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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 WWOX levels in prostate carcinoma cells.LNCaP cells and HUVECs in coculture were labeled using (panel A) DAPI, to identify nuclei, (panel B) anti-CDw75 antibodies, to identify LNCaP cells, and (panel C) anti-WWOX antibodies. In panel D, cells classified as HUVEC are shown as red squares, adjacent LNCaP cells as yellow squares and non-adjacent LNCaP cells as blue squares.
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pone-0006822-g003: Identification of WWOX levels in prostate carcinoma cells.LNCaP cells and HUVECs in coculture were labeled using (panel A) DAPI, to identify nuclei, (panel B) anti-CDw75 antibodies, to identify LNCaP cells, and (panel C) anti-WWOX antibodies. In panel D, cells classified as HUVEC are shown as red squares, adjacent LNCaP cells as yellow squares and non-adjacent LNCaP cells as blue squares.

Mentions: To test the response of WWOX levels to coculture, the methodology needed to be altered. Specifically, the method is currently not robust in four channels, due to spectral overlap in the blue to orange range of the spectra and some weakness in the intensity in the red range. As such, labeling of one antigen needed to be omitted to include WWOX. We tried omitting the CD31 antigen and testing whether CDw75 staining was sufficient to differentiate between LNCaP cells and HUVECs. Monocultures and cocultures were evaluated to determine the distribution of CDw75 staining levels in each cell type and to determine the point at which HUVECs began to be called as LNCaPs. The range of levels for the two cell types was similar to what was observed in the initial phase of the study (Figure 1), where LNCaP cells showed a broad range of staining, but only a few cells per field were stained lightly enough that they could be classified as HUVECs, and therefore this method of distinguishing the two cell types appeared robust enough to allow for staining of WWOX or phospho-WWOX as well. The identification of cell types and quantitation of WWOX levels are shown in Figure 3. Association of the signaling intensities with the correct cell type was improved over initial studies (described above) by limiting the number of cells plated, particularly for the 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)

Identification of WWOX levels in prostate carcinoma cells.LNCaP cells and HUVECs in coculture were labeled using (panel A) DAPI, to identify nuclei, (panel B) anti-CDw75 antibodies, to identify LNCaP cells, and (panel C) anti-WWOX antibodies. In panel D, cells classified as HUVEC are shown as red squares, adjacent LNCaP cells as yellow squares and non-adjacent LNCaP cells as blue squares.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006822-g003: Identification of WWOX levels in prostate carcinoma cells.LNCaP cells and HUVECs in coculture were labeled using (panel A) DAPI, to identify nuclei, (panel B) anti-CDw75 antibodies, to identify LNCaP cells, and (panel C) anti-WWOX antibodies. In panel D, cells classified as HUVEC are shown as red squares, adjacent LNCaP cells as yellow squares and non-adjacent LNCaP cells as blue squares.
Mentions: To test the response of WWOX levels to coculture, the methodology needed to be altered. Specifically, the method is currently not robust in four channels, due to spectral overlap in the blue to orange range of the spectra and some weakness in the intensity in the red range. As such, labeling of one antigen needed to be omitted to include WWOX. We tried omitting the CD31 antigen and testing whether CDw75 staining was sufficient to differentiate between LNCaP cells and HUVECs. Monocultures and cocultures were evaluated to determine the distribution of CDw75 staining levels in each cell type and to determine the point at which HUVECs began to be called as LNCaPs. The range of levels for the two cell types was similar to what was observed in the initial phase of the study (Figure 1), where LNCaP cells showed a broad range of staining, but only a few cells per field were stained lightly enough that they could be classified as HUVECs, and therefore this method of distinguishing the two cell types appeared robust enough to allow for staining of WWOX or phospho-WWOX as well. The identification of cell types and quantitation of WWOX levels are shown in Figure 3. Association of the signaling intensities with the correct cell type was improved over initial studies (described above) by limiting the number of cells plated, particularly for the 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