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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

Distribution of c-Jun and p73 levels in LNCaP cells cocultured with HUVECs.Cumulative plots of c-Jun (panel A) and p73 (panel B) in adjacent and non-adjacent LNCaP cells (red and black, respectively). Data are from single wells, the ratio of c-Jun expression for the well analyzed in this figure was 0.9952, and that of p73 expression was 0.9155.
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pone-0006822-g006: Distribution of c-Jun and p73 levels in LNCaP cells cocultured with HUVECs.Cumulative plots of c-Jun (panel A) and p73 (panel B) in adjacent and non-adjacent LNCaP cells (red and black, respectively). Data are from single wells, the ratio of c-Jun expression for the well analyzed in this figure was 0.9952, and that of p73 expression was 0.9155.

Mentions: The data from these experiments were also evaluated at the population level through the KS statistic. The results for c-Jun shows that the small difference observed in the adjacent/non-adjacent ratio in Table 2 (0.9952) are reflected in Figure 6A as highly coincident curves. While some of tests of coculture on c-Jun levels appear to show a difference (ratios<0.95 and a p value of 0.482), the effect is difficult to establish through multiple testing. In the case of p73, shown in Figure 6B, the difference of 0.9155 (p value<1×10−3) in the overall ratio is seen in the separation of the distribution curves. While subtle, the difference is reflected in the entire population rather than a specific subset of cells, such as a group of apoptotic cells that are specific to the adjacent cells. Statistical summaries for the comparisons in Figure 5 and Figure 6 are shown in Table 3.


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)

Distribution of c-Jun and p73 levels in LNCaP cells cocultured with HUVECs.Cumulative plots of c-Jun (panel A) and p73 (panel B) in adjacent and non-adjacent LNCaP cells (red and black, respectively). Data are from single wells, the ratio of c-Jun expression for the well analyzed in this figure was 0.9952, and that of p73 expression was 0.9155.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006822-g006: Distribution of c-Jun and p73 levels in LNCaP cells cocultured with HUVECs.Cumulative plots of c-Jun (panel A) and p73 (panel B) in adjacent and non-adjacent LNCaP cells (red and black, respectively). Data are from single wells, the ratio of c-Jun expression for the well analyzed in this figure was 0.9952, and that of p73 expression was 0.9155.
Mentions: The data from these experiments were also evaluated at the population level through the KS statistic. The results for c-Jun shows that the small difference observed in the adjacent/non-adjacent ratio in Table 2 (0.9952) are reflected in Figure 6A as highly coincident curves. While some of tests of coculture on c-Jun levels appear to show a difference (ratios<0.95 and a p value of 0.482), the effect is difficult to establish through multiple testing. In the case of p73, shown in Figure 6B, the difference of 0.9155 (p value<1×10−3) in the overall ratio is seen in the separation of the distribution curves. While subtle, the difference is reflected in the entire population rather than a specific subset of cells, such as a group of apoptotic cells that are specific to the adjacent cells. Statistical summaries for the comparisons in Figure 5 and Figure 6 are shown in Table 3.

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