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Claudin-containing exosomes in the peripheral circulation of women with ovarian cancer.

Li J, Sherman-Baust CA, Tsai-Turton M, Bristow RE, Roden RB, Morin PJ - BMC Cancer (2009)

Bottom Line: The CA125 marker was also assessed in these samples and compared with claudin-4 positivity.In our small panel, at a specificity of 98%, the claudin-4 and CA125 tests had sensitivities of 51% and 71%, respectively.The two tests did not appear to be independent and were strongly correlated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cellular and Molecular Biology, National Institute on Aging, Baltimore, MD, USA. lijiang@grc.nia.nih.gov

ABSTRACT

Background: The absence of highly sensitive and specific serum biomarkers makes mass screening for ovarian cancer impossible. The claudin proteins are frequently overexpressed in ovarian cancers, but their potential as prognostic, diagnostic, or detection markers remains unclear. Here, we have explored the possible use of these proteins as screening biomarkers for ovarian cancer detection.

Methods: Claudin protein shedding from cells was examined by immunoblotting of conditioned culture media. The presence of claudins in exosomes released from ovarian cancer cells was demonstrated by sucrose gradient separation and immunogold electron microscopy experiments. Claudin-4-containing exosomes in the plasma of ovarian cancer patients were evaluated in a pilot panel of 63 ovarian cancer patients and 50 healthy volunteers. The CA125 marker was also assessed in these samples and compared with claudin-4 positivity.

Results: We show that full-length claudins can be shed from ovarian cancer cells in culture and found in the media as part of small lipid vesicles known as exosomes. Moreover, 32 of 63 plasma samples from ovarian cancer patients exhibited the presence of claudin-4-containing exosomes. In contrast, only one of 50 samples from individuals without cancer exhibited claudin-4-positive exosomes. In our small panel, at a specificity of 98%, the claudin-4 and CA125 tests had sensitivities of 51% and 71%, respectively. The two tests did not appear to be independent and were strongly correlated.

Conclusion: Our work shows for the first time that claudin-4 can be released from ovarian cancer cells and can be detected in the peripheral circulation of ovarian cancer patients. The development of sensitive assays for the detection of claudin-4 in blood will be crucial in determining whether this approach can be useful, alone or in combination with other screening methods, for the detection of ovarian cancer.

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Claudin-4 expression in the plasma of ovarian cancer patients. A, Representative examples claudin-4 detection in plasma samples by immunoblotting. N21 to N28 are non-cancer plasma controls. P21 to P30 are plasma from ovarian cancer patients. C is a positive control (BG-1 culture media). B, CA125 levels in plasma of ovarian cancer patients are compared to claudin-4 expression levels. ++, claudin-4 level is greater than 0.15 μl of BG-1 24 h culture media; +, claudin-4 level is greater than 0.07 μl of BG-1 24 h culture media; -, no detectable expression of claudin-4. CA125 is plotted on a log scale and the threshold of 125 U/ml is shown by a dotted line.
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Figure 4: Claudin-4 expression in the plasma of ovarian cancer patients. A, Representative examples claudin-4 detection in plasma samples by immunoblotting. N21 to N28 are non-cancer plasma controls. P21 to P30 are plasma from ovarian cancer patients. C is a positive control (BG-1 culture media). B, CA125 levels in plasma of ovarian cancer patients are compared to claudin-4 expression levels. ++, claudin-4 level is greater than 0.15 μl of BG-1 24 h culture media; +, claudin-4 level is greater than 0.07 μl of BG-1 24 h culture media; -, no detectable expression of claudin-4. CA125 is plotted on a log scale and the threshold of 125 U/ml is shown by a dotted line.

Mentions: Our finding that claudins can be released from ovarian cancer cells suggests that these proteins may become useful for the detection of ovarian cancer in patients. We first sought to determine whether exosome-associated claudins could indeed be detected in the peripheral circulation of ovarian cancer patients. Plasma samples from 63 high-grade serous ovarian cancer patients treated at Johns Hopkins University, as well as 50 healthy volunteers were assessed. Exosomes were purified from the plasma by ultracentrifugation and assayed for the presence of claudin-4 and claudin-3 by immunoblotting. Several plasma samples from patients with ovarian cancer exhibited high levels of claudin-4, while blood from control individuals showed very low or undetectable levels (Figure 4A). The pattern obtained with claudin-3 was somewhat similar, but the background levels in the plasma of unaffected women were much higher (data not shown). Claudin-3 was therefore considered less likely than claudin-4 to represent a useful plasma-based ovarian cancer biomarker and was not studied further. Among the 63 ovarian cancer patients, 23 samples demonstrated a strong claudin-4 band with 9 additional samples exhibiting low to moderate levels (Table 1). Thirty one samples had no detectable claudin-4 protein. In contrast, among the control group (n = 50), only one sample was positive (at a low level) and all the other plasma samples were negative (Table 2). Based on these figures, claudin-4 has a sensitivity of 51% (32/63) and specificity of 98% (49/50) for the detection of ovarian cancer.


Claudin-containing exosomes in the peripheral circulation of women with ovarian cancer.

Li J, Sherman-Baust CA, Tsai-Turton M, Bristow RE, Roden RB, Morin PJ - BMC Cancer (2009)

Claudin-4 expression in the plasma of ovarian cancer patients. A, Representative examples claudin-4 detection in plasma samples by immunoblotting. N21 to N28 are non-cancer plasma controls. P21 to P30 are plasma from ovarian cancer patients. C is a positive control (BG-1 culture media). B, CA125 levels in plasma of ovarian cancer patients are compared to claudin-4 expression levels. ++, claudin-4 level is greater than 0.15 μl of BG-1 24 h culture media; +, claudin-4 level is greater than 0.07 μl of BG-1 24 h culture media; -, no detectable expression of claudin-4. CA125 is plotted on a log scale and the threshold of 125 U/ml is shown by a dotted line.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719664&req=5

Figure 4: Claudin-4 expression in the plasma of ovarian cancer patients. A, Representative examples claudin-4 detection in plasma samples by immunoblotting. N21 to N28 are non-cancer plasma controls. P21 to P30 are plasma from ovarian cancer patients. C is a positive control (BG-1 culture media). B, CA125 levels in plasma of ovarian cancer patients are compared to claudin-4 expression levels. ++, claudin-4 level is greater than 0.15 μl of BG-1 24 h culture media; +, claudin-4 level is greater than 0.07 μl of BG-1 24 h culture media; -, no detectable expression of claudin-4. CA125 is plotted on a log scale and the threshold of 125 U/ml is shown by a dotted line.
Mentions: Our finding that claudins can be released from ovarian cancer cells suggests that these proteins may become useful for the detection of ovarian cancer in patients. We first sought to determine whether exosome-associated claudins could indeed be detected in the peripheral circulation of ovarian cancer patients. Plasma samples from 63 high-grade serous ovarian cancer patients treated at Johns Hopkins University, as well as 50 healthy volunteers were assessed. Exosomes were purified from the plasma by ultracentrifugation and assayed for the presence of claudin-4 and claudin-3 by immunoblotting. Several plasma samples from patients with ovarian cancer exhibited high levels of claudin-4, while blood from control individuals showed very low or undetectable levels (Figure 4A). The pattern obtained with claudin-3 was somewhat similar, but the background levels in the plasma of unaffected women were much higher (data not shown). Claudin-3 was therefore considered less likely than claudin-4 to represent a useful plasma-based ovarian cancer biomarker and was not studied further. Among the 63 ovarian cancer patients, 23 samples demonstrated a strong claudin-4 band with 9 additional samples exhibiting low to moderate levels (Table 1). Thirty one samples had no detectable claudin-4 protein. In contrast, among the control group (n = 50), only one sample was positive (at a low level) and all the other plasma samples were negative (Table 2). Based on these figures, claudin-4 has a sensitivity of 51% (32/63) and specificity of 98% (49/50) for the detection of ovarian cancer.

Bottom Line: The CA125 marker was also assessed in these samples and compared with claudin-4 positivity.In our small panel, at a specificity of 98%, the claudin-4 and CA125 tests had sensitivities of 51% and 71%, respectively.The two tests did not appear to be independent and were strongly correlated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Cellular and Molecular Biology, National Institute on Aging, Baltimore, MD, USA. lijiang@grc.nia.nih.gov

ABSTRACT

Background: The absence of highly sensitive and specific serum biomarkers makes mass screening for ovarian cancer impossible. The claudin proteins are frequently overexpressed in ovarian cancers, but their potential as prognostic, diagnostic, or detection markers remains unclear. Here, we have explored the possible use of these proteins as screening biomarkers for ovarian cancer detection.

Methods: Claudin protein shedding from cells was examined by immunoblotting of conditioned culture media. The presence of claudins in exosomes released from ovarian cancer cells was demonstrated by sucrose gradient separation and immunogold electron microscopy experiments. Claudin-4-containing exosomes in the plasma of ovarian cancer patients were evaluated in a pilot panel of 63 ovarian cancer patients and 50 healthy volunteers. The CA125 marker was also assessed in these samples and compared with claudin-4 positivity.

Results: We show that full-length claudins can be shed from ovarian cancer cells in culture and found in the media as part of small lipid vesicles known as exosomes. Moreover, 32 of 63 plasma samples from ovarian cancer patients exhibited the presence of claudin-4-containing exosomes. In contrast, only one of 50 samples from individuals without cancer exhibited claudin-4-positive exosomes. In our small panel, at a specificity of 98%, the claudin-4 and CA125 tests had sensitivities of 51% and 71%, respectively. The two tests did not appear to be independent and were strongly correlated.

Conclusion: Our work shows for the first time that claudin-4 can be released from ovarian cancer cells and can be detected in the peripheral circulation of ovarian cancer patients. The development of sensitive assays for the detection of claudin-4 in blood will be crucial in determining whether this approach can be useful, alone or in combination with other screening methods, for the detection of ovarian cancer.

Show MeSH
Related in: MedlinePlus