Limits...
Impedimetric detection of mutant p53 biomarker-driven metastatic breast cancers under hyposmotic pressure.

Shi M, Shtraizent N, Polotskaia A, Bargonetti J, Matsui H - PLoS ONE (2014)

Bottom Line: The results showed that knockdown of mtp53 leads to decrease in cell swelling.In addition, by means of two types of impedimetric detection systems we consistently detected enhancement of impedance signal in mtp53-expressing breast cancer cells.The identification via the electric measurement can be accomplished within 10 minutes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Hunter College and the Graduate Center, City University of New York, New York, New York, United States of America.

ABSTRACT
In cancer cells, the oncogenic mutant p53 (mtp53) protein is present at high levels and gain-of-function (GOF) activities with more expression of mtp53 proteins contribute to tumor growth and metastasis. Robust analytical approaches that probe the degree of metastasis of cancer cells in connection with the mtp53 activity will be extremely useful not only for establishing a better cancer prognosis but also understanding the fundamental mechanism of mtp53 oncogenic action. Here we assessed the influence of mtp53 in breast cancers to the mechanical property of breast cancer cells. Recently, ovarian and kidney cancer cell lines have been shown to have higher cellular elasticity as compared to normal cells assessed by monitoring the degree of deformation under hyposmotic pressure. To make fast detection in large scale, the impedance measurement was applied to monitor the swelling ratio of cells with time. The results showed that knockdown of mtp53 leads to decrease in cell swelling. In addition, by means of two types of impedimetric detection systems we consistently detected enhancement of impedance signal in mtp53-expressing breast cancer cells. Based on this observation we hypothesize that highly expressed mtp53 in metastatic mutant breast cancers can promote tumor progression by making cells more deformable and easier to spread out through extracellular matrix. The identification via the electric measurement can be accomplished within 10 minutes. All results in this report suggest that electric probing for the extent of the mtp53 expression of breast cancer cells may serve as a meaningful fingerprint for the cancer diagnostics, and this outcome will also have an important clinical implication for the development of mtp53-based targeting for tumor detection and treatment.

Show MeSH

Related in: MedlinePlus

Variations of impedance (Z’) as the function of time resulted from the swelling under hyposmotic stress.With regard to all the samples, 1000 cancer cells with dox (solid lines) or without dox (dotted lines) were incubated with electrodes for 30 min. A. MDA-231.shp53 (clone 1D10), B. MDA-231.shp53 (clone 2C9), C. MDA-468.shp53 (clone 2F3), D. MDA-MB-231 parental, E. MDA-MB-231 STGM, and F. MDA-MB-468 STGM breast cancer cells.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4060997&req=5

pone-0099351-g004: Variations of impedance (Z’) as the function of time resulted from the swelling under hyposmotic stress.With regard to all the samples, 1000 cancer cells with dox (solid lines) or without dox (dotted lines) were incubated with electrodes for 30 min. A. MDA-231.shp53 (clone 1D10), B. MDA-231.shp53 (clone 2C9), C. MDA-468.shp53 (clone 2F3), D. MDA-MB-231 parental, E. MDA-MB-231 STGM, and F. MDA-MB-468 STGM breast cancer cells.

Mentions: To confirm this trend of structural transformation with a larger number of cells, we investigated the impedance variation of mtp53-containing breast cancer cells on the order of 1,000 cells before and after applying hyposmotic pressure. Previously, ovarian and kidney cancer cell lines were confirmed to increase impedance values as these cells were swelled by mixing with water [12] and here we used the same protocol to correlate the elastic structural characteristic with the degree of mtp53 expression and the metastatic feature. After interdigitated electrodes were coated with polylysine for cell adhesion, 5 µL of cell suspension was incubated on top of the electrode and then deionized water was added to trigger the swelling via the increase of hyposmotic stress. Impedance changes in MDA-231.shp53 and MDA-468.shp53 cells were measured over time at 20 kHz, the same protocol previously optimized for the sensitive detection of cancer cells [12]. The real part of the impedance (Z’) increased rapidly for all of these cells, indicating that these cells are elastic. However, after addition of doxycycline their impedance values decreased and the difference became clear after 60 s of swelling time due to the reduced elasticity (Figures 4 -A, B, and C). All control cell lines, MDA-MB-231 parental, MDA-MB-231 STGM and MDA-MB-468 STGM, also showed the same impedance values through the swelling time with and without doxycycline (Figures 4-D, E, and F), meaning that these cells swelled in the same volume expansion % regardless of the presence of doxycycline. The detection limit of breast cancer cells with and without mtp53 expression is 2 cells/µL with two times the standard deviation of blank signal in the swelling time of 120 s. Thus, this comparison suggests that breast cancer cells with different levels of mtp53 could be screened by the impedance variation induced by the volume change under the hyposmotic pressure in the range of swelling time between 60 s and 120 s where the impedance difference between mutant breast cancer cells and the one with mtp53 knockdown is significant. It should be noted that the size of MDA-231.shp53 increased with the incubation time and reached the plateau at 60 s of the incubation time (Figure S1), agreeing with the impedance change profile in Figure 4. From this observation, the cell expansion limit in an average size change % was determined as 38%. The agreement between the size change and the impedance profiles indicates that there is no major loss of cancer cells via lysis in the range of impedance measurement times; because no significant size change is observed after 60 s of incubation time under hyposmotic pressure, the impedance values of cells should be decreased if many cells are lost by lysis. However, the decrease of impedance values is not observed as shown in Figure 4, supporting that lysis does not interfere with the impedance measurement. In addition, a major loss of cells is not recognized in a series of fluorescence images under osmosis. The feature for no size change of cells after a few minutes of the osmosis is consistent with cells returning to iso-osmotic medium, which was also previously observed in other cells and mutants [16].


Impedimetric detection of mutant p53 biomarker-driven metastatic breast cancers under hyposmotic pressure.

Shi M, Shtraizent N, Polotskaia A, Bargonetti J, Matsui H - PLoS ONE (2014)

Variations of impedance (Z’) as the function of time resulted from the swelling under hyposmotic stress.With regard to all the samples, 1000 cancer cells with dox (solid lines) or without dox (dotted lines) were incubated with electrodes for 30 min. A. MDA-231.shp53 (clone 1D10), B. MDA-231.shp53 (clone 2C9), C. MDA-468.shp53 (clone 2F3), D. MDA-MB-231 parental, E. MDA-MB-231 STGM, and F. MDA-MB-468 STGM breast cancer cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4060997&req=5

pone-0099351-g004: Variations of impedance (Z’) as the function of time resulted from the swelling under hyposmotic stress.With regard to all the samples, 1000 cancer cells with dox (solid lines) or without dox (dotted lines) were incubated with electrodes for 30 min. A. MDA-231.shp53 (clone 1D10), B. MDA-231.shp53 (clone 2C9), C. MDA-468.shp53 (clone 2F3), D. MDA-MB-231 parental, E. MDA-MB-231 STGM, and F. MDA-MB-468 STGM breast cancer cells.
Mentions: To confirm this trend of structural transformation with a larger number of cells, we investigated the impedance variation of mtp53-containing breast cancer cells on the order of 1,000 cells before and after applying hyposmotic pressure. Previously, ovarian and kidney cancer cell lines were confirmed to increase impedance values as these cells were swelled by mixing with water [12] and here we used the same protocol to correlate the elastic structural characteristic with the degree of mtp53 expression and the metastatic feature. After interdigitated electrodes were coated with polylysine for cell adhesion, 5 µL of cell suspension was incubated on top of the electrode and then deionized water was added to trigger the swelling via the increase of hyposmotic stress. Impedance changes in MDA-231.shp53 and MDA-468.shp53 cells were measured over time at 20 kHz, the same protocol previously optimized for the sensitive detection of cancer cells [12]. The real part of the impedance (Z’) increased rapidly for all of these cells, indicating that these cells are elastic. However, after addition of doxycycline their impedance values decreased and the difference became clear after 60 s of swelling time due to the reduced elasticity (Figures 4 -A, B, and C). All control cell lines, MDA-MB-231 parental, MDA-MB-231 STGM and MDA-MB-468 STGM, also showed the same impedance values through the swelling time with and without doxycycline (Figures 4-D, E, and F), meaning that these cells swelled in the same volume expansion % regardless of the presence of doxycycline. The detection limit of breast cancer cells with and without mtp53 expression is 2 cells/µL with two times the standard deviation of blank signal in the swelling time of 120 s. Thus, this comparison suggests that breast cancer cells with different levels of mtp53 could be screened by the impedance variation induced by the volume change under the hyposmotic pressure in the range of swelling time between 60 s and 120 s where the impedance difference between mutant breast cancer cells and the one with mtp53 knockdown is significant. It should be noted that the size of MDA-231.shp53 increased with the incubation time and reached the plateau at 60 s of the incubation time (Figure S1), agreeing with the impedance change profile in Figure 4. From this observation, the cell expansion limit in an average size change % was determined as 38%. The agreement between the size change and the impedance profiles indicates that there is no major loss of cancer cells via lysis in the range of impedance measurement times; because no significant size change is observed after 60 s of incubation time under hyposmotic pressure, the impedance values of cells should be decreased if many cells are lost by lysis. However, the decrease of impedance values is not observed as shown in Figure 4, supporting that lysis does not interfere with the impedance measurement. In addition, a major loss of cells is not recognized in a series of fluorescence images under osmosis. The feature for no size change of cells after a few minutes of the osmosis is consistent with cells returning to iso-osmotic medium, which was also previously observed in other cells and mutants [16].

Bottom Line: The results showed that knockdown of mtp53 leads to decrease in cell swelling.In addition, by means of two types of impedimetric detection systems we consistently detected enhancement of impedance signal in mtp53-expressing breast cancer cells.The identification via the electric measurement can be accomplished within 10 minutes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Hunter College and the Graduate Center, City University of New York, New York, New York, United States of America.

ABSTRACT
In cancer cells, the oncogenic mutant p53 (mtp53) protein is present at high levels and gain-of-function (GOF) activities with more expression of mtp53 proteins contribute to tumor growth and metastasis. Robust analytical approaches that probe the degree of metastasis of cancer cells in connection with the mtp53 activity will be extremely useful not only for establishing a better cancer prognosis but also understanding the fundamental mechanism of mtp53 oncogenic action. Here we assessed the influence of mtp53 in breast cancers to the mechanical property of breast cancer cells. Recently, ovarian and kidney cancer cell lines have been shown to have higher cellular elasticity as compared to normal cells assessed by monitoring the degree of deformation under hyposmotic pressure. To make fast detection in large scale, the impedance measurement was applied to monitor the swelling ratio of cells with time. The results showed that knockdown of mtp53 leads to decrease in cell swelling. In addition, by means of two types of impedimetric detection systems we consistently detected enhancement of impedance signal in mtp53-expressing breast cancer cells. Based on this observation we hypothesize that highly expressed mtp53 in metastatic mutant breast cancers can promote tumor progression by making cells more deformable and easier to spread out through extracellular matrix. The identification via the electric measurement can be accomplished within 10 minutes. All results in this report suggest that electric probing for the extent of the mtp53 expression of breast cancer cells may serve as a meaningful fingerprint for the cancer diagnostics, and this outcome will also have an important clinical implication for the development of mtp53-based targeting for tumor detection and treatment.

Show MeSH
Related in: MedlinePlus