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

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.

Figure 1

Claudin proteins are shed from cancer cell lines. A, Cell lysates and culture media were collected from ovarian cancer cell lines, and analyzed by immunoblotting for the expression of claudin-1, claudin-3, claudin-4 and claudin-5. Ovarian cancer cell lines used: OVCAR-2 (1), OVCAR-3 (2), OVCA420 (3), OVCA433 (4), BG-1 (5), Hey (6), UCI101 (7), and A2780 (8). B, Cell lysates and culture media were collected from various cancer cell lines, and analyzed by immunoblotting for the expression of claudin-1, claudin-3, claudin-4 and claudin-5. Cell lines analyzed are: colon cancer HCT-116 (1), colon cancer SW-480 (2), breast cancer MCF-7 (3), and ovarian cancer OVCAR-3 (4). Claudins expressed in the various cell lines were released in the media.

Figure 2

Released claudin proteins are present in exosome particles. A, BG-1 culture media was centrifuged sequentially and analyzed by immunoblotting for the expression of claudin-4. Claudin-4 was sedimented by a 100,000 g centrifugation (100 K Centrif.) but not by 10,000 g centrifugation (10 K Centrif.). B, Sucrose gradient analysis of the 100,000 g pellet. Following separation of the pellet on a sucrose gradient, ten successive fractions of increasing densities were collected and analyzed by immunoblotting for the expression of the indicated claudin proteins. The density of the various fractions was measured and is indicated on top of the lanes. C, Transmission electron microscopy image of exosomes released from ovarian cancer cells. Exosomes isolated from BG-1 culture media by the 100,000 g centrifugation were fixed, negatively stained, and observed by TEM. The exosomes observed are 40–100 nm in diameter and exhibit the typical cup-shaped morphology. D, Indirect immunogold labeling with anti-claudin-4 antibody followed by a secondary antibody coupled to 6-nm gold particles. The gold particles (dark dots on the figure) clearly localize to the exosomes.

Figure 3

The kinetics of claudin release. A, BG-1 cells were pulsed-labeled with [³⁵S]methionine and [³⁵S]cysteine, chased for various times as indicated, before the media was collected. Claudin-4 was immunoprecipited from the media or from the [³⁵S]-labeled whole cell lysate (as control for the total amount of labeled claudin-4) and analyzed by SDS-PAGE. Claudin-4 is released rapidly in the media, with radiolabeled claudin-4 detectable as early as 2 hours post-labeling. B, The radiolabeled claudin-4 released into the culture media was quantitated by phosphorimaging of the blot shown in (A) and plotted against time. Claudin-4 increase in the media is linear with time.

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.