 in adjacent and non-adjacent LNCaP cells are shown in Figure 7. The mosaics support the analytical methods identification of p73 levels being lower inLNCaP cells that contact HUVECs.


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)

Image mosaic analysis of p73 levels in prostate carcinoma cells.Antigen intensities in LNCaP cells are clustered by proximity to HUVECs to allow overall comparisons of aggregate intensity levels between groups. Top: LNCaP cells adjacent to HUVECs; bottom: LNCaP cells non-adjacent to HUVECs.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006822-g007: Image mosaic analysis of p73 levels in prostate carcinoma cells.Antigen intensities in LNCaP cells are clustered by proximity to HUVECs to allow overall comparisons of aggregate intensity levels between groups. Top: LNCaP cells adjacent to HUVECs; bottom: LNCaP cells non-adjacent to HUVECs.
Mentions: As noted previously, one of the major challenges to this approach is the subtly of the measurements. Specifically, changes in two groups of cells are detected using population-based methods, such as the KS statistic, and need to be reconciled with direct observation wherever possible. This is difficult to achieve by examining single fields of cells, where the variability in each sample group can make group-wide trends difficult to detect. To address this, we have grouped images of cells into mosaics, which provide a stronger visual effect based on larger groups of cells within each class. The results for the p73 in adjacent and non-adjacent LNCaP cells are shown in Figure 7. The mosaics support the analytical methods identification of p73 levels being lower inLNCaP cells that contact HUVECs.

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