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Ovarian tumours, cell isolation, cell survival and sex steroids
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Ovarian cancer is the most deadly malignancy of the female reproductive tract. Ovarian epithelial tumours are considered to be endocrine related, but the factors that govern their genesis and progression remain unclear. Radical surgery and recent advances in chemotherapy have only moderately improved survival rates. Biological and endocrine approaches may help to satisfy the pressing need for alternative therapies.

Aims: The aims of the studies included in this thesis were to isolate and culture ovarian tumour cells with a high epithelial cell fraction (ECF) and to evaluate the fluorometric microculture cytotoxity assay (FMCA) as a tool in the development of alternative therapies. We also aimed to measure the effects of bispecific antibodies (BsAbs) directed against three ovarian tumour markers, separately and in combination, and to study the effects of sex steroids on tumour cell survival, on autonomous steroid secretion and on the expression of estrogen (ER) and progesterone receptors (PR).

Methods: Tumour cells from a total of 75 patient were isolated and cultured in the experiment described by the four papers. The cells were incubated in the presence of BsAbs and peripheral blood mononuclear cells (PBMCs), and at varying concentrations of sex steroids. The FMCA and the neutral red cytotoxity assay were used to measure tumour cell survival. The time-resolved fluoroimmunoassay technique - DELFIA was used to measure steroid concentrations and immunohistochemistry was used to detect tumour markers, ER and PR.

Results and conclusions: It was shown that it is possible to isolate tumour cells from benign as well as malignant tumours, and that the fraction of malignant and atypical cells from carcinoma and borderline tumours can be determined in the cell preparation. Reliable predictive tests such as the FMCA performed on primary cell cultures can make it possible to create individual patinet profiles, thus optimising drug treatment.

Analysis of the BsAbs cultures showed that a combination of the three BsAbs used in our study was the most efficient in tumour cell elimination. In some cases however, the effect of one single BsAb was better than the combination treatment, and this suggests that an optimal therapeutic approach based on BsAbs in a clinical setting requires a panel of BsAbs directed against the commonly expressed ovarian tumour markers.

Tumour cells cultured in 17 β-estradiol and testosterone showed a reduced cell survival and this reduction was inversely proportional to hormone concentrations within the range studied. It was found that 17 β-estradiol was secreted from the primary cell cultures and, interestingly, the amount of 17 β-estradiol secreted increased with increasing levels of 17 β-estradiol in the environment. The majority of the isolated cells analysed for ER and PR expressed both of these receptors to some degree, i.e. the combination ER+/PR+ was the most common. Both ER and PR expression decreased after 72 h culture, revealing an unexpectedly dynamic system. Lowering levels of 17 β-estradiol in cell cultures reduced cell survival, but it appears that this observation depends on factors other than ER. The survival rates of cells cultured in progesterone seemed to be inversely related to their PR expression. It is believed that 17 β-estradiol has an antiapoptotic effect on ovarian surface epithelial (OSE) cells. Reduction of 17 β-estradiol in the environment may inhibit this effect, resulting in reduced cell survival. The ovarian epithelial tumour cell's ability to secrete 17 β-estradiol  suggests that epithelial ovarian tumours play an active role in altering their own hormonal environment, promoting tumour progression.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2002. , 54 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 799
National Category
Obstetrics, Gynecology and Reproductive Medicine
Research subject
Obstetrics and Gynaecology
Identifiers
URN: urn:nbn:se:umu:diva-44796ISBN: 91-7305-271-X (print)OAI: oai:DiVA.org:umu-44796DiVA: diva2:421948
Public defence
2002-09-13, Hörsal E04, byggnad 6E, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Available from: 2011-06-10 Created: 2011-06-10 Last updated: 2011-06-10Bibliographically approved
List of papers
1. Isolation and culture of ovarian tumour cells, cytological and cell survival evaluation
Open this publication in new window or tab >>Isolation and culture of ovarian tumour cells, cytological and cell survival evaluation
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1999 (English)In: Anticancer Research, ISSN 0250-7005, E-ISSN 1791-7530, Vol. 19, no 6B, 5069-5073 p.Article in journal (Refereed) Published
Abstract [en]

The purpose of this study was to evaluate the reproducibility and reliability of the fluorometric microculture cytotoxicity assay (FMCA). Emphasis was placed on obtaining pure tumour cell cultures which were subjected to careful cytological evaluation. Preparations of 39 ovarian tumours, malignant, borderline and benign were made, of which 37 were successfully cultured. In 34 of the 37 tumour cell cultures, the epithelial cell fraction was > 90%, and in 30 of 39 cultures the epithelial cell fraction was > 95%. Transportation within 24 h and the 72 h incubation did not change the yield or epithelial cell fraction. There was a linear relationship between fluorescence and the number of viable cells. The fluorescence increased with time, making only comparisons within each assay plate possible. The sensitivity of the method makes it possible to perform many analyses on a small amount of material. The method also makes it possible to study cells derived from all stages of the disease, including benign tumours.

Identifiers
urn:nbn:se:umu:diva-44794 (URN)10697512 (PubMedID)
Available from: 2011-06-10 Created: 2011-06-10 Last updated: 2017-12-11Bibliographically approved
2. Bispecific antibody-mediated lysis of primary cultures of ovarian carcinoma cells using multiple target antigens
Open this publication in new window or tab >>Bispecific antibody-mediated lysis of primary cultures of ovarian carcinoma cells using multiple target antigens
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1999 (English)In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 83, no 2, 270-277 p.Article in journal (Refereed) Published
Abstract [en]

We have shown previously that a bispecific antibody (BsAb) directed against both germ-cell alkaline phosphatase (GCAP) and the CD3 complex on mouse T cells could effectively eliminate GCAP-positive tumor cells in vivo using an immunocompetent mouse model. However, some GCAP-negative tumor cells were still able to grow, suggesting that BsAb therapy, when used in a clinical setting, could benefit from targeting several tumor markers to prevent outgrowth of tumor cells lacking a targeted marker. To test this hypothesis, we developed an in vitro model based on primary human ovarian carcinoma (OC) cultures and BsAbs directed against human T cells and several tumor markers [placental alkaline phosphatase (PLAP), GCAP, folate-binding protein (FBP) and CA19.9]. OC cells, isolated from primary tumors, were co-cultured with human peripheral blood mononuclear cells in the presence or absence of various concentrations of BsAbs against PLAP/GCAP, FBP and CA19.9 administered separately or in combination. Results derived from 18 primary OC samples showed that the combination treatment was better than or equally effective as the best single BsAB treatment in 60% of cases. Sometimes targeting FBP, PLAP/GCAP or CA19.9 alone was superior to targeting all simultaneously. Combining each BsAb with a low dose of IL-2 was always beneficial. These results indicate that before using a specific BsAb in the clinic, it is important to determine the optimal BsAb for each patient using this in vitro assay on cells from the removed tumor mass.

Identifiers
urn:nbn:se:umu:diva-44795 (URN)10.1002/(SICI)1097-0215(19991008) (DOI)10471538 (PubMedID)
Available from: 2011-06-10 Created: 2011-06-10 Last updated: 2017-12-11Bibliographically approved
3. Low sex steroid environment affects survival and steroid secretion of ovarian tumour cells in primary cultures
Open this publication in new window or tab >>Low sex steroid environment affects survival and steroid secretion of ovarian tumour cells in primary cultures
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2002 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 20, no 3, 589-594 p.Article in journal (Refereed) Published
Abstract [en]

Ovarian epithelial tumours are considered to be endocrine related. The effects of an environment with low levels of the steroid hormones 17 beta-estradiol, testosterone or progesterone on cell survival and steroid secretion were studied in primary cell cultures derived from 25 patients suffering from epithelial ovarian tumours. Tumour cells cultured in 17 beta-estradiol and testosterone showed a reduced cell survival (-10.3 +/- 2.3% and -15.6 +/- 2.7% minimum survival respectively). This reduction was inversely proportional to hormone concentrations within the range studied. No similar effect was observed in the progesterone cultures. It was found that 17 beta-estradiol was secreted from the primary cell cultures and, interestingly, the amount of 17 beta-estradiol secreted increased with increasing levels of 17 beta-estradiol in the environment. Neither progesterone nor testosterone production was observed in any of the cultures studied. It is believed that 17 beta-estradiol has an antiapoptotic effect on ovarian surface epithelial (OSE) cells. Reduction of 17 beta-estradiol in the environment may inhibit this effect, resulting in reduced cell survival. The ability of ovarian epithelial tumour cells to secrete 17 beta-estradiol suggests that epithelial ovarian tumours play an active role in altering their own hormonal environment, promoting tumour progression.

Identifiers
urn:nbn:se:umu:diva-44792 (URN)11836573 (PubMedID)
Available from: 2011-06-10 Created: 2011-06-10 Last updated: 2017-12-11Bibliographically approved
4. Effects of sex steroids on survival and receptor expression in ovarian epithelial tumour cells
Open this publication in new window or tab >>Effects of sex steroids on survival and receptor expression in ovarian epithelial tumour cells
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2003 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 22, no 6, 1257-1262 p.Article in journal (Refereed) Published
Abstract [en]

The factors that govern the genesis and progression of ovarian cancer remain unclear. It is thought that ovarian tumours are endocrine related and hormone dependent. We therefore investigated the effects of the sex steroids progesterone, testosterone and 17 beta-estradiol on tumour cell survival and the expression of estrogen receptors (ER) and progesterone receptors (PR) in tumour cells. The study was performed on primary cell cultures derived from patients suffering from epithelial ovarian cancer. The majority of the cells isolated expressed ER and PR to some degree, the combination ER+/PR+ was the most common. Both ER and PR expression decreased after 72-h culture, revealing an unexpectedly dynamic system. The survival rates of cells cultured in progesterone seemed to be inversely related to their PR expression. Lowering levels of 17 beta-estradiol and testosterone in cell cultures reduced cell survival, but it appears that this observation depends on factors other than ER.

Identifiers
urn:nbn:se:umu:diva-44793 (URN)12738991 (PubMedID)
Available from: 2011-06-10 Created: 2011-06-10 Last updated: 2017-12-11Bibliographically approved

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