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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

Quantitation of WWOX and phospho-WWOX levels in LNCaP cells.The amount of WWOX protein (in panel A) and phospho-WWOX protein (in panel B), are shown for LNCaP cells adjacent to HUVECs (in red) and those cells non-adjacent to HUVECs (in black). Data points represent (phospho-) protein levels per cell as a they contribute to the total antigen intensity for the entire field.
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pone-0006822-g005: Quantitation of WWOX and phospho-WWOX levels in LNCaP cells.The amount of WWOX protein (in panel A) and phospho-WWOX protein (in panel B), are shown for LNCaP cells adjacent to HUVECs (in red) and those cells non-adjacent to HUVECs (in black). Data points represent (phospho-) protein levels per cell as a they contribute to the total antigen intensity for the entire field.

Mentions: A separate observation is that although the differences in many wells are statistically significant (with p values below 1×10−3), the magnitude of the effect appears modest. At this stage we considered a different numerical test of the difference between target protein levels in LNCaP populations. The test we used was the Kolmogorov-Smirnov (or KS) statistic. The test compares two populations as fractional contributions to the total amount of antigen. Results for WWOX levels are shown in Figure 5A and for phosphorylated WWOX, which is mediated by Src, as shown in Figure 5B. The difference in the two curves shows that LNCaP cells adjacent to HUVECs have less WWOX protein than LNCaP cells that are non-adjacent to HUVECs when compared in a ranked listing.


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)

Quantitation of WWOX and phospho-WWOX levels in LNCaP cells.The amount of WWOX protein (in panel A) and phospho-WWOX protein (in panel B), are shown for LNCaP cells adjacent to HUVECs (in red) and those cells non-adjacent to HUVECs (in black). Data points represent (phospho-) protein levels per cell as a they contribute to the total antigen intensity for the entire field.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006822-g005: Quantitation of WWOX and phospho-WWOX levels in LNCaP cells.The amount of WWOX protein (in panel A) and phospho-WWOX protein (in panel B), are shown for LNCaP cells adjacent to HUVECs (in red) and those cells non-adjacent to HUVECs (in black). Data points represent (phospho-) protein levels per cell as a they contribute to the total antigen intensity for the entire field.
Mentions: A separate observation is that although the differences in many wells are statistically significant (with p values below 1×10−3), the magnitude of the effect appears modest. At this stage we considered a different numerical test of the difference between target protein levels in LNCaP populations. The test we used was the Kolmogorov-Smirnov (or KS) statistic. The test compares two populations as fractional contributions to the total amount of antigen. Results for WWOX levels are shown in Figure 5A and for phosphorylated WWOX, which is mediated by Src, as shown in Figure 5B. The difference in the two curves shows that LNCaP cells adjacent to HUVECs have less WWOX protein than LNCaP cells that are non-adjacent to HUVECs when compared in a ranked listing.

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